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# Future prospects for CD8 + regulatory T cells in immune tolerance

Léa Flippe, Séverine Bézie, Ignacio Anegon, Carole Guillonneau

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Léa Flippe, Séverine Bézie, Ignacio Anegon, Carole Guillonneau. Future prospects for CD8 + regulatory T cells in immune tolerance. Immunological Reviews, 2019, 10.1111/imr.12812. inserm-02341615

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DOI: 10.1111/imr.12812

INVITED REVIEW

Immunological Reviews

WILEY

# Future prospects for CD8⁺ regulatory T cells in immune tolerance

Léa Flippe<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> | Séverine Bézie<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> | Ignacio Anegon<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> | Carole Guillonneau<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

## Correspondence

Carole Guillonneau and Ignacio Anegon, Centre de Recherche en Transplantation et Immunologie INSERM 1064-ITUN, 30 Bd. Jean Monnet, 44093 Nantes Cedex 01, France.

Email: carole.guillonneau@univ-nantes.fr (C. G.) and ignacio.anegon@univ-nantes.fr (I. A.)

## Funding information

National Research Agency, Grant/Award Number: ANR-11-LABX-0016-01 and ANR-10-IBHU-005; Nantes Metropole; Pays de la Loire Region; Progreffe; Fondation pour la Recherche Médicale; Etoiles Montantes of Pays de la Loire; Fondation du rein; European Union, Grant/Award Number: 825392

## Abstract

CD8⁺ Tregs have been long described and significant progresses have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4⁺ Tregs. They are now at the dawn of their clinical use. In this review, we will summarize their phenotypic characteristics, their mechanisms of action, the similarities, differences and synergies between CD8⁺ and CD4⁺ Tregs, and we will discuss the biology, development and induction of CD8⁺ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible improvements notably through genetic modifications.

## KEYWORDS

CD8⁺ Treg, therapy, tolerance

## 1 | INTRODUCTION

CD8⁺ Tregs were the first suppressive cells reported but studies were then focused on CD4⁺ Tregs.<fnref n="1" /> Over the last two decades, we, along with others, deeply investigated the CD8⁺ Tregs.<fnref n="2" /> To date, significant progress have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4⁺ Tregs.<fnref n="3" /> Through this in-depth fundamental research, it has been learned more about their origin and development and such advances will lead to improvement in their generation including from stem cells (unpublished work discussed in this review).

Cell therapy has developed in recent years in the field of transplantation and autoimmune diseases with promising results. Current clinical trials use T cells and non-T cells, and among Tregs, FOXP3⁺ and FOXP3⁻ CD4⁺ Tregs (phase I ONE, ALT-TEN, phase I/IIa CATS1), but not CD8⁺ Tregs. We are at the dawn of the first in human clinical trial using CD8⁺ Treg-cell therapy in transplanted patients (https://www.reshape-h2020.eu/). Experience from previous and current clinical trials using T cell therapy, combined with our extensive knowledge of CD8⁺ Tregs, incremented by the technical revolution of T Cell Receptor (TCR) and chimeric antigen receptor (CAR) engineering, in synergy with Treg promoting drugs, led us to the Eight-Treg first in human clinical trial.

This article is part of a series of reviews covering Tolerance and Exhaustion in Peripheral T and B cells appearing in Volume 292 of *Immunological Reviews*.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
© 2019 The Authors. *Immunological Reviews* published by John Wiley & Sons Ltd.

<footnote n="1">¹Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes, Nantes, France</footnote>

<footnote n="2">²Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France</footnote>

<footnote n="3">³LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France</footnote>
<figure><img src="image_3.png" /></figure>

DOI: 10.1111/imr.12812

INVITED REVIEW

Immunological Reviews

WILEY

# Future prospects for CD8⁺ regulatory T cells in immune tolerance

Léa Flippe<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Séverine Bézie<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Ignacio Anegon<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Carole Guillonneau<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

### Correspondence

Carole Guillonneau and Ignacio Anegon, Centre de Recherche en Transplantation et Immunologie INSERM 1064-ITUN, 30 Bd. Jean Monnet, 44093 Nantes Cedex 01, France.

Email: carole.guillonneau@univ-nantes.fr (C. G.) and ignacio.anegon@univ-nantes.fr (I. A.)

### Funding information

National Research Agency, Grant/Award Number: ANR-11-LABX-0016-01 and ANR-10-IBHU-005; Nantes Metropole; Pays de la Loire Region; Progreffe; Fondation pour la Recherche Médicale; Etoiles Montantes of Pays de la Loire; Fondation du rein; European Union, Grant/Award Number: 825392

### Abstract

CD8⁺ Tregs have been long described and significant progresses have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4⁺ Tregs. They are now at the dawn of their clinical use. In this review, we will summarize their phenotypic characteristics, their mechanisms of action, the similarities, differences and synergies between CD8⁺ and CD4⁺ Tregs, and we will discuss the biology, development and induction of CD8⁺ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible improvements notably through genetic modifications.

### KEYWORDS

CD8⁺ Treg, therapy, tolerance

## 1 | INTRODUCTION

CD8⁺ Tregs were the first suppressive cells reported but studies were then focused on CD4⁺ Tregs.<fnref n="1" /> Over the last two decades, we, along with others, deeply investigated the CD8⁺ Tregs.<fnref n="2" /> To date, significant progress have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4⁺ Tregs.<fnref n="3" /> Through this in-depth fundamental research, it has been learned more about their origin and development and such advances will lead to improvement in their generation including from stem cells (unpublished work discussed in this review).

Cell therapy has developed in recent years in the field of transplantation and autoimmune diseases with promising results. Current clinical trials use T cells and non-T cells, and among Tregs, FOXP3⁺ and FOXP3⁻ CD4⁺ Tregs (phase I ONE, ALT-TEN, phase I/IIa CATS1), but not CD8⁺ Tregs. We are at the dawn of the first in human clinical trial using CD8⁺ Treg-cell therapy in transplanted patients (https://www.reshape-h2020.eu/). Experience from previous and current clinical trials using T cell therapy, combined with our extensive knowledge of CD8⁺ Tregs, incremented by the technical revolution of T Cell Receptor (TCR) and chimeric antigen receptor (CAR) engineering, in synergy with Treg promoting drugs, led us to the Eight-Treg first in human clinical trial.

This article is part of a series of reviews covering Tolerance and Exhaustion in Peripheral T and B cells appearing in Volume 292 of *Immunological Reviews*.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2019 The Authors. *Immunological Reviews* published by John Wiley & Sons Ltd.

<footnote n="1">¹Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes, Nantes, France</footnote>

<footnote n="2">²Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France</footnote>

<footnote n="3">³LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France</footnote>
In this review, we will discuss the biology and development of CD8⁺ Tregs, their contribution to the tolerance status apart from CD4⁺ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible betterments notably through genetic modifications.

## 2 | MAIN CHARACTERISTICS OF CD8⁺ REGULATORY T CELLS

Despite a streamline work from several groups, the definition of CD8⁺ Tregs remains the major problem of this cell subset population and the reason for them not being assessed in clinical protocols. This lack in assessment is making marker discovery even more problematic and slow, leading to a vicious circle. The fact that, at steady state, expression of FOXP3, a sensitive marker of Treg, is lower in the CD8⁺ T cell population compared to the CD4⁺ T cell population in mouse, rat and human,³ has contributed to this disregard of CD8⁺ Treg's potential and interest, despite in vitro and in vivo data demonstrating their suppressive capacity. In addition, the methylation status of FOXP3 in human CD8⁺ Tregs showed an intermediate level of demethylation compared to one of the CD4⁺ Tregs for some locus of the FOXP3 gene.³ We showed that following ex vivo stimulation, human CD8⁺ Tregs sorted based on the expression of CD45RC, a splicing variant of the CD45 molecule key in the signaling of T cells that we use as a marker to isolate CD8⁺ Tregs⁴⁻⁶; FOXP3 is expressed by all expanded CD8⁺ Tregs that display highly upregulated levels of expression vs. blood unstimulated CD8⁺FOXP3⁺, suggesting that FOXP3 can correlate with the in vivo and in vitro suppressive potential of human CD8⁺ Tregs.³ In contrast, in a rat model of transplantation tolerance following costimulation blockade inducing in vivo CD8⁺ Tregs, FOXP3 was not upregulated,⁴ nor was it in a model of tolerance in rat using donor antigen therapy.⁷ FOXP3 is also not found particularly expressed in mouse CD8⁺ Tregs, and rather the transcription factor HELIOS or the surface marker CD122⁺ or CD28⁻ are used to identify CD8⁺ Tregs in mice.⁸,⁹ Other surface markers were also suggested and among the most frequently used CD25, CD127, GITR, CD39, Lag3, and CTLA-4 that are used in different contexts and different species to identify CD8⁺ Tregs and analyze their suppressive activity in vitro and in vivo in autoimmune diseases, immune reactions to non-self, cancer and materno-fetal tolerance.²,¹⁰ Indeed, using a combination of these markers to identify CD8⁺ Tregs, their suppressive action has been demonstrated in autoimmune diseases such as in the experimental autoimmune encephalomyelitis model,¹¹⁻¹³ multiple sclerosis,¹⁴⁻¹⁶ systemic lupus erythematosus (SLE)¹⁷,¹⁸ and primary biliary cirrhosis,¹⁹ in infection in humans with mycobacteria, human immunodeficiency virus or epstein-barr virus,²⁰⁻²³ in cancer in humans and mice²⁴⁻²⁶ or in transplantation in humans, mice and rats.³,⁴,²⁷⁻³² CXCR5 was used to identify antibody-suppressor FOXP3⁺CD8⁺ cells in mice and essential in their function of suppression of humoral immunity, reducing germinal center B cells and CD4⁺ T follicular helper cells in the context of hepatocyte transplantation.³³

A very recent study of single cell RNA sequencing on CD4⁺ Tregs has highlighted the importance of some transcription factors

and extra-membrane molecules.³⁴ This work showed that CD4⁺ Tregs and CD4⁺ Tconvs can be distinguished by the ubiquitous expression of FOXP3, IKZF2 (IKAROS), TNFR2, IL2RA and IL2RB in mice and humans. Analysis using DGE-RNA sequencing of fresh and activated CD8⁺CD45RC<sup>low/-</sup> Tregs allowed us to show the increase in FOXP3 expression and suppressive molecules after activation. However, nowadays no single cell RNA sequencing study is being published on CD8⁺ Tregs. Our analysis of single cell RNA sequencing data on fresh CD8⁺CD45RC<sup>low/-</sup> cells from healthy volunteers confirms the expression of molecules known in CD4⁺ Tregs such as TNFR2 but never clearly showed before in CD8⁺ Tregs (unpublished data).

Soluble factors can also be used to identify and even sort CD8⁺ Tregs since the development of kit using bispecific antibodies allows the sorting of live cytokine-secreting cells. We have shown that sorted human IFNγ⁺IL-10⁺CD8⁺CD45RC<sup>low/-</sup> Tregs were more potent suppressor cells than the rest of the CD8⁺CD45RC<sup>low/-</sup> Tregs³ and this was also indicative of the role of these cytokines in CD8⁺ Treg suppressive activity, in particular IFNγ (Figure 1). Our interest in IFNγ came from the observations that blocking its activity abrogated the CD8⁺CD45RC<sup>low/-</sup> Treg suppressive activity in a model of allogeneic cardiac transplantation.⁴ We further demonstrated that indeed, IFNγ induced indoleamine 2,3-dioxygenase (IDO) production by endothelial cells (ECs) of the graft and plasmacytoid dendritic cells, an enzyme catabolizing tryptophan essential for effector T cells proliferation.⁴,³⁵ IFNγ was also involved in fibroleukin-2 (FGL2) induction, a cytokine acting further through regulatory B cell induction.³⁶ We have also shown that IL-34, a recently discovered cytokine involved in monocyte/macrophage differentiation, was secreted by approximately half of FOXP3⁺CD8⁺ and CD4⁺ Tregs.³⁷ We showed that IL-34 was involved in FOXP3⁺ Treg suppressive activity and acted as a suppressive cytokine since administration in a model of allogeneic cardiac transplantation in combination with a suboptimal dose of rapamycin-induced transplant tolerance. This was the first description of a role for this cytokine in T cell biology and transplantation.³⁸ Thus, a complex regulatory network of cells around CD8⁺ Tregs is acting to sustain tolerance.³⁹ Other more classical cytokines such as TGFβ have also been described as playing a role in CD8⁺ Treg suppressive activity.²

Some of these molecules or mechanisms of action have been evidenced by study of the fetal-maternal interface.⁴⁰,⁴¹ Indeed, in maternal tolerance where the allogeneic fetal trophoblast invades maternal tissues and interacts with maternal leukocytes, the immune system needs to be regulated to tolerate the presence of the fetus. CD8⁺ T cells are the main component of decidual T cells and CD8⁺ Tregs have been evidenced as important in the maintenance of a normal pregnancy.⁴²⁻⁴⁴ Decreased number and altered function of Tim-3⁺CTLA-4⁺CD8⁺ T cells correlated with miscarriage and blockade of Tim-3 and CTLA-4 inhibited production of anti-inflammatory cytokines and were detrimental in the maintenance of the pregnancy.⁴² Allogeneic fetal trophoblasts express the non-classical major histocompatibility complex (MHC) class I molecule human leukocyte antigen G which is needed for immune tolerance establishment and was shown to increase the number of Tregs.⁴⁵,⁴⁶
In this review, we will discuss the biology and development of CD8⁺ Tregs, their contribution to the tolerance status apart from CD4⁺ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible betterments notably through genetic modifications.

## 2 | MAIN CHARACTERISTICS OF CD8⁺ REGULATORY T CELLS

Despite a streamline work from several groups, the definition of CD8⁺ Tregs remains the major problem of this cell subset population and the reason for them not being assessed in clinical protocols. This lack in assessment is making marker discovery even more problematic and slow, leading to a vicious circle. The fact that, at steady state, expression of FOXP3, a sensitive marker of Treg, is lower in the CD8⁺ T cell population compared to the CD4⁺ T cell population in mouse, rat and human,³ has contributed to this disregard of CD8⁺ Treg's potential and interest, despite in vitro and in vivo data demonstrating their suppressive capacity. In addition, the methylation status of FOXP3 in human CD8⁺ Tregs showed an intermediate level of demethylation compared to one of the CD4⁺ Tregs for some locus of the FOXP3 gene.³ We showed that following ex vivo stimulation, human CD8⁺ Tregs sorted based on the expression of CD45RC, a splicing variant of the CD45 molecule key in the signaling of T cells that we use as a marker to isolate CD8⁺ Tregs⁴⁻⁶; FOXP3 is expressed by all expanded CD8⁺ Tregs that display highly upregulated levels of expression vs. blood unstimulated CD8⁺FOXP3⁺, suggesting that FOXP3 can correlate with the in vivo and in vitro suppressive potential of human CD8⁺ Tregs.³ In contrast, in a rat model of transplantation tolerance following costimulation blockade inducing in vivo CD8⁺ Tregs, FOXP3 was not upregulated,⁴ nor was it in a model of tolerance in rat using donor antigen therapy.⁷ FOXP3 is also not found particularly expressed in mouse CD8⁺ Tregs, and rather the transcription factor HELIOS or the surface marker CD122⁺ or CD28⁻ are used to identify CD8⁺ Tregs in mice.⁸,⁹ Other surface markers were also suggested and among the most frequently used CD25, CD127, GITR, CD39, Lag3, and CTLA-4 that are used in different contexts and different species to identify CD8⁺ Tregs and analyze their suppressive activity in vitro and in vivo in autoimmune diseases, immune reactions to non-self, cancer and materno-fetal tolerance.²,¹⁰ Indeed, using a combination of these markers to identify CD8⁺ Tregs, their suppressive action has been demonstrated in autoimmune diseases such as in the experimental autoimmune encephalomyelitis model,¹¹⁻¹³ multiple sclerosis,¹⁴⁻¹⁶ systemic lupus erythematosus (SLE)¹⁷,¹⁸ and primary biliary cirrhosis,¹⁹ in infection in humans with mycobacteria, human immunodeficiency virus or epstein-barr virus,²⁰⁻²³ in cancer in humans and mice²⁴⁻²⁶ or in transplantation in humans, mice and rats.³,⁴,²⁷⁻³² CXCR5 was used to identify antibody-suppressor FOXP3⁺CD8⁺ cells in mice and essential in their function of suppression of humoral immunity, reducing germinal center B cells and CD4⁺ T follicular helper cells in the context of hepatocyte transplantation.³³

A very recent study of single cell RNA sequencing on CD4⁺ Tregs has highlighted the importance of some transcription factors

and extra-membrane molecules.³⁴ This work showed that CD4⁺ Tregs and CD4⁺ Tconvs can be distinguished by the ubiquitous expression of FOXP3, IKZF2 (IKAROS), TNFR2, IL2RA and IL2RB in mice and humans. Analysis using DGE-RNA sequencing of fresh and activated CD8⁺CD45RC<sup>low/-</sup> Tregs allowed us to show the increase in FOXP3 expression and suppressive molecules after activation. However, nowadays no single cell RNA sequencing study is being published on CD8⁺ Tregs. Our analysis of single cell RNA sequencing data on fresh CD8⁺CD45RC<sup>low/-</sup> cells from healthy volunteers confirms the expression of molecules known in CD4⁺ Tregs such as TNFR2 but never clearly showed before in CD8⁺ Tregs (unpublished data).

Soluble factors can also be used to identify and even sort CD8⁺ Tregs since the development of kit using bispecific antibodies allows the sorting of live cytokine-secreting cells. We have shown that sorted human IFNγ⁺IL-10⁺CD8⁺CD45RC<sup>low/-</sup> Tregs were more potent suppressor cells than the rest of the CD8⁺CD45RC<sup>low/-</sup> Tregs³ and this was also indicative of the role of these cytokines in CD8⁺ Treg suppressive activity, in particular IFNγ (Figure 1). Our interest in IFNγ came from the observations that blocking its activity abrogated the CD8⁺CD45RC<sup>low/-</sup> Treg suppressive activity in a model of allogeneic cardiac transplantation.⁴ We further demonstrated that indeed, IFNγ induced indoleamine 2,3-dioxygenase (IDO) production by endothelial cells (ECs) of the graft and plasmacytoid dendritic cells, an enzyme catabolizing tryptophan essential for effector T cells proliferation.⁴,³⁵ IFNγ was also involved in fibroleukin-2 (FGL2) induction, a cytokine acting further through regulatory B cell induction.³⁶ We have also shown that IL-34, a recently discovered cytokine involved in monocyte/macrophage differentiation, was secreted by approximately half of FOXP3⁺CD8⁺ and CD4⁺ Tregs.³⁷ We showed that IL-34 was involved in FOXP3⁺ Treg suppressive activity and acted as a suppressive cytokine since administration in a model of allogeneic cardiac transplantation in combination with a suboptimal dose of rapamycin-induced transplant tolerance. This was the first description of a role for this cytokine in T cell biology and transplantation.³⁸ Thus, a complex regulatory network of cells around CD8⁺ Tregs is acting to sustain tolerance.³⁹ Other more classical cytokines such as TGFβ have also been described as playing a role in CD8⁺ Treg suppressive activity.²

Some of these molecules or mechanisms of action have been evidenced by study of the fetal-maternal interface.⁴⁰,⁴¹ Indeed, in maternal tolerance where the allogeneic fetal trophoblast invades maternal tissues and interacts with maternal leukocytes, the immune system needs to be regulated to tolerate the presence of the fetus. CD8⁺ T cells are the main component of decidual T cells and CD8⁺ Tregs have been evidenced as important in the maintenance of a normal pregnancy.⁴²⁻⁴⁴ Decreased number and altered function of Tim-3⁺CTLA-4⁺CD8⁺ T cells correlated with miscarriage and blockade of Tim-3 and CTLA-4 inhibited production of anti-inflammatory cytokines and were detrimental in the maintenance of the pregnancy.⁴² Allogeneic fetal trophoblasts express the non-classical major histocompatibility complex (MHC) class I molecule human leukocyte antigen G which is needed for immune tolerance establishment and was shown to increase the number of Tregs.⁴⁵,⁴⁶
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Schematic depicting identified mechanisms of action and markers of rat, mouse and human CD8⁺CD45RC<sup>low/-</sup> Tregs. Breg, regulatory B cell; Co-stim, costimulatory molecules; DC, dendritic cell; EC, endothelial cell; IDO, indoleamine 2,3-dioxygenase; Kyn, kynurenin; Mreg, regulatory macrophages; pDC, plasmacytoid dendritic cell; Trp, tryptophan. Bended arrows indicate conversion or induction. Up and down arrows indicate increase and decrease, respectively</figcaption></figure>

MHC restriction preference of CD8⁺ Tregs has been shown as important for their suppressive activity and for some sub-populations of CD8⁺ Tregs different from what has been described for CD8⁺ Teff cells. Particularly in mice and humans, the best described population of mouse CD8αα⁺ TCRαβ⁺ Tregs or human CD8⁺ Tregs (no specific phenotype associated except pMHC restriction) has been described to preferentially recognize non-classical MHC class I molecules Qa-1 or HLA-E that are orthologous genes.<sup>47-50</sup> These non-classical MHC class I restricted populations have the property to recognize TCR, MHC or heat shock protein derived peptides (ie Qdm, HSP60sp) presented by Qa-1 or HLA-E.<sup>50,51</sup> We have also shown that this capacity to recognize MHC-derived peptide is not restricted to non-classical

MHC class I molecule but also true for classical MHC class I molecule. We showed that peptides derived from a polymorphic region of the β1domain of MHC class II molecules from the donor and presented by recipient classical MHC I molecules can expand CD8⁺CD45RC<sup>low/-</sup> Tregs and induce tolerance in transplantation in an antigen-specific manner in rats<sup>7,52</sup> and humans (unpublished data).

In this context, the identification of new membrane markers for CD8⁺ Treg populations to better define a consensus phenotype is a major challenge and objective for the future. In addition, a better understanding of the mechanisms involved in their development and function would allow to better understand and optimize their clinical potential.

<table><caption>**TABLE 1** Differences between CD8⁺ and CD4⁺ Tregs</caption><thead><tr><th></th><th>CD8<sup>+</sup> Treg</th><th>CD4<sup>+</sup> Treg</th></tr></thead><tbody><tr><td>MHC</td><td>Class I, classical<sup>7</sup> and non-classical<sup>47</sup></td><td>Class II<sup>53</sup></td></tr><tr><td>Composition</td><td>Mostly memory<sup>3</sup></td><td>Naive and memory<sup>213</sup></td></tr><tr><td>Overall suppressive capacity</td><td>CD8<sup>+</sup> or =CD4<sup>3,32,54</sup></td><td></td></tr><tr><td>Suppression of memory and/or naive T cell responses</td><td>Memory and naive<sup>58</sup></td><td>Naive<sup>57</sup></td></tr><tr><td>Interferon gamma mediated suppression</td><td>Repeatedly described<sup>3,4,65,73-77</sup></td><td>Rarely described<sup>70,78</sup></td></tr><tr><td>IL-2 induced proliferation</td><td>CD8<sup>+</sup> or =CD4<sup>32,103-107</sup></td><td></td></tr><tr><td>IL-15 induced proliferation</td><td>CD8<sup>+</sup> or =CD4<sup>3,54,61,111-114</sup></td><td></td></tr><tr><td>Preferred polyclonal stimulation</td><td>mAbs<sup>3</sup></td><td>Beads<sup>146-149</sup></td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**FIGURE 1** Schematic depicting identified mechanisms of action and markers of rat, mouse and human CD8⁺CD45RC<sup>low/-</sup> Tregs. Breg, regulatory B cell; Co-stim, costimulatory molecules; DC, dendritic cell; EC, endothelial cell; IDO, indoleamine 2,3-dioxygenase; Kyn, kynurenin; Mreg, regulatory macrophages; pDC, plasmacytoid dendritic cell; Trp, tryptophan. Bended arrows indicate conversion or induction. Up and down arrows indicate increase and decrease, respectively</figcaption></figure>

MHC restriction preference of CD8⁺ Tregs has been shown as important for their suppressive activity and for some sub-populations of CD8⁺ Tregs different from what has been described for CD8⁺ Teff cells. Particularly in mice and humans, the best described population of mouse CD8αα⁺ TCRαβ⁺ Tregs or human CD8⁺ Tregs (no specific phenotype associated except pMHC restriction) has been described to preferentially recognize non-classical MHC class I molecules Qa-1 or HLA-E that are orthologous genes.<sup>47-50</sup> These non-classical MHC class I restricted populations have the property to recognize TCR, MHC or heat shock protein derived peptides (ie Qdm, HSP60sp) presented by Qa-1 or HLA-E.<sup>50,51</sup> We have also shown that this capacity to recognize MHC-derived peptide is not restricted to non-classical

MHC class I molecule but also true for classical MHC class I molecule. We showed that peptides derived from a polymorphic region of the β1domain of MHC class II molecules from the donor and presented by recipient classical MHC I molecules can expand CD8⁺CD45RC<sup>low/-</sup> Tregs and induce tolerance in transplantation in an antigen-specific manner in rats<sup>7,52</sup> and humans (unpublished data).

In this context, the identification of new membrane markers for CD8⁺ Treg populations to better define a consensus phenotype is a major challenge and objective for the future. In addition, a better understanding of the mechanisms involved in their development and function would allow to better understand and optimize their clinical potential.

<table><caption>**TABLE 1** Differences between CD8⁺ and CD4⁺ Tregs</caption><thead><tr><th></th><th>CD8<sup>+</sup> Treg</th><th>CD4<sup>+</sup> Treg</th></tr></thead><tbody><tr><td>MHC</td><td>Class I, classical<sup>7</sup> and non-classical<sup>47</sup></td><td>Class II<sup>53</sup></td></tr><tr><td>Composition</td><td>Mostly memory<sup>3</sup></td><td>Naive and memory<sup>213</sup></td></tr><tr><td>Overall suppressive capacity</td><td>CD8<sup>+</sup> or =CD4<sup>3,32,54</sup></td><td></td></tr><tr><td>Suppression of memory and/or naive T cell responses</td><td>Memory and naive<sup>58</sup></td><td>Naive<sup>57</sup></td></tr><tr><td>Interferon gamma mediated suppression</td><td>Repeatedly described<sup>3,4,65,73-77</sup></td><td>Rarely described<sup>70,78</sup></td></tr><tr><td>IL-2 induced proliferation</td><td>CD8<sup>+</sup> or =CD4<sup>32,103-107</sup></td><td></td></tr><tr><td>IL-15 induced proliferation</td><td>CD8<sup>+</sup> or =CD4<sup>3,54,61,111-114</sup></td><td></td></tr><tr><td>Preferred polyclonal stimulation</td><td>mAbs<sup>3</sup></td><td>Beads<sup>146-149</sup></td></tr></tbody></table>
# 3 | SIMILARITIES, DIFFERENCES AND SYNERGIES BETWEEN CD8⁺ AND CD4⁺ TREGS

A major aspect that remains to be properly addressed given the lack of assessment of CD8⁺ Tregs in diseases is the similarities, differences and synergies that could exist between CD8⁺ and CD4⁺ Tregs that could emphasize the interest in CD8⁺ Tregs (Table 1).

## 3.1 | Function and mechanisms of action

A very obvious and important difference is that CD8⁺ and CD4⁺ Tregs recognize their cognate antigens presented by MHC-I or MHC-II molecules, respectively. Therefore, CD8⁺ Tregs will activate their suppressive activity on virtually all cells whereas CD4⁺ Tregs activation will occur only on cells expressing MHC-II molecules. In the case of solid organ transplantation, CD8⁺ Tregs will activate on not only all donor graft cells but also on dendritic cells through indirect donor alloantigen presentation on recipient MHC-I molecules.⁷ Thus, for both CD8⁺ and CD4⁺ Tregs, bystander inhibition of T and B cell activation will occur through antigen presenting cells (APCs) which express MHC-I⁺ and MHC-II⁺ molecules. In inflamed tissues non-immune cells upon activation by IFNγ and/or TNFα may express MHC-II molecules, but MHC-I expression is constitutive by virtually all cells and its expression is also upregulated by IFNγ and/or TNFα. Thus, CD8⁺ Tregs will be advantageous, particularly in organ transplantation or graft versus host disease (GVHD) since suppression will additionally occur in all allogeneic cells, including ECs of vascularized grafts or host organs in GVHD. Another potential area in which MHC-I antigen recognition might be useful is in the case of using in the future allogeneic off-the shelf Tregs. Recipient cells attacking allogeneic CD8⁺ Tregs may be suppressed through MHC-I recognition whereas CD4⁺ Tregs could only do it if the recipient cells express MHC-II molecules, which can be the case for human T cells but this demands high levels of IFNγ-mediated activation. Although a group recently published the structure of an induced CD4⁺ Tregs' TCR binding a pMHC,⁵³ little is known about CD4⁺ Tregs pMHC restriction in contrast to CD8⁺ Tregs. This is probably due to the easiness to generate MHC class I/peptide tetramers that led to the opportunity to better identify and understand the TCR constraints and antigen recognition of CD8⁺ Tregs.

A direct comparison of CD8⁺ and CD4⁺ Tregs' overall suppressive capacity has not been done in many settings but in solid organ transplant³⁴ and GVHD³² models in mice, suppression by CD8⁺ Tregs was superior to the one of CD4⁺ Tregs. We have observed that fresh, in vitro expanded human polyclonal and donor-specific CAR engineered CD8⁺ Tregs were all superior to their CD4⁺ Treg counterparts (unpublished data and Ref. 3). Importantly, in certain tolerance models one or the other or both Treg subsets can be induced and play a role. As examples, in the same rat heart allotransplantation tolerance model, CD8⁺ and/or CD4⁺ Tregs were differently involved depending on the treatment used: blocking CD40-CD40L induced tolerance that was entirely dependent and transferable by CD8⁺ Tregs,⁴

whereas donor-specific blood transfusion as well as anti-CD45RC MAb or IL-34 administration tolerance was dependent on both CD8⁺ and CD4⁺ Tregs.³⁷,⁵⁵ In these two last models adoptive transfer of each of the CD8⁺ or CD4⁺ Treg subsets resulted in tolerance in a fraction of the recipients that was lower than the one obtained when all Tregs were transferred.³⁷,⁵⁵ Similarly, in a mouse model, GVHD was most efficiently inhibited when both CD4⁺ and CD8⁺ Tregs were used associated and the presence of CD8⁺ Tregs preserved a T eff-mediated graft versus leukemia effect that was lost when CD4⁺ Treg cells were used alone.⁵⁶ Interestingly in the perspective of simultaneously using CD8⁺ and CD4⁺ Tregs, both Treg subsets could be produced in vitro at the same time, such as when they are induced in the presence of APCs treated with IL-34.³⁷ Finally, the suppressive capacity of CD8⁺ and CD4⁺ Tregs to suppress naive and memory CD4⁺ T effector response has been compared in the transplantation field by different groups. This is a very clinically relevant situation to analyze the role of naive vs. memory CD4⁺ T effector responses, since both are activated at different time points during transplantation and memory T cell response is described as particularly difficult to control with usual immunotherapeutic or even immunosuppression protocols. Both naive and memory CD4⁺ Treg cells only suppressed naive and not memory CD4⁺ and CD8⁺ T responses.⁵⁷ In contrast, CD8⁺ Treg cells suppressed both naive and memory effector CD4⁺ T cell responses.⁵⁸

As far as mechanisms of action are concerned, several cytokines have been described as playing a role for both CD8⁺ and CD4⁺ Tregs, although not necessarily in all models or in vivo or in vitro situations. As an example, IL-10-mediated suppression has been described for CD8⁺CD122⁺ Tregs in mice⁵⁴ but not for human CD8⁺CD45RC<sup>low/-</sup> Tregs.³

Similarly, TGFβ has been implicated in the suppressive function of some CD4⁺ Tregs and at least some types of CD8⁺ Tregs, such as mouse peptide-specific CD8⁺ iTreg cells,⁵⁹ CD8⁺FOXP3⁺CD25⁺ Tregs induced by corneal ECs⁶⁰ and CD8⁺CD45RC<sup>low/-</sup> human Treg cells³ but not of CD8⁺CD45RA⁺CCR7⁺FOXP3⁺ Treg cells.⁶¹

IL-34 is a cytokine with immunoregulatory properties by at least inducing M2 macrophages³⁸ and that is expressed by both CD8⁺ and CD4⁺ Tregs and is able to prolong heart allograft survival.³⁷

FGL2 immunoregulatory properties were first described in CD4⁺ Treg cells,⁶² FGL2 expression was then reported in mouse CD8αα Treg cells⁶³ and in rat CD40Ig-induced CD8⁺CD45RC<sup>low/-</sup> Treg cells functions. FGL2 function is at least mediated through the FcγRIIB receptor inhibiting dendritic cell maturation, inducing B cell apoptosis⁶⁴ and generating regulatory B cells through unknown mechanisms.³⁶ IFNγ is a mechanism of suppression not only of CD8⁺ Tregs³,⁶⁵⁻⁶⁷ but also of CD4⁺ Tregs.⁶⁸⁻⁷⁰ The mechanism of suppression by IFNγ includes actions on endothelial and antigen presenting cells, such as induction of IDO and FGL2.

IL-35 has been previously described as expressed by CD4⁺ Tregs⁷¹ but it has also been described as expressed and playing a suppressive role for tumor-associated CD8⁺ Tregs.⁶⁶,⁷²

Although IFNγ has proinflammatory consequences it has also been described as having pro-tolerogenic actions, through the
3 | SIMILARITIES, DIFFERENCES AND SYNERGIES BETWEEN CD8⁺ AND CD4⁺ TREGS

A major aspect that remains to be properly addressed given the lack of assessment of CD8⁺ Tregs in diseases is the similarities, differences and synergies that could exist between CD8⁺ and CD4⁺ Tregs that could emphasize the interest in CD8⁺ Tregs (Table 1).

3.1 | Function and mechanisms of action

A very obvious and important difference is that CD8⁺ and CD4⁺ Tregs recognize their cognate antigens presented by MHC-I or MHC-II molecules, respectively. Therefore, CD8⁺ Tregs will activate their suppressive activity on virtually all cells whereas CD4⁺ Tregs activation will occur only on cells expressing MHC-II molecules. In the case of solid organ transplantation, CD8⁺ Tregs will activate on not only all donor graft cells but also on dendritic cells through indirect donor alloantigen presentation on recipient MHC-I molecules.⁷ Thus, for both CD8⁺ and CD4⁺ Tregs, bystander inhibition of T and B cell activation will occur through antigen presenting cells (APCs) which express MHC-I⁺ and MHC-II⁺ molecules. In inflamed tissues non-immune cells upon activation by IFNγ and/or TNFα may express MHC-II molecules, but MHC-I expression is constitutive by virtually all cells and its expression is also upregulated by IFNγ and/or TNFα. Thus, CD8⁺ Tregs will be advantageous, particularly in organ transplantation or graft versus host disease (GVHD) since suppression will additionally occur in all allogeneic cells, including ECs of vascularized grafts or host organs in GVHD. Another potential area in which MHC-I antigen recognition might be useful is in the case of using in the future allogeneic off-the shelf Tregs. Recipient cells attacking allogeneic CD8⁺ Tregs may be suppressed through MHC-I recognition whereas CD4⁺ Tregs could only do it if the recipient cells express MHC-II molecules, which can be the case for human T cells but this demands high levels of IFNγ-mediated activation. Although a group recently published the structure of an induced CD4⁺ Tregs' TCR binding a pMHC,⁵³ little is known about CD4⁺ Tregs pMHC restriction in contrast to CD8⁺ Tregs. This is probably due to the easiness to generate MHC class I/peptide tetramers that led to the opportunity to better identify and understand the TCR constraints and antigen recognition of CD8⁺ Tregs.

A direct comparison of CD8⁺ and CD4⁺ Tregs' overall suppressive capacity has not been done in many settings but in solid organ transplant<sup>54</sup> and GVHD<sup>32</sup> models in mice, suppression by CD8⁺ Tregs was superior to the one of CD4⁺ Tregs. We have observed that fresh, in vitro expanded human polyclonal and donor-specific CAR engineered CD8⁺ Tregs were all superior to their CD4⁺ Treg counterparts (unpublished data and Ref. 3). Importantly, in certain tolerance models one or the other or both Treg subsets can be induced and play a role. As examples, in the same rat heart allotransplantation tolerance model, CD8⁺ and/or CD4⁺ Tregs were differently involved depending on the treatment used: blocking CD40-CD40L induced tolerance that was entirely dependent and transferable by CD8⁺ Tregs,⁴

whereas donor-specific blood transfusion as well as anti-CD45RC MAb or IL-34 administration tolerance was dependent on both CD8⁺ and CD4⁺ Tregs.³⁷,⁵⁵ In these two last models adoptive transfer of each of the CD8⁺ or CD4⁺ Treg subsets resulted in tolerance in a fraction of the recipients that was lower than the one obtained when all Tregs were transferred.³⁷,⁵⁵ Similarly, in a mouse model, GVHD was most efficiently inhibited when both CD4⁺ and CD8⁺ Tregs were used associated and the presence of CD8⁺ Tregs preserved a T eff-mediated graft versus leukemia effect that was lost when CD4⁺ Treg cells were used alone.⁵⁶ Interestingly in the perspective of simultaneously using CD8⁺ and CD4⁺ Tregs, both Treg subsets could be produced in vitro at the same time, such as when they are induced in the presence of APCs treated with IL-34.³⁷ Finally, the suppressive capacity of CD8⁺ and CD4⁺ Tregs to suppress naive and memory CD4⁺ T effector response has been compared in the transplantation field by different groups. This is a very clinically relevant situation to analyze the role of naive vs. memory CD4⁺ T effector responses, since both are activated at different time points during transplantation and memory T cell response is described as particularly difficult to control with usual immunotherapeutic or even immunosuppression protocols. Both naive and memory CD4⁺ Treg cells only suppressed naive and not memory CD4⁺ and CD8⁺ T responses.⁵⁷ In contrast, CD8⁺ Treg cells suppressed both naive and memory effector CD4⁺ T cell responses.⁵⁸

As far as mechanisms of action are concerned, several cytokines have been described as playing a role for both CD8⁺ and CD4⁺ Tregs, although not necessarily in all models or in vivo or in vitro situations. As an example, IL-10-mediated suppression has been described for CD8⁺CD122⁺ Tregs in mice⁵⁴ but not for human CD8⁺CD45RC<sup>low/-</sup> Tregs.³

Similarly, TGFβ has been implicated in the suppressive function of some CD4⁺ Tregs and at least some types of CD8⁺ Tregs, such as mouse peptide-specific CD8⁺ iTreg cells,⁵⁹ CD8⁺FOXP3⁺CD25⁺ Tregs induced by corneal ECs⁶⁰ and CD8⁺CD45RC<sup>low/-</sup> human Treg cells³ but not of CD8⁺CD45RA⁺CCR7⁺FOXP3⁺ Treg cells.⁶¹

IL-34 is a cytokine with immunoregulatory properties by at least inducing M2 macrophages³⁸ and that is expressed by both CD8⁺ and CD4⁺ Tregs and is able to prolong heart allograft survival.³⁷

FGL2 immunoregulatory properties were first described in CD4⁺ Treg cells,⁶² FGL2 expression was then reported in mouse CD8αα Treg cells⁶³ and in rat CD40Ig-induced CD8⁺CD45RC<sup>low/-</sup> Treg cells functions. FGL2 function is at least mediated through the FcγRIIB receptor inhibiting dendritic cell maturation, inducing B cell apoptosis⁶⁴ and generating regulatory B cells through unknown mechanisms.³⁶ IFNγ is a mechanism of suppression not only of CD8⁺ Tregs³,⁶⁵⁻⁶⁷ but also of CD4⁺ Tregs.⁶⁸⁻⁷⁰ The mechanism of suppression by IFNγ includes actions on endothelial and antigen presenting cells, such as induction of IDO and FGL2.

IL-35 has been previously described as expressed by CD4⁺ Tregs⁷¹ but it has also been described as expressed and playing a suppressive role for tumor-associated CD8⁺ Tregs.⁶⁶,⁷²

Although IFNγ has proinflammatory consequences it has also been described as having pro-tolerogenic actions, through the
production of molecules such as IDO, iNOs and FGL2.<sup>68</sup> IFNγ has been described as produced by several CD8<sup>+</sup> Treg types <sup>3,4,65,73-77</sup> although the function of IFNγ in CD8<sup>+</sup>FOXP3<sup>+</sup> cells has not been specifically analyzed. In contrast, and although suppression through IFNγ has been described by CD4<sup>+</sup> Tregs in certain models<sup>70,78</sup> CD4<sup>+</sup>FOXP3<sup>+</sup> Tregs in general produce low levels of IFNγ.

PD-1 has been described in mice to be expressed at lower levels in CD8+ Tregs vs CD4+ Tregs<sup>32</sup> but PD-1 expression is important for CD8+ Treg function,<sup>79,80</sup> as it is for CD4+ Tregs.<sup>81,82</sup> Additionally, PD-L1 expressed by CD4+ Tregs<sup>83</sup> and by human CD8+ Tregs<sup>84</sup> suppress CD4+ Teff responses directly through PD-1 or also through PD-L1 expressed by DC and macrophages. Therefore, the use of PD-L1-Fc or agonistic anti-PD-1 antibodies would inhibit immune responses by both promoting Treg activity and by inhibiting Teff responses directly, as well as by inducing tolerogenic APCs.

Although cytotoxicity could be expected to be a widespread mechanism of suppression by CD8+ Treg cells and not of CD4+ Treg cells, only in some cases CD8+ Tregs have been described as cytotoxic<sup>85-88</sup> and CD4+ Tregs have also been shown in some instances as cytotoxic,<sup>89-94</sup> thus this mechanism is shared by both types of Tregs.

## 3.2 | Metabolic activity

Mechanistic target of rapamycin (mTOR) through mTOR complex 1 (mTORC1) and mTORC2 regulates activation and proliferation through the phosphorylation of several transcription factors.<sup>95</sup> Mechanistic target of rapamycin signaling is high and increases the inflammatory response in Teff cells maintaining glycolysis whereas mTOR activity is low in CD4<sup>+</sup> Tregs.<sup>96</sup> Inhibitors of mTOR such as rapamycin directly bind and inhibit mTORC1. Activity of mTORC1 is necessary for CD4<sup>+</sup> Treg cell function to maintain their metabolic status through lipogenesis.<sup>97</sup> Therefore, rapamycin at moderate concentrations inhibits CD4<sup>+</sup> Teff growth but promotes CD4<sup>+</sup> Treg growth and function.<sup>98,99</sup> Although metabolic activity and mTOR function have not been directly analyzed in CD8<sup>+</sup> Tregs, the metabolic status of memory CD8<sup>+</sup> cells is similar to that of CD4<sup>+</sup> Tregs<sup>100,101</sup> and different types of human CD8<sup>+</sup> Tregs having a memory phenotype have been shown to expand in the presence of rapamycin,<sup>3,102</sup> thus, inclusion of rapamycin in the in vitro expansion of clinically applicable human CD8<sup>+</sup> Tregs as for CD4<sup>+</sup> Tregs is a logical approach to increase safety.

## 3.3 | Proliferation in vitro and in vivo

Freshly analyzed human and mouse CD8⁺FOXP3⁺ Tregs have been reported as IL2Rα/CD25high and CD127low as CD4⁺FOXP3⁺ Tregs<sup>103</sup> whereas human CD8⁺CD45RClow/- have a more complex phenotype.<sup>3</sup> Transgenic rats with green fluorescent protein under control of the FOXP3 promoter are also CD25highCD127⁻ (unpublished data). The other chains of the IL2R are expressed in a large proportion of fresh human CD8⁺FOXP3⁺ Tregs comparable to the ones of CD4⁺FOXP3⁺ Tregs (unpublished data). With this expression of the IL2R chains

similar to those of CD4⁺FOXP3⁺ Tregs it is logical that low dose IL-2 administration in vivo to mice and human healthy volunteers expanded CD8⁺FOXP3⁺ Tregs in both species and even in a higher proportion in humans (6.7 ± 4.3 fold) as compared to CD4⁺FOXP3⁺ Tregs (1.8 ± 0.7 fold). The suppression capacity after IL-2 treatment was also higher in CD8⁺FOXP3⁺ Tregs vs. CD4⁺FOXP3⁺ Tregs.<sup>103</sup> Administration of low dose IL-2 in cynomolgus monkeys expanded both CD8⁺FOXP3⁺ and CD4⁺FOXP3⁺ Tregs 10-fold and 15-fold, respectively when compared to the baseline levels and the suppressive effect after IL-2 treatment was comparable for CD8⁺ and CD4⁺FOXP3⁺ Tregs.<sup>104</sup> Similar increase in CD8⁺ and CD4⁺ Tregs (11 to 14-fold) was observed when using an IL-2 mutein molecule.<sup>105</sup> Increase in CD8⁺FOXP3⁺ Tregs along with CD4⁺FOXP3⁺ Tregs was also observed in patients with hepatitis C virus-induced vasculitis<sup>106</sup> and type 1 diabetic patients<sup>107</sup> treated with low doses of IL-2. In a model of GVHD in mice, administration of IL-2 antibody complexes and rapamycin was synergic to CD8⁺FOXP3⁺ Tregs increase up to 30-fold whereas CD4⁺FOXP3⁺ Tregs increased 4-fold.<sup>32</sup> In other studies, in patients treated with low doses of IL-2, CD8⁺ Tregs were not reported,<sup>108,109</sup> although only a few of them described extensive immunophenotyping.<sup>109</sup>

Although IL-15 favors thymic development of CD4⁺ Tregs along with IL-2 through the IL-2Rβ/γc cytokine receptor complex,<sup>110</sup> IL-15Ra is not expressed by CD4⁺ Tregs and these cells respond to IL-15 only when soluble IL-15Ra binds IL-15 and presents it in trans.<sup>111</sup> This explains why adult CD4⁺ Tregs do not respond to IL-15 in vitro while they do in vivo.<sup>111,112</sup> In contrast, IL-15 is necessary for optimal CD8⁺ Tregs expansion in vivo<sup>113,114</sup> and in vitro.<sup>3,54,61</sup>

In vitro expansion of CD8⁺ Tregs in the same conditions used to expand CD4⁺ Tregs has shown lower suppression activity in some cases but this may be related to the use of culture conditions that are optimal for CD4⁺ and not CD8⁺ Tregs.<sup>115</sup> In this regard, we have observed that the use of beads coated with anti-CD3 and anti-CD28 MAbs ideal for the culture of CD4⁺ Tregs is toxic for human CD8⁺ Tregs while being well-tolerated by mouse CD8⁺ Tregs (manuscript in preparation). It is then important when comparing in vitro cultured CD8⁺ and CD4⁺ Tregs to use the best culture conditions for each cell type.

# 4 | BIOLOGY AND DEVELOPMENT OF CD8+ TREGS

## 4.1 | Origin

The thymic origin of CD8+ nTregs is however still poorly described. In 2016, the presence of CD8⁺CD28low Treg cells in peripheral blood mononuclear cells (PBMCs) and in children's thymuses was described and also their thymic origin demonstrated.<sup>9,116</sup> CD8⁺ Tregs are associated with different phenotypes depending on the studies (CD122, CD28, CD45RC, CD103, PD-1) but mostly their phenotype is rather associated to a differentiation status of central memory cells or effectors memory as characterized by the absence of CD28, CD62L or CD122 expression.<sup>3,117</sup> Other teams describe them rather
production of molecules such as IDO, iNOs and FGL2.<sup>68</sup> IFNγ has been described as produced by several CD8<sup>+</sup> Treg types <sup>3,4,65,73-77</sup> although the function of IFNγ in CD8<sup>+</sup>FOXP3<sup>+</sup> cells has not been specifically analyzed. In contrast, and although suppression through IFNγ has been described by CD4<sup>+</sup> Tregs in certain models<sup>70,78</sup> CD4<sup>+</sup>FOXP3<sup>+</sup> Tregs in general produce low levels of IFNγ.

PD-1 has been described in mice to be expressed at lower levels in CD8+ Tregs vs CD4+ Tregs<sup>32</sup> but PD-1 expression is important for CD8+ Treg function,<sup>79,80</sup> as it is for CD4+ Tregs.<sup>81,82</sup> Additionally, PD-L1 expressed by CD4+ Tregs<sup>83</sup> and by human CD8+ Tregs<sup>84</sup> suppress CD4+ Teff responses directly through PD-1 or also through PD-L1 expressed by DC and macrophages. Therefore, the use of PD-L1-Fc or agonistic anti-PD-1 antibodies would inhibit immune responses by both promoting Treg activity and by inhibiting Teff responses directly, as well as by inducing tolerogenic APCs.

Although cytotoxicity could be expected to be a widespread mechanism of suppression by CD8+ Treg cells and not of CD4+ Treg cells, only in some cases CD8+ Tregs have been described as cytotoxic<sup>85-88</sup> and CD4+ Tregs have also been shown in some instances as cytotoxic,<sup>89-94</sup> thus this mechanism is shared by both types of Tregs.

## 3.2 | Metabolic activity

Mechanistic target of rapamycin (mTOR) through mTOR complex 1 (mTORC1) and mTORC2 regulates activation and proliferation through the phosphorylation of several transcription factors.<sup>95</sup> Mechanistic target of rapamycin signaling is high and increases the inflammatory response in Teff cells maintaining glycolysis whereas mTOR activity is low in CD4<sup>+</sup> Tregs.<sup>96</sup> Inhibitors of mTOR such as rapamycin directly bind and inhibit mTORC1. Activity of mTORC1 is necessary for CD4<sup>+</sup> Treg cell function to maintain their metabolic status through lipogenesis.<sup>97</sup> Therefore, rapamycin at moderate concentrations inhibits CD4<sup>+</sup> Teff growth but promotes CD4<sup>+</sup> Treg growth and function.<sup>98,99</sup> Although metabolic activity and mTOR function have not been directly analyzed in CD8<sup>+</sup> Tregs, the metabolic status of memory CD8<sup>+</sup> cells is similar to that of CD4<sup>+</sup> Tregs<sup>100,101</sup> and different types of human CD8<sup>+</sup> Tregs having a memory phenotype have been shown to expand in the presence of rapamycin,<sup>3,102</sup> thus, inclusion of rapamycin in the in vitro expansion of clinically applicable human CD8<sup>+</sup> Tregs as for CD4<sup>+</sup> Tregs is a logical approach to increase safety.

## 3.3 | Proliferation in vitro and in vivo

Freshly analyzed human and mouse CD8⁺FOXP3⁺ Tregs have been reported as IL2Rα/CD25high and CD127low as CD4⁺FOXP3⁺ Tregs<sup>103</sup> whereas human CD8⁺CD45RClow/- have a more complex phenotype.<sup>3</sup> Transgenic rats with green fluorescent protein under control of the FOXP3 promoter are also CD25highCD127⁻ (unpublished data). The other chains of the IL2R are expressed in a large proportion of fresh human CD8⁺FOXP3⁺ Tregs comparable to the ones of CD4⁺FOXP3⁺ Tregs (unpublished data). With this expression of the IL2R chains

similar to those of CD4⁺FOXP3⁺ Tregs it is logical that low dose IL-2 administration in vivo to mice and human healthy volunteers expanded CD8⁺FOXP3⁺ Tregs in both species and even in a higher proportion in humans (6.7 ± 4.3 fold) as compared to CD4⁺FOXP3⁺ Tregs (1.8 ± 0.7 fold). The suppression capacity after IL-2 treatment was also higher in CD8⁺FOXP3⁺ Tregs vs. CD4⁺FOXP3⁺ Tregs.<sup>103</sup> Administration of low dose IL-2 in cynomolgus monkeys expanded both CD8⁺FOXP3⁺ and CD4⁺FOXP3⁺ Tregs 10-fold and 15-fold, respectively when compared to the baseline levels and the suppressive effect after IL-2 treatment was comparable for CD8⁺ and CD4⁺FOXP3⁺ Tregs.<sup>104</sup> Similar increase in CD8⁺ and CD4⁺ Tregs (11 to 14-fold) was observed when using an IL-2 mutein molecule.<sup>105</sup> Increase in CD8⁺FOXP3⁺ Tregs along with CD4⁺FOXP3⁺ Tregs was also observed in patients with hepatitis C virus-induced vasculitis<sup>106</sup> and type 1 diabetic patients<sup>107</sup> treated with low doses of IL-2. In a model of GVHD in mice, administration of IL-2 antibody complexes and rapamycin was synergic to CD8⁺FOXP3⁺ Tregs increase up to 30-fold whereas CD4⁺FOXP3⁺ Tregs increased 4-fold.<sup>32</sup> In other studies, in patients treated with low doses of IL-2, CD8⁺ Tregs were not reported,<sup>108,109</sup> although only a few of them described extensive immunophenotyping.<sup>109</sup>

Although IL-15 favors thymic development of CD4⁺ Tregs along with IL-2 through the IL-2Rβ/γc cytokine receptor complex,<sup>110</sup> IL-15Ra is not expressed by CD4⁺ Tregs and these cells respond to IL-15 only when soluble IL-15Ra binds IL-15 and presents it in trans.<sup>111</sup> This explains why adult CD4⁺ Tregs do not respond to IL-15 in vitro while they do in vivo.<sup>111,112</sup> In contrast, IL-15 is necessary for optimal CD8⁺ Tregs expansion in vivo<sup>113,114</sup> and in vitro.<sup>3,54,61</sup>

In vitro expansion of CD8⁺ Tregs in the same conditions used to expand CD4⁺ Tregs has shown lower suppression activity in some cases but this may be related to the use of culture conditions that are optimal for CD4⁺ and not CD8⁺ Tregs.<sup>115</sup> In this regard, we have observed that the use of beads coated with anti-CD3 and anti-CD28 MAbs ideal for the culture of CD4⁺ Tregs is toxic for human CD8⁺ Tregs while being well-tolerated by mouse CD8⁺ Tregs (manuscript in preparation). It is then important when comparing in vitro cultured CD8⁺ and CD4⁺ Tregs to use the best culture conditions for each cell type.

# 4 | BIOLOGY AND DEVELOPMENT OF CD8+ TREGS

## 4.1 | Origin

The thymic origin of CD8⁺nTregs is however still poorly described. In 2016, the presence of CD8⁺CD28low Treg cells in peripheral blood mononuclear cells (PBMCs) and in children's thymuses was described and also their thymic origin demonstrated.<sup>9,116</sup> CD8⁺ Tregs are associated with different phenotypes depending on the studies (CD122, CD28, CD45RC, CD103, PD-1) but mostly their phenotype is rather associated to a differentiation status of central memory cells or effectors memory as characterized by the absence of CD28, CD62L or CD122 expression.<sup>3,117</sup> Other teams describe them rather
through the expression of CD44, CCR7 and CD62L which are mark-
ers of central memory cells ³,⁷³ or CD122⁺ and CD28⁺.³²,⁵⁴ When
activated, CD8⁺ Tregs lose the expression of the CD45RA, distin-
guishing the naive state, to express CD45RO, a marker of mem-
ory state, and are dependent on IL-2 and IL-15. Naive CD8⁺ Tregs
leave the thymus and such natural CD8⁺ Tregs have been shown
by us with a limited suppressive capacity in both rat and human.³,⁴
Indeed, adoptively transferred naive rat CD8⁺CD45RC<sup>low</sup>/- Tregs
cannot inhibit acute transplant rejection in a fully allogeneic model
of transplantation⁴ and human CD8⁺CD45RC<sup>low</sup>/- Tregs from blood
of healthy individuals are more potent suppressor cells in vitro fol-
lowing anti-CD3 and anti-CD28 stimulation.³ Effector and memory
Tregs have a much more potent suppressive capacity than naive
Tregs in mice and humans.¹¹⁸ Effector Tregs are found in the blood
and in secondary lymphoid organs, while memory Tregs are found
mainly in peripheral tissues and in secondary lymphoid organs.

Transcription factors can identify the thymic origin of CD8⁺ Tregs. Some were suggested for a long time as reliable markers of Tregs from thymic origin. Indeed, HELIOS, a member of Ikaros family of zinc finger transcription factor, is expressed by 100% of CD4⁺FOXP3⁺ Tregs in the thymus and 70% of CD4⁺FOXP3⁺ Tregs in peripheral lymphoid tissues.¹¹⁹ HELIOS seems to be involved in the stabilization of the phenotype of CD4⁺ and CD8⁺ Tregs in an inflammatory context.⁸ In a mouse model deficient for HELIOS, CD8⁺ and CD4⁺ Tregs were not able to control effector T cells responses. This demonstrates HELIOS' involvement in suppressive functions, differentiation and survival of Tregs.⁸,¹²⁰ Human CD8⁺CD45RC<sup>low</sup>/- cells co-express HELIOS and FOXP3.³ FOXP3 seems to be a key transcription factor in the development of Tregs including CD8⁺ Tregs since silencing FOXP3 with siRNA abrogated the ability of CD8⁺ Tregs to suppress anti-DNA antibodies in a lupus model in mice.¹²¹ Although adoptive transfer of CD4⁺FOXP3⁺ Tregs can cure immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in FOXP3-deficient mice¹²² this does not exclude the possibility that transfer of CD8⁺FOXP3⁺ Tregs could also have a similar effect. This is an important experiment that has not been reported and would confirm a physiological role in immune homeostasis for CD8⁺FOXP3⁺ Treg. Induced CD8⁺ Tregs express less FOXP3 than CD4⁺ Tregs.¹²³ However, we have shown a specific expression of FOXP3 in natural CD8⁺CD45RC<sup>low</sup>/- Tregs while we do not find expression in CD8⁺CD45RC<sup>high</sup> effector T cells. This expression of FOXP3 is all the more important after cell activation.³ This expression is correlated with the expression of suppressive molecules such as CTLA-4, GITR, IFNγ and TGFβ. In mice, other transcription factors have been described as involved in the development and function of CD4⁺ Tregs but they have not yet been described for CD8⁺ Tregs. IFR4 was shown essential for the expression of Blimp-1 in the differentiation of CD4⁺ effector Tregs¹²⁴; the canonical pathway nuclear factor-kappa B is also involved in the development and function of CD4⁺ Tregs by performing inducible deletions of some subunits of the complex. The c-Rel subunit was critical in thymic development, while the p65 subunit was essential for maturing Tregs and maintaining immune tolerance.¹²⁵

## 4.2 | Targeted therapies modulate or synergize with CD8+ Tregs

Tregs have been well described to be induced or synergize with antibody or cytokine therapies, to be the main effector regulatory mechanisms boosting the efficacy of such therapies and being key to long-term sustained effect. Thus, new therapies should strategically aim to promote Tregs to induce a satisfactory control of immune responses. Strategies of costimulation blockade have been shown to efficiently induce and mediate their tolerogenic effect through CD8⁺ Tregs. Blockade of the CD40/CD40L costimulatory pathway is a well-known strategy in transplantation for its potential in animal models in tolerance induction, although its translation to the clinic has been slowed down due to thromboembolic events.¹²⁶ We have shown that blockade of the CD40/CD40L pathway using an adenovirus encoding CD40lg, a chimeric molecule, in a model of fully incompatible cardiac allotransplantation in rats resulted in tolerance induction dependent on CD8⁺CD45RC<sup>low/-</sup> Tregs.²,⁴,³⁵,¹²⁷ Blockade of the 4-1BB/4-1BBL costimulatory pathway together with immunization in mice was also shown to induce antigen-specific CD8⁺ Tregs acting in a IFNγ and TGFβ dependent manner.⁵⁹ In this model IFNγ directly stimulates CD8⁺ Tregs to induce a TGFβ-based suppression.⁵⁹ In contradiction, 4-1BB has been shown as important for the suppressive activity of CD8⁺ Tregs in a model of allergic inflammation.¹²⁸ We and others showed that IFNγ and TGFβ were key mediators of the suppressive activity of CD8⁺ Tregs.⁴,⁶⁸,¹²⁹,¹³⁰ Blockade of ICOS/B7h costimulation also showed potent induction of alloantigen-specific CD8⁺PD1⁺ Tregs in vivo in a model of heart grafts in mice.¹³¹ More surprisingly, targeting CD3, a molecule expressed by definition by Treg, has shown effectiveness in inducing both CD4⁺ and CD8⁺ Tregs in a collagen induced arthritis model of rheumatoid arthritis in mice.¹³²,¹³³ In this model, CD8⁺ Tregs, unlike CD4⁺ Tregs, potently inhibited Th17 responses, thereby inhibiting a wider range of inflammatory pathways. The authors also showed that monocyte membrane bound TNFα increased FOXP3 expression in CD8⁺ T cells. Administration of hOKT3g1 (Ala-Ala) in patients with type 1 diabetes mellitus halted disease progression for >1 year and was associated with increased CD8⁺ FOXP3⁺ Tregs.¹³⁴ Induction of CD8⁺ Tregs was dependent on TNF and associated with TNFR2 expression by the CD8⁺ Tregs.¹³⁵ There is a ying and yang action observed by several groups for cytokines such as TNFα and IFNγ. Anti-TNF antibodies are used in Crohn's disease and have shown effectiveness in management of the disease but also common failure.¹³⁶ The importance of action of these cytokines on and by CD8⁺ Tregs, but also more recently by CD4⁺ Tregs,¹³⁷ should certainly be taken into consideration in the potential limited effect of cytokine-targeting drugs in autoimmune diseases.

Co-administration of CD8⁺ Treg therapy with costimulation blockade is also showing synergistic efficacy. In a model of skin transplantation in mice, CD8⁺CD122⁺PD-1⁺ Tregs expanded ex vivo combined with costimulation blockade of CD40/CD154, but not of B7/CD28, synergizing to prolong the allograft survival in an IL-10-dependent manner.¹¹³ In fact, blockade of the B7/CD28
through the expression of CD44, CCR7 and CD62L which are mark-
ers of central memory cells ³,⁷³ or CD122⁺ and CD28⁺.³²,⁵⁴ When
activated, CD8⁺ Tregs lose the expression of the CD45RA, distin-
guishing the naive state, to express CD45RO, a marker of mem-
ory state, and are dependent on IL-2 and IL-15. Naive CD8⁺ Tregs
leave the thymus and such natural CD8⁺ Tregs have been shown
by us with a limited suppressive capacity in both rat and human.³,⁴
Indeed, adoptively transferred naive rat CD8⁺CD45RC<sup>low</sup>/- Tregs
cannot inhibit acute transplant rejection in a fully allogeneic model
of transplantation⁴ and human CD8⁺CD45RC<sup>low</sup>/- Tregs from blood
of healthy individuals are more potent suppressor cells in vitro fol-
lowing anti-CD3 and anti-CD28 stimulation.³ Effector and memory
Tregs have a much more potent suppressive capacity than naive
Tregs in mice and humans.¹¹⁸ Effector Tregs are found in the blood
and in secondary lymphoid organs, while memory Tregs are found
mainly in peripheral tissues and in secondary lymphoid organs.

Transcription factors can identify the thymic origin of CD8⁺
Tregs. Some were suggested for a long time as reliable markers
of Tregs from thymic origin. Indeed, HELIOS, a member of Ikaros
family of zinc finger transcription factor, is expressed by 100% of
CD4⁺FOXP3⁺ Tregs in the thymus and 70% of CD4⁺FOXP3⁺ Tregs
in peripheral lymphoid tissues.¹¹⁹ HELIOS seems to be involved in
the stabilization of the phenotype of CD4⁺ and CD8⁺ Tregs in an in-
flammatory context.⁸ In a mouse model deficient for HELIOS, CD8⁺
and CD4⁺ Tregs were not able to control effector T cells responses.
This demonstrates HELIOS' involvement in suppressive functions,
differentiation and survival of Tregs.⁸,¹²⁰ Human CD8⁺CD45RC<sup>low</sup>-
cells co-express HELIOS and FOXP3.³ FOXP3 seems to be a key
transcription factor in the development of Tregs including CD8⁺
Tregs since silencing FOXP3 with siRNA abrogated the ability of
CD8⁺ Tregs to suppress anti-DNA antibodies in a lupus model in
mice.¹²¹ Although adoptive transfer of CD4⁺FOXP3⁺ Tregs can cure
immune dysregulation, polyendocrinopathy, enteropathy, X-linked
syndrome in FOXP3-deficient mice¹²² this does not exclude the
possibility that transfer of CD8⁺FOXP3⁺ Tregs could also have a
similar effect. This is an important experiment that has not been
reported and would confirm a physiological role in immune ho-
meostasis for CD8⁺FOXP3⁺ Treg. Induced CD8⁺ Tregs express less
FOXP3 than CD4⁺ Tregs.¹²³ However, we have shown a specific
expression of FOXP3 in natural CD8⁺CD45RC<sup>low</sup>/- Tregs while we
do not find expression in CD8⁺CD45RC<sup>high</sup> effector T cells. This ex-
pression of FOXP3 is all the more important after cell activation.³
This expression is correlated with the expression of suppressive
molecules such as CTLA-4, GITR, IFNγ and TGFβ. In mice, other
transcription factors have been described as involved in the de-
velopment and function of CD4⁺ Tregs but they have not yet been
described for CD8⁺ Tregs. IFR4 was shown essential for the expres-
sion of Blimp-1 in the differentiation of CD4⁺ effector Tregs¹²⁴; the
canonical pathway nuclear factor-kappa B is also involved in the
development and function of CD4⁺ Tregs by performing inducible
deletions of some subunits of the complex. The c-Rel subunit was
critical in thymic development, while the p65 subunit was essential
for maturing Tregs and maintaining immune tolerance.¹²⁵

## 4.2 | Targeted therapies modulate or synergize with CD8⁺ Tregs

Tregs have been well described to be induced or synergize with
antibody or cytokine therapies, to be the main effector regulatory
mechanisms boosting the efficacy of such therapies and being key
to long-term sustained effect. Thus, new therapies should strategi-
cally aim to promote Tregs to induce a satisfactory control of im-
mune responses. Strategies of costimulation blockade have been
shown to efficiently induce and mediate their tolerogenic effect
through CD8⁺ Tregs. Blockade of the CD40/CD40L costimulatory
pathway is a well-known strategy in transplantation for its potential
in animal models in tolerance induction, although its translation to
the clinic has been slowed down due to thromboembolic events.¹²⁶
We have shown that blockade of the CD40/CD40L pathway using
an adenovirus encoding CD40lg, a chimeric molecule, in a model
of fully incompatible cardiac allotransplantation in rats resulted in
tolerance induction dependent on CD8⁺CD45RC<sup>low/-</sup> Tregs.²,⁴,³⁵,¹²⁷
Blockade of the 4-1BB/4-1BBL costimulatory pathway together
with immunization in mice was also shown to induce antigen-specific
CD8⁺ Tregs acting in a IFNγ and TGFβ dependent manner.⁵⁹ In this
model IFNγ directly stimulates CD8⁺ Tregs to induce a TGFβ-based
suppression.⁵⁹ In contradiction, 4-1BB has been shown as impor-
tant for the suppressive activity of CD8⁺ Tregs in a model of allergic
inflammation.¹²⁸ We and others showed that IFNγ and TGFβ were
key mediators of the suppressive activity of CD8⁺ Tregs.⁴,⁶⁸,¹²⁹,¹³⁰
Blockade of ICOS/B7h costimulation also showed potent induction
of alloantigen-specific CD8⁺PD1⁺ Tregs in vivo in a model of heart
grafts in mice.¹³¹ More surprisingly, targeting CD3, a molecule ex-
pressed by definition by Treg, has shown effectiveness in inducing
both CD4⁺ and CD8⁺ Tregs in a collagen induced arthritis model of
rheumatoid arthritis in mice.¹³²,¹³³ In this model, CD8⁺ Tregs, unlike
CD4⁺ Tregs, potently inhibited Th17 responses, thereby inhibiting
a wider range of inflammatory pathways. The authors also showed
that monocyte membrane bound TNFα increased FOXP3 expres-
sion in CD8⁺ T cells. Administration of hOKT3g1 (Ala-Ala) in pa-
tients with type 1 diabetes mellitus halted disease progression for
>1 year and was associated with increased CD8⁺ FOXP3⁺ Tregs.¹³⁴
Induction of CD8⁺ Tregs was dependent on TNF and associated
with TNFR2 expression by the CD8⁺ Tregs.¹³⁵ There is a ying and
yang action observed by several groups for cytokines such as TNFα
and IFNγ. Anti-TNF antibodies are used in Crohn's disease and have
shown effectiveness in management of the disease but also com-
mon failure.¹³⁶ The importance of action of these cytokines on and
by CD8⁺ Tregs, but also more recently by CD4⁺ Tregs,¹³⁷ should cer-
tainly be taken into consideration in the potential limited effect of
cytokine-targeting drugs in autoimmune diseases.

Co-administration of CD8⁺ Treg therapy with costimulation
blockade is also showing synergistic efficacy. In a model of skin
transplantation in mice, CD8⁺CD122⁺PD-1⁺ Tregs expanded ex
vivo combined with costimulation blockade of CD40/CD154, but
not of B7/CD28, synergizing to prolong the allograft survival in
an IL-10-dependent manner.¹¹³ In fact, blockade of the B7/CD28
costimulation pathway has been also shown by us as being detrimental to CD8⁺ Treg-mediated tolerance. Simultaneous blockade of the CD40/CD40L and CD28/B7 interactions using CD40Ig and anti-CD28 mAbs abrogated in 50% of the recipients tolerance, inhibited CD8⁺ Treg induction and modified the regulatory mechanisms taking place in the remaining recipients that did not reject their allograft,¹³⁸ suggesting that the B7/CD28 costimulation is required for CD8⁺ Treg expansion and function.

CD8⁺ Treg induction following administration of depleting, modulating or blocking drugs has been very poorly addressed. There are several strategies under investigation for their potential in modulating the Teff/Treg balance, that are not directly targeting costimulation blockade but rather eliminating Teff of activating Tregs. We have shown that modulation of the Teff/Treg balance using anti-CD45RC mAbs depleting CD45RC<sup>high</sup> cells, ie naive and TEMRA cells, but preserving CD45RC<sup>low/-</sup> cells, ie Tregs, can efficiently induce transplant tolerance in rats and humanized immune mice.⁵⁵ The mechanisms of tolerance involved the transient depletion of naive and TEMRA cells at the time of transplantation and during 10-20 days associated with a transient increase of CD4⁺ and CD8⁺ Tregs. In this model we demonstrated that upon arrest of the treatment and return to normal level, CD4⁺ and CD8⁺ Tregs gained antigen specificity and were functionally superior as shown by in vitro suppressive assays, in vivo adoptive

cell transfer in transplanted recipients and transcriptomic analysis. We have similar unpublished results in a model of GVHD in rats and nod scid gamma (NSG) mice and in a model of lupus (unpublished data). In a lupus model in mice, administration of T cell targeting nanoparticles loaded with IL-2 and TGFβ significantly expanded CD4⁺ and CD8⁺ Tregs that could reduce the disease.¹³⁹ Cytokine therapies such as the one using IL-34 has been shown to also modulate the Treg/Teff balance by differentiating monocytes into regulatory macrophages that could in turn induce CD8⁺ and CD4⁺ Tregs responsible for the long-term tolerogenic effect.³⁷

## 5 | CD8⁺ TREG CLINICAL TRIAL IN KIDNEY TRANSPLANTED PATIENTS

While CD8⁺ T cells have been used in clinic as effector cells to treat cancer and infectious diseases (NCT00791037, NCT01325636, NCT01475058, NCT00110578), CD8⁺ Tregs have never been used as suppressive cells to treat autoimmune diseases or to control transplant rejection. Indeed, the lack of interest for CD8⁺ Tregs due to the difficulties of identification and characterization of these cells, in contrast with the extensive knowledge acquired for years on CD4⁺ Tregs, and the urgency for HSC-grafted patients with fatal outcome supported rapid translation of CD4⁺

<figure><img src="image_3.png" /><figcaption>**FIGURE 2** CD8⁺ Treg-based therapy process. In vitro steps are indicated in pink background, optional steps in blank background and clinical steps in blue background. CNI, calcineurin inhibitor; KI, knock-in; KO, knock-out; MMF, mycophenolate mofetil; Prdn, prednisolone; TAC, tacrolimus; UCB, umbilical cord blood</figcaption></figure>
costimulation pathway has been also shown by us as being detrimental to CD8⁺ Treg-mediated tolerance. Simultaneous blockade of the CD40/CD40L and CD28/B7 interactions using CD40Ig and anti-CD28 mAbs abrogated in 50% of the recipients tolerance, inhibited CD8⁺ Treg induction and modified the regulatory mechanisms taking place in the remaining recipients that did not reject their allograft,¹³⁸ suggesting that the B7/CD28 costimulation is required for CD8⁺ Treg expansion and function.

CD8⁺ Treg induction following administration of depleting, modulating or blocking drugs has been very poorly addressed. There are several strategies under investigation for their potential in modulating the Teff/Treg balance, that are not directly targeting costimulation blockade but rather eliminating Teff of activating Tregs. We have shown that modulation of the Teff/Treg balance using anti-CD45RC mAbs depleting CD45RC<sup>high</sup> cells, ie naive and TEMRA cells, but preserving CD45RC<sup>low/-</sup> cells, ie Tregs, can efficiently induce transplant tolerance in rats and humanized immune mice.⁵⁵ The mechanisms of tolerance involved the transient depletion of naive and TEMRA cells at the time of transplantation and during 10-20 days associated with a transient increase of CD4⁺ and CD8⁺ Tregs. In this model we demonstrated that upon arrest of the treatment and return to normal level, CD4⁺ and CD8⁺ Tregs gained antigen specificity and were functionally superior as shown by in vitro suppressive assays, in vivo adoptive

cell transfer in transplanted recipients and transcriptomic analysis. We have similar unpublished results in a model of GVHD in rats and nod scid gamma (NSG) mice and in a model of lupus (unpublished data). In a lupus model in mice, administration of T cell targeting nanoparticles loaded with IL-2 and TGFβ significantly expanded CD4⁺ and CD8⁺ Tregs that could reduce the disease.¹³⁹ Cytokine therapies such as the one using IL-34 has been shown to also modulate the Treg/Teff balance by differentiating monocytes into regulatory macrophages that could in turn induce CD8⁺ and CD4⁺ Tregs responsible for the long-term tolerogenic effect.³⁷

## 5 | CD8⁺ TREG CLINICAL TRIAL IN KIDNEY TRANSPLANTED PATIENTS

While CD8⁺ T cells have been used in clinic as effector cells to treat cancer and infectious diseases (NCT00791037, NCT01325636, NCT01475058, NCT00110578), CD8⁺ Tregs have never been used as suppressive cells to treat autoimmune diseases or to control transplant rejection. Indeed, the lack of interest for CD8⁺ Tregs due to the difficulties of identification and characterization of these cells, in contrast with the extensive knowledge acquired for years on CD4⁺ Tregs, and the urgency for HSC-grafted patients with fatal outcome supported rapid translation of CD4⁺

<figure><img src="image_5.png" /><figcaption>**FIGURE 2** CD8⁺ Treg-based therapy process. In vitro steps are indicated in pink background, optional steps in blank background and clinical steps in blue background. CNI, calcineurin inhibitor; KI, knock-in; KO, knock-out; MMF, mycophenolate mofetil; Prdn, prednisolone; TAC, tacrolimus; UCB, umbilical cord blood</figcaption></figure>
Treg to the clinic for cell-based therapies.¹ The new interest for CD8⁺ Tregs over the past two decades has strengthened their therapeutic potential, leading to a future first in human phase I CD8⁺ Treg-cell therapy in kidney transplanted patients, named Eight-Treg, supported by the ReSHAPE consortium, and that will take place in 2021² (https://www.reshape-h2020.eu/) (Figure 2). The primary objective of the Eight-Treg Phase I trial will be to determine the safety and dose of CD8⁺ Treg cells. CD8⁺ Tregs showed no cytotoxicity in vitro or in vivo in NSG mouse models and we observed comparable suppressive activity for CD8⁺ Tregs and CD4⁺ Tregs in vitro.³ Currently, the phase I clinical trials using CD4⁺ Tregs evaluate the cell toxicity by a dose escalation from 5 × 10⁵ to 2 × 10⁷ cells/kg in solid organ transplanted (SOT) patients and from 1 × 10⁵ to 3 × 10⁷ cells/kg in hematopoietic stem cell transplantation (HSCT) patients (NCT02371434 ONEnTreg13, NCT03444064, NCT01050764, NCT00725062, NCT03198234). In particular, the previous ONETreg1 clinical trial showed the safety of a 1 × 10⁷/kg CD4⁺ Tregs infusion in patients, and the downstream Two Study is underway to evaluate the efficacy of this dose in the treatment of kidney transplanted patients (NCT02129881, 2017-001421-41). Even the highly cytotoxic tumor infiltrating lymphocytes (TIL) CD8⁺ cells infused up to 3×10⁹ cells/kg in patients with melanoma did not induce serious adverse events (NCT01118091, NCT01236573).¹⁴⁰ Altogether, these results suggest a low risk in receiving a high dose of CD8⁺ Tregs for patients. Therefore, the Eight-Treg protocol is designed for the administration of escalating cell dose every three patients, from 3 × 10⁵ to 2 × 10⁷ CD8⁺ Tregs/kg in 12 kidney transplanted patients (https://www.reshape-h2020.eu).

The type and dose of immunosuppressive drug treatments are critical to ensure patient safety whether the cell therapy is not effective, but should not be deleterious to the adoptively transferred CD8⁺ Treg cells. We have recently shown that mycophenolic acid stopped their proliferation in contrast to rapamycin.³ In addition, we observed that rapamycin promoted CD8⁺ Treg proliferation and suppression capacities.³ Similarly, calcineurin inhibitor drugs (CNIs) were deleterious to the persistence of CD4⁺ Tregs in patients up to 1 year after kidney transplantation, while rapamycin monotherapy promoted CD4⁺ Tregs in SOT and HSCT patients¹⁴¹⁻¹⁴⁴ (NCT00803010). Nowadays, some clinical trials focus on the impact of different IS drugs, including rapamycin, on the frequency and function of CD4⁺ Tregs in SOT patients, and the inclusion of the analysis of CD8⁺ Tregs counterpart would be of great interest (NCT01014234, NCT01640743). In ongoing clinical trials in SOT, induction treatment is generally composed of tacrolimus + MMF+prednisolone for 1 month. Meanwhile, CD4⁺ Tregs are transferred adoptively rather after the critical rejection period to avoid any erroneous conclusion about any cytotoxic effect induced by the injected cells, between 1 week and 6 months after transplantation. Thus, ongoing clinical trials switch from tacrolimus to rapamycin one month after SOT to spare CD4⁺ Tregs at least and maybe to promote them (TASK, DelTa).¹⁴⁵ This rational strategy will be applied to the Eight-Treg clinical trial.

It is important to note that the Treg cells product, with or without genetic modifications, is classified as an Advanced Therapy Medicinal Product and regulated by the directive 1394/2007, aiming at standardizing the manufacturing process. The purity of cell product, the expression of key markers, and the cytotoxic and suppressive functions will be controlled. Fortunately, the manufacturing processes of CD4⁺ Tregs and CD8⁺ TILs used in ongoing clinical trials are helpful to set up the good manufacturing practices manufacturing of CD8⁺ Tregs product.¹⁴⁰,¹⁴⁶⁻¹⁵⁰ In addition, these processes can be upgraded by technical advances, such as clinical grade flow cytometry cell sorting. With this equipment, we can isolate 8.0 × 10⁴ CD8⁺ Tregs/mL blood, based on negative expression of CD161, low/neg expression of CD45RC and positive expression of CD8 marker from peripheral blood lymphocytes. Then, the cells can be highly expanded, about 200 fold in 2 weeks, while maintaining their suppressive capacity and without acquiring cytotoxic function.³ Although beads coated with anti-CD3/anti-CD28 mAbs are generally used to expand CD4⁺ Tregs, CD8⁺ Tregs are rather stimulated using the OKT3 clone, as CD8⁺ TIL for cell therapy in cancer, associated with CD28 stimulation using the CD28.2 mAb clone.¹⁴⁰ Besides, many ongoing clinical trials are evaluating the therapeutic potential of antigen-specific Tregs, such as donor alloantigen-specific Tregs in SOT (DarTregs in the delta, NJLT001, DART, ARTEMIS, LITTMUS-MGH) or tumor cells specific Teffs (TILs) in cancer (NCT01118091, NCT01236573, NCT03610490, NCT03068624), and TCR and CAR engineering is a promising alternative strategy for antigen-specific cell therapy in transplantation and autoimmune diseases.¹⁵¹ Actually, we observed higher efficacy of donor MHC-I-specific CAR CD8⁺ Tregs than polyclonal ones in MHC-I restricted transplantation models in NSG mice in which this MHC-I antigen was incompatible (unpublished data). Our current manufacturing process would allow us to infuse up to 1 × 10⁸ polyclonal CD8⁺ Tregs/kg or 1 × 10⁷ CAR-CD8⁺ Tregs/kg in patients.

An important problem for the success of Treg cell-based therapies is the persistence of the infused cells in the patient. However, the lack of specific markers for CD8⁺ Tregs is a limitation for their tracking in blood and protocol biopsies of patients. Indeed, FOXP3 has been shown to be transiently overexpressed in Teffs when stimulated while there are technical problems in analyzing treg-specific demethylated region methylations in biopsies.¹⁵²,¹⁵³ There are also technical problems in detecting key cytokines to track IL-34⁺IFNγ⁺TGFβ⁺CD8⁺ Tregs. Thus, new methods of systematic proteomic and transcriptomic analyses are emerging. Notably, deuterium is currently used in the TASK clinical trial (NCT02088931) to track adoptively transferred CD4⁺ Tregs in blood and in the grafted kidney, and this method might be considered to track CD8⁺ Tregs in patients.

Early exhaustion of adoptively transferred Tregs would affect their persistence and thus limit their therapeutic effect. Exhaustion of Tconv is reported as the expression of a combination of inhibitory receptors, activation and memory markers, such as PD-1, TIM-3, LAG-3, CTLA-4, CD160, TIGIT, BTLA-4, and correlates with improved outcome of transplanted patients.¹⁵⁴,¹⁵⁵ However, PD-1 is not only a discriminant marker but also plays a functional role in the suppressive
Treg to the clinic for cell-based therapies.¹ The new interest for CD8⁺ Tregs over the past two decades has strengthened their therapeutic potential, leading to a future first in human phase I CD8⁺ Treg-cell therapy in kidney transplanted patients, named Eight-Treg, supported by the ReSHAPE consortium, and that will take place in 2021² (https://www.reshape-h2020.eu/) (Figure 2). The primary objective of the Eight-Treg Phase I trial will be to determine the safety and dose of CD8⁺ Treg cells. CD8⁺ Tregs showed no cytotoxicity in vitro or in vivo in NSG mouse models and we observed comparable suppressive activity for CD8⁺ Tregs and CD4⁺ Tregs in vitro.³ Currently, the phase I clinical trials using CD4⁺ Tregs evaluate the cell toxicity by a dose escalation from 5 × 10⁵ to 2 × 10⁷ cells/kg in solid organ transplanted (SOT) patients and from 1 × 10⁵ to 3 × 10⁷ cells/kg in hematopoietic stem cell transplantation (HSCT) patients (NCT02371434 ONEnTreg13, NCT03444064, NCT01050764, NCT00725062, NCT03198234). In particular, the previous ONETreg1 clinical trial showed the safety of a 1 × 10⁷/kg CD4⁺ Tregs infusion in patients, and the downstream Two Study is underway to evaluate the efficacy of this dose in the treatment of kidney transplanted patients (NCT02129881, 2017-001421-41). Even the highly cytotoxic tumor infiltrating lymphocytes (TIL) CD8⁺ cells infused up to 3×10⁹ cells/kg in patients with melanoma did not induce serious adverse events (NCT01118091, NCT01236573).¹⁴⁰ Altogether, these results suggest a low risk in receiving a high dose of CD8⁺ Tregs for patients. Therefore, the Eight-Treg protocol is designed for the administration of escalating cell dose every three patients, from 3 × 10⁵ to 2 × 10⁷ CD8⁺ Tregs/kg in 12 kidney transplanted patients (https://www.reshape-h2020.eu).

The type and dose of immunosuppressive drug treatments are critical to ensure patient safety whether the cell therapy is not effective, but should not be deleterious to the adoptively transferred CD8⁺ Treg cells. We have recently shown that mycophenolic acid stopped their proliferation in contrast to rapamycin.³ In addition, we observed that rapamycin promoted CD8⁺ Treg proliferation and suppression capacities.³ Similarly, calcineurin inhibitor drugs (CNIs) were deleterious to the persistence of CD4⁺ Tregs in patients up to 1 year after kidney transplantation, while rapamycin monotherapy promoted CD4⁺ Tregs in SOT and HSCT patients¹⁴¹⁻¹⁴⁴ (NCT00803010). Nowadays, some clinical trials focus on the impact of different IS drugs, including rapamycin, on the frequency and function of CD4⁺ Tregs in SOT patients, and the inclusion of the analysis of CD8⁺ Tregs counterpart would be of great interest (NCT01014234, NCT01640743). In ongoing clinical trials in SOT, induction treatment is generally composed of tacrolimus + MMF+prednisolone for 1 month. Meanwhile, CD4⁺ Tregs are transferred adoptively rather after the critical rejection period to avoid any erroneous conclusion about any cytotoxic effect induced by the injected cells, between 1 week and 6 months after transplantation. Thus, ongoing clinical trials switch from tacrolimus to rapamycin one month after SOT to spare CD4⁺ Tregs at least and maybe to promote them (TASK, DelTa).¹⁴⁵ This rational strategy will be applied to the Eight-Treg clinical trial.

It is important to note that the Treg cells product, with or without genetic modifications, is classified as an Advanced Therapy Medicinal Product and regulated by the directive 1394/2007, aiming at standardizing the manufacturing process. The purity of cell product, the expression of key markers, and the cytotoxic and suppressive functions will be controlled. Fortunately, the manufacturing processes of CD4⁺ Tregs and CD8⁺ TILs used in ongoing clinical trials are helpful to set up the good manufacturing practices manufacturing of CD8⁺ Tregs product.¹⁴⁰,¹⁴⁶⁻¹⁵⁰ In addition, these processes can be upgraded by technical advances, such as clinical grade flow cytometry cell sorting. With this equipment, we can isolate 8.0 × 10⁴ CD8⁺ Tregs/mL blood, based on negative expression of CD161, low/neg expression of CD45RC and positive expression of CD8 marker from peripheral blood lymphocytes. Then, the cells can be highly expanded, about 200 fold in 2 weeks, while maintaining their suppressive capacity and without acquiring cytotoxic function.³ Although beads coated with anti-CD3/anti-CD28 mAbs are generally used to expand CD4⁺ Tregs, CD8⁺ Tregs are rather stimulated using the OKT3 clone, as CD8⁺ TIL for cell therapy in cancer, associated with CD28 stimulation using the CD28.2 mAb clone.¹⁴⁰ Besides, many ongoing clinical trials are evaluating the therapeutic potential of antigen-specific Tregs, such as donor alloantigen-specific Tregs in SOT (DarTregs in the delta, NJLT001, DART, ARTEMIS, LITTMUS-MGH) or tumor cells specific Teffs (TILs) in cancer (NCT01118091, NCT01236573, NCT03610490, NCT03068624), and TCR and CAR engineering is a promising alternative strategy for antigen-specific cell therapy in transplantation and autoimmune diseases.¹⁵¹ Actually, we observed higher efficacy of donor MHC-I-specific CAR CD8⁺ Tregs than polyclonal ones in MHC-I restricted transplantation models in NSG mice in which this MHC-I antigen was incompatible (unpublished data). Our current manufacturing process would allow us to infuse up to 1 × 10⁸ polyclonal CD8⁺ Tregs/kg or 1 × 10⁷ CAR-CD8⁺ Tregs/kg in patients.

An important problem for the success of Treg cell-based therapies is the persistence of the infused cells in the patient. However, the lack of specific markers for CD8⁺ Tregs is a limitation for their tracking in blood and protocol biopsies of patients. Indeed, FOXP3 has been shown to be transiently overexpressed in Teffs when stimulated while there are technical problems in analyzing treg-specific demethylated region methylations in biopsies.¹⁵²,¹⁵³ There are also technical problems in detecting key cytokines to track IL-34⁺IFNγ⁺TGFβ⁺CD8⁺ Tregs. Thus, new methods of systematic proteomic and transcriptomic analyses are emerging. Notably, deuterium is currently used in the TASK clinical trial (NCT02088931) to track adoptively transferred CD4⁺ Tregs in blood and in the grafted kidney, and this method might be considered to track CD8⁺ Tregs in patients.

Early exhaustion of adoptively transferred Tregs would affect their persistence and thus limit their therapeutic effect. Exhaustion of Tconv is reported as the expression of a combination of inhibitory receptors, activation and memory markers, such as PD-1, TIM-3, LAG-3, CTLA-4, CD160, TIGIT, BTLA-4, and correlates with improved outcome of transplanted patients.¹⁵⁴,¹⁵⁵ However, PD-1 is not only a discriminant marker but also plays a functional role in the suppressive
activity of both CD4⁺ and CD8⁺ Treg subsets,⁸⁴ while the CTLA-4 and LAG3 markers of human CD8⁺ Tregs may have a role.²⁷,²⁸,¹⁵⁶,¹⁵⁷ It is important to note that the CD8⁺ Tregs used for the Eight-Treg trial show no phenotypic sign of exhaustion after 2 weeks of culture, and survive and still proliferate for at least 1 month in vitro.³

Co-treatment with IL-2 is considered to promote the persistence of CD8⁺ Tregs in patients, as required for ex vivo culture.³,¹⁰³⁻¹⁰⁷ Encouraging results were obtained in HSCT patients treated with low dose IL-2, expanding a functional CD4⁺Foxp3⁺ Treg subset associated with a lower incidence of GVHD while maintaining a low viral infection incidence¹⁵⁸,¹⁵⁹ (NCT00539695). However, soluble circulating IL2-R can mediate sequestration of IL-2 and limit its effect¹⁶⁰ (NCT01927120). Further investigations regarding the dose and duration of IL-2 administration are required. Here again, rapamycin can promote the persistence and stability of CD8⁺ Tregs face to an inflammatory environment.³,¹⁶¹

To cope with the lack of persistence of the infused Tregs, we could multiply the injections of CD8⁺ Tregs as in CD4⁺ Treg-based clinical trials (RSMU-001: NCT01446484, Treg: NCT01624077, NCT02749084). This process requires the production of a large amount of Tregs and reliable storage with a stable suppressive function.¹⁶² Long-term culture and stable cryopreservation of CD8⁺ Tregs are possible.³

## 6 | NEW SOURCES OF TREGS: DIFFERENTIATION FROM PLURIPOTENT STEM CELLS

Although cell therapy with Tregs is very promising for treating GVHD, rejection after transplantation and autoimmune diseases, one of the limitations is the source of cells to be injected. The cell source must be easily accessible and allow a large number of cells to be donated. PBMCs and cord blood cells are the most traditional sources. They also allow autologous treatments. However, the prevalence of Tregs in these sources is quite low and requires more rounds cell expansion.¹⁶³

Recently a study has shown the possibility of using Tregs from the thymus of children recovered after cardiac surgery. Indeed, cardiac intervention in children routinely requires the removal of the thymus and these thymuses are then thrown away. However, there are more Tregs cells in a child's thymus than in an adult's entire blood volume and 100 times more than in a unit of cord blood, making it a very interesting source.¹⁶⁴ Nevertheless, this source requires a very complex organization between surgical teams and teams of biologists to collect the thymus and extract the cells and the Treg product would be from allogeneic sources to the recipient.

Since 1998, human embryonic stem cell has been the ultimate source of cells for regenerative medicine. Thanks to their pluripotent characteristic, they can differentiate into any cell in the body and are able to renew themselves indefinitely. More recently, induced pluripotent stem cells (IPSC) were obtained by

reprogramming of adult somatic cells using transduction of four factors essential for pluripotency in mice and then in humans.¹⁶⁵,¹⁶⁶ This IPSC could lead to tailor-made regenerative medicine. This avoids alloreactivity and therefore potentially reduces the doses of immunosuppressive treatment, which has many side effects. In recent years, studies on the differentiation of T cells from embryonic stem (ES) or induced pluripotent stem (iPS) cells have been carried out mainly in the field of oncology. In 2009, Timmermans et al were the first to demonstrate T cell differentiation from ES using an OP9 cell co-culture protocol.¹⁶⁷ They reported CD3 and TCR expression and IFNγ secretion after stimulation of their cells via TCR. Nowadays, several groups focus on obtaining T cells with antigenic specificity and have shown the possibility of reprogramming specific antigen T cells into IPSC: T-IPS. During the differentiation of these T-IPS the antigenic specificity is preserved.¹⁶⁸⁻¹⁷¹ All these studies showed the great potential of IPS-derived T cells as an alternative source for T cell-based immunotherapy in cancer treatments. This is why the use of ES- or IPSC-derived Tregs is also considered with great interest in the treatment of GVHD or in autoimmune diseases. In 2012, R. Haque et al showed that the transduction of FOXP3 during the differentiation of IPSC into T cells led to the production of functional CD4⁺FOXP3⁺ Tregs in mice.¹⁷² Indeed, these CD4⁺FOXP3⁺ Tregs derived from IPSC were able to secrete suppressive molecules such as IL-10 and TGFβ and they controlled autoimmunity in an arthritic mouse model. The same group showed in 2016 the use of Tregs derived from antigen-specific IPSC in the same arthritic mouse model.¹⁷³ Specific tissues or organ antigen targeting by IPSC-derived CD4⁺ Tregs allowed a faster cell response on the inflammation site. They also showed that the adoptive transfer of autoantigen-specific IPSC-CD4⁺ Tregs significantly reduced the CD8⁺/CD4⁺ ratio in the pancreas of diabetic mice.¹⁷⁴ Our own protocol of fully functional CD4⁺ and CD8⁺ Treg differentiation from human ES or IPS using FOXP3 transduction during the differentiation process allowed efficient generation of FOXP3⁺CD4⁺ and FOXP3⁺CD8⁺ Tregs (unpublished data). In addition, recently the knockdown of the polycomb group protein EZH1 was shown to activate lymphoid differentiation potential from pluripotent stem cells (PSCs) and this combined to FOXP3 transduction could improve Treg differentiation from PSCs.¹⁷⁵

All these studies demonstrate the proof of concept that Tregs derived from ES cells or IPSC are an alternative source of T and Treg cells in immunotherapy.

## 7 | ENGINEERING OF CD8⁺ TREGS

### 7.1 | TCR/CAR and others

In recent years, many clinical trials have used polyclonal Tregs or expanded Tregs.¹⁷⁶ However, polyclonal Tregs are non-specific and can potentially cause global immunosuppression. Several studies have demonstrated that the use of antigen-specific Tregs was much more effective than polyclonal Tregs in animal models.⁷,⁵²,¹⁷⁷,¹⁷⁸
activity of both CD4⁺ and CD8⁺ Treg subsets,⁸⁴ while the CTLA-4 and LAG3 markers of human CD8⁺ Tregs may have a role.²⁷,²⁸,¹⁵⁶,¹⁵⁷ It is important to note that the CD8⁺ Tregs used for the Eight-Treg trial show no phenotypic sign of exhaustion after 2 weeks of culture, and survive and still proliferate for at least 1 month in vitro.³

Co-treatment with IL-2 is considered to promote the persistence of CD8⁺ Tregs in patients, as required for ex vivo culture.³,¹⁰³⁻¹⁰⁷ Encouraging results were obtained in HSCT patients treated with low dose IL-2, expanding a functional CD4⁺Foxp3⁺ Treg subset associated with a lower incidence of GVHD while maintaining a low viral infection incidence¹⁵⁸,¹⁵⁹ (NCT00539695). However, soluble circulating IL2-R can mediate sequestration of IL-2 and limit its effect¹⁶⁰ (NCT01927120). Further investigations regarding the dose and duration of IL-2 administration are required. Here again, rapamycin can promote the persistence and stability of CD8⁺ Tregs face to an inflammatory environment.³,¹⁶¹

To cope with the lack of persistence of the infused Tregs, we could multiply the injections of CD8⁺ Tregs as in CD4⁺ Treg-based clinical trials (RSMU-001: NCT01446484, Treg: NCT01624077, NCT02749084). This process requires the production of a large amount of Tregs and reliable storage with a stable suppressive function.¹⁶² Long-term culture and stable cryopreservation of CD8⁺ Tregs are possible.³

## 6 | NEW SOURCES OF TREGS: DIFFERENTIATION FROM PLURIPOTENT STEM CELLS

Although cell therapy with Tregs is very promising for treating GVHD, rejection after transplantation and autoimmune diseases, one of the limitations is the source of cells to be injected. The cell source must be easily accessible and allow a large number of cells to be donated. PBMCs and cord blood cells are the most traditional sources. They also allow autologous treatments. However, the prevalence of Tregs in these sources is quite low and requires more rounds cell expansion.¹⁶³

Recently a study has shown the possibility of using Tregs from the thymus of children recovered after cardiac surgery. Indeed, cardiac intervention in children routinely requires the removal of the thymus and these thymuses are then thrown away. However, there are more Tregs cells in a child's thymus than in an adult's entire blood volume and 100 times more than in a unit of cord blood, making it a very interesting source.¹⁶⁴ Nevertheless, this source requires a very complex organization between surgical teams and teams of biologists to collect the thymus and extract the cells and the Treg product would be from allogeneic sources to the recipient.

Since 1998, human embryonic stem cell has been the ultimate source of cells for regenerative medicine. Thanks to their pluripotent characteristic, they can differentiate into any cell in the body and are able to renew themselves indefinitely. More recently, induced pluripotent stem cells (IPSC) were obtained by

reprogramming of adult somatic cells using transduction of four factors essential for pluripotency in mice and then in humans.¹⁶⁵,¹⁶⁶ This IPSC could lead to tailor-made regenerative medicine. This avoids alloreactivity and therefore potentially reduces the doses of immunosuppressive treatment, which has many side effects. In recent years, studies on the differentiation of T cells from embryonic stem (ES) or induced pluripotent stem (iPS) cells have been carried out mainly in the field of oncology. In 2009, Timmermans et al were the first to demonstrate T cell differentiation from ES using an OP9 cell co-culture protocol.¹⁶⁷ They reported CD3 and TCR expression and IFNγ secretion after stimulation of their cells via TCR. Nowadays, several groups focus on obtaining T cells with antigenic specificity and have shown the possibility of reprogramming specific antigen T cells into IPSC: T-IPS. During the differentiation of these T-IPS the antigenic specificity is preserved.¹⁶⁸⁻¹⁷¹ All these studies showed the great potential of IPS-derived T cells as an alternative source for T cell-based immunotherapy in cancer treatments. This is why the use of ES- or IPSC-derived Tregs is also considered with great interest in the treatment of GVHD or in autoimmune diseases. In 2012, R. Haque et al showed that the transduction of FOXP3 during the differentiation of IPSC into T cells led to the production of functional CD4⁺FOXP3⁺ Tregs in mice.¹⁷² Indeed, these CD4⁺FOXP3⁺ Tregs derived from IPSC were able to secrete suppressive molecules such as IL-10 and TGFβ and they controlled autoimmunity in an arthritic mouse model. The same group showed in 2016 the use of Tregs derived from antigen-specific IPSC in the same arthritic mouse model.¹⁷³ Specific tissues or organ antigen targeting by IPSC-derived CD4⁺ Tregs allowed a faster cell response on the inflammation site. They also showed that the adoptive transfer of autoantigen-specific IPSC-CD4⁺ Tregs significantly reduced the CD8⁺/CD4⁺ ratio in the pancreas of diabetic mice.¹⁷⁴ Our own protocol of fully functional CD4⁺ and CD8⁺ Treg differentiation from human ES or IPS using FOXP3 transduction during the differentiation process allowed efficient generation of FOXP3⁺CD4⁺ and FOXP3⁺CD8⁺ Tregs (unpublished data). In addition, recently the knockdown of the polycomb group protein EZH1 was shown to activate lymphoid differentiation potential from pluripotent stem cells (PSCs) and this combined to FOXP3 transduction could improve Treg differentiation from PSCs.¹⁷⁵

All these studies demonstrate the proof of concept that Tregs derived from ES cells or IPSC are an alternative source of T and Treg cells in immunotherapy.

## 7 | ENGINEERING OF CD8⁺ TREGS

### 7.1 | TCR/CAR and others

In recent years, many clinical trials have used polyclonal Tregs or expanded Tregs.¹⁷⁶ However, polyclonal Tregs are non-specific and can potentially cause global immunosuppression. Several studies have demonstrated that the use of antigen-specific Tregs was much more effective than polyclonal Tregs in animal models.⁷,⁵²,¹⁷⁷,¹⁷⁸
Specific antigen Tregs can be generated by cultivating them with APCs containing specific antigens or using TCR engineering techniques. However, expansion with APCs can remain quite ineffective because of the few specific antigen Tregs present in the original polyclonal cells and difficulty to translate to the clinic. Therefore, TCR engineering appears as a promising technique to obtain antigen-specific Tregs.<sup>179-181</sup> Nevertheless, the use of TCRs remains restricted to a given MHC and TCR cannot be identified in all diseases, this is why another strategy providing antigen specificity emerged a few years ago with spectacular results in cancer: the CAR. The extracellular part of this synthetic receptor binds to surface molecules on the cells independently of the MHC, unlike TCRs, and independently of the patient's HLA haplotype since the specificity is provided by antibody sequences.<sup>182</sup> The manufacture of CARs has evolved over the years, today there are four generations of CARs. The 1st generation contains a single chain variable fragment of an extracellular monoclonal antibody, a transmembrane domain and finally an intracellular domain CD3z to conduct the signal.<sup>183</sup> For the second generation, a costimulation domain linked to CD3z has been added: CD28 (cytotoxic potential) or 4-1BB (persistence and decrease exhaustion).<sup>184</sup> The third generation contains the CD3z, CD28 and 4-1BB domains which improve persistence and anti-tumor activity.<sup>185</sup> Finally, the fourth generation called T cell redirected for universal cytokine-mediated killing (TRUCKS) for TRUCKS allows the production of inducible cytokines after the recognition of the molecule of interest by the CAR. This system could make it possible to recruit non-CAR cells on the tumor site, for example through the secretion of chemokines to promote their elimination.<sup>186</sup> Cells are now mainly used for cancer immunotherapy. CAR-T cells targeting CD19 are effective in the treatment of malignant hematological diseases in preclinical and clinical trials.<sup>182,187,188</sup> They were approved by the US food and drug administration for clinical treatments in 2017. This technology has shown its full potential in cancer therapy, it is now extended to therapies using Tregs. CAR Tregs' ability to control an inflammatory bowel disease in mice<sup>189</sup> and in another model of colitis in mice<sup>190</sup> has been demonstrated. Clinical studies show the use of Tregs to prevent GVHD after allogeneic hematopoietic stem cell transplantation.<sup>191-193</sup> However, one study shows that the transfer of Tregs after hematopoietic stem cell transplantation can lead to overall immunosuppression and thus an increase in viral infections in the patient.<sup>194</sup> This is why a more specific therapy is being sought and the use of antigen-specific CAR Tregs is on the rise. HLA-A2 has been shown to be a common antigen of incompatibility in transplantation with a prevalence of approximately 50% in European phenotype patients. Over the last 3 years, three groups have used anti-HLA-A2 antibody sequences to generate CARs and shown that anti-HLA-A2 CAR-transduced CD4<sup>+</sup> Tregs in xeno-GVHD and skin grafts in NSG mice humanized with PBMCs had a higher suppressive potential than control CD4<sup>+</sup> Tregs.<sup>178,195-197</sup> This year, our group has shown for the first time the proof of concept that CD8<sup>+</sup>CD45RC<sup>low/-</sup> Tregs expressing a CAR directed against HLA-A2 are significantly more suppressive on CD4<sup>+</sup> and CD8<sup>+</sup> effector T cell

in vitro than polyclonal Tregs and are able to inhibit graft rejection and GVHD more effectively than polyclonal Tregs in vivo in a model of HLA-A2⁺ skin graft rejection and HLA-A2⁺ PBMC GVHD in NSG mice (manuscript submitted). The transfer of antigen-specific Tregs offers a promising strategy against pathologies characterized by aberrant immune activation such as transplant rejection and autoimmune diseases. In addition, the antigenic specificity provided by the CAR allows a much more targeted therapy and thus avoids the side effects of an overall immunosuppression of the patient.

In addition, insertion of a CNI resistance gene into Tregs is an interesting option considered for future clinical trials (the TacRes trial). Indeed, the stability of CD8⁺ Treg function may be optimized by genetic modifications. The CRISPR/Cas9 system opens new possibilities for stabilizing Foxp3 expression in Tregs.<sup>198</sup> Besides, demethylation of Foxp1 could help Foxp3 DNA binding and increase CD8⁺ Treg suppressive function as reported for CD4⁺ Tregs.<sup>199</sup> Otherwise, targeting genes such as HIF-1alpha KO would switch Treg metabolism from glycolytic-driven migration to an oxidative phosphorylation-driven immunosuppression.<sup>200</sup> On the other hand, overexpression of Hdr1 could help Tregs to face to ER stress response to an inflammatory environment.<sup>201</sup> Further studies are required to assess the relevance of targeting these genes to improve the stability of CD8⁺ Tregs.

## 7.2 | Safety system

To avoid any adverse event, genetic modifications to introduce a safety switch into the cells prior to the adoptive transfer have been developed. There are several techniques involving suicide genes. The two most commonly used techniques are Herpes Simplex virus thymidine kinase (HSV-TK) and human inducible caspase 9 (iCasp9). HSV-TK is the first suicide gene studied. The interaction of HSV-TK with Ganciclovir leads to cell suicide by polymerase and DNA synthesis disruption.<sup>202</sup> This technique has been tested in clinical phases I and II in France.<sup>203</sup> While effective, this approach is limited by the immunogenicity of HSV-TK expression with resultant rejection of modified cells.<sup>204</sup> iCasp9 is very effective, 30 minutes after the injection of the molecule allowing his dimerization (CID) there is elimination of 90% of the T cells modified for iCasp9. The dimerization of iCasp9 with CID results in a cascade of caspases that is responsible for cell death by apoptosis.<sup>204-207</sup> However, this approach also has disadvantages such as the possible toxicity of the remaining cells and the possible autonomous dimerization of iCasp9 leading to the elimination of the cells before their action. An iCasp9 gene has been introduced into the CAR construct for effector T cell-based therapy clinical trials in cancer (NCT03696784, NCT03016377, NCT03721068, NCT01822652).<sup>208</sup>

Other approaches such as controlling the intensity or toxicity of T or Treg cells by adding an "on switch" system via the CAR are being studied. The T cell response can be controlled by the recognition of two antigens simultaneously.<sup>209-211</sup> Kloss et al showed in a mouse prostate cancer model that T cells co-transduced with a CAR against
Specific antigen Tregs can be generated by cultivating them with APCs containing specific antigens or using TCR engineering techniques. However, expansion with APCs can remain quite ineffective because of the few specific antigen Tregs present in the original polyclonal cells and difficulty to translate to the clinic. Therefore, TCR engineering appears as a promising technique to obtain antigen-specific Tregs.<sup>179-181</sup> Nevertheless, the use of TCRs remains restricted to a given MHC and TCR cannot be identified in all diseases, this is why another strategy providing antigen specificity emerged a few years ago with spectacular results in cancer: the CAR. The extracellular part of this synthetic receptor binds to surface molecules on the cells independently of the MHC, unlike TCRs, and independently of the patient's HLA haplotype since the specificity is provided by antibody sequences.<sup>182</sup> The manufacture of CARs has evolved over the years, today there are four generations of CARs. The 1st generation contains a single chain variable fragment of an extracellular monoclonal antibody, a transmembrane domain and finally an intracellular domain CD3z to conduct the signal.<sup>183</sup> For the second generation, a costimulation domain linked to CD3z has been added: CD28 (cytotoxic potential) or 4-1BB (persistence and decrease exhaustion).<sup>184</sup> The third generation contains the CD3z, CD28 and 4-1BB domains which improve persistence and anti-tumor activity.<sup>185</sup> Finally, the fourth generation called T cell redirected for universal cytokine-mediated killing (TRUCKs) for TRUCKs allows the production of inducible cytokines after the recognition of the molecule of interest by the CAR. This system could make it possible to recruit non-CAR cells on the tumor site, for example through the secretion of chemokines to promote their elimination.<sup>186</sup> Cells are now mainly used for cancer immunotherapy. CAR-T cells targeting CD19 are effective in the treatment of malignant hematological diseases in preclinical and clinical trials.<sup>182,187,188</sup> They were approved by the US food and drug administration for clinical treatments in 2017. This technology has shown its full potential in cancer therapy, it is now extended to therapies using Tregs. CAR Tregs' ability to control an inflammatory bowel disease in mice<sup>189</sup> and in another model of colitis in mice<sup>190</sup> has been demonstrated. Clinical studies show the use of Tregs to prevent GVHD after allogeneic hematopoietic stem cell transplantation.<sup>191-193</sup> However, one study shows that the transfer of Tregs after hematopoietic stem cell transplantation can lead to overall immunosuppression and thus an increase in viral infections in the patient.<sup>194</sup> This is why a more specific therapy is being sought and the use of antigen-specific CAR Tregs is on the rise. HLA-A2 has been shown to be a common antigen of incompatibility in transplantation with a prevalence of approximately 50% in European phenotype patients. Over the last 3 years, three groups have used anti-HLA-A2 antibody sequences to generate CARs and shown that anti-HLA-A2 CAR-transduced CD4<sup>+</sup> Tregs in xeno-GVHD and skin grafts in NSG mice humanized with PBMCs had a higher suppressive potential than control CD4<sup>+</sup> Tregs.<sup>178,195-197</sup> This year, our group has shown for the first time the proof of concept that CD8<sup>+</sup>CD45RC<sup>low/-</sup> Tregs expressing a CAR directed against HLA-A2 are significantly more suppressive on CD4<sup>+</sup> and CD8<sup>+</sup> effector T cell

in vitro than polyclonal Tregs and are able to inhibit graft rejection and GVHD more effectively than polyclonal Tregs in vivo in a model of HLA-A2⁺ skin graft rejection and HLA-A2⁺ PBMC GVHD in NSG mice (manuscript submitted). The transfer of antigen-specific Tregs offers a promising strategy against pathologies characterized by aberrant immune activation such as transplant rejection and autoimmune diseases. In addition, the antigenic specificity provided by the CAR allows a much more targeted therapy and thus avoids the side effects of an overall immunosuppression of the patient.

In addition, insertion of a CNI resistance gene into Tregs is an interesting option considered for future clinical trials (the TacRes trial). Indeed, the stability of CD8⁺ Treg function may be optimized by genetic modifications. The CRISPR/Cas9 system opens new possibilities for stabilizing Foxp3 expression in Tregs.<sup>198</sup> Besides, demethylation of Foxp1 could help Foxp3 DNA binding and increase CD8⁺ Treg suppressive function as reported for CD4⁺ Tregs.<sup>199</sup> Otherwise, targeting genes such as HIF-1alpha KO would switch Treg metabolism from glycolytic-driven migration to an oxidative phosphorylation-driven immunosuppression.<sup>200</sup> On the other hand, overexpression of Hdr1 could help Tregs to face to ER stress response to an inflammatory environment.<sup>201</sup> Further studies are required to assess the relevance of targeting these genes to improve the stability of CD8⁺ Tregs.

## 7.2 | Safety system

To avoid any adverse event, genetic modifications to introduce a safety switch into the cells prior to the adoptive transfer have been developed. There are several techniques involving suicide genes. The two most commonly used techniques are Herpes Simplex virus thymidine kinase (HSV-TK) and human inducible caspase 9 (iCasp9). HSV-TK is the first suicide gene studied. The interaction of HSV-TK with Ganciclovir leads to cell suicide by polymerase and DNA synthesis disruption.<sup>202</sup> This technique has been tested in clinical phases I and II in France.<sup>203</sup> While effective, this approach is limited by the immunogenicity of HSV-TK expression with resultant rejection of modified cells.<sup>204</sup> iCasp9 is very effective, 30 minutes after the injection of the molecule allowing his dimerization (CID) there is elimination of 90% of the T cells modified for iCasp9. The dimerization of iCasp9 with CID results in a cascade of caspases that is responsible for cell death by apoptosis.<sup>204-207</sup> However, this approach also has disadvantages such as the possible toxicity of the remaining cells and the possible autonomous dimerization of iCasp9 leading to the elimination of the cells before their action. An iCasp9 gene has been introduced into the CAR construct for effector T cell-based therapy clinical trials in cancer (NCT03696784, NCT03016377, NCT03721068, NCT01822652).<sup>208</sup>

Other approaches such as controlling the intensity or toxicity of T or Treg cells by adding an "on switch" system via the CAR are being studied. The T cell response can be controlled by the recognition of two antigens simultaneously.<sup>209-211</sup> Kloss et al showed in a mouse prostate cancer model that T cells co-transduced with a CAR against
two antigenic prostate tumors (prostate-specific membrane antigen and prostate stem cell antigen) were able to destroy tumors expressing both antigens. However, these cells have no effect on cells with only one of the two antigens.²¹⁰ Conversely, it is possible to transduce T cells with an inhibitory CTLA-4- or PD-1-based iCAR to limit cytokine secretion, cytotoxicity, and proliferation.²¹² Overall, all this work provides an opportunity to specify selectivity and mimic the activity of modified T cells in order to reconcile processing power and safety.

## 8 | CONCLUSION

Altogether, we believe that CD8⁺ Tregs are powerful players in immune tolerance that should be developed further through careful and systematic analyses in rodent models and human diseases. Although there is always some way to go, all these recent advances and upheavals will bring us to a better understanding of the CD8⁺ Tregs and the next generation CD8⁺ Tregs usable for cell therapy in transplantation and autoimmune diseases.

## ACKNOWLEDGMENT

We thank the Labex IGO program supported by the National Research Agency via the investment of the future program ANR-11-LABX-0016-01 and the IHU-Cesti project program ANR-10-IBHU-005. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. We also thank the foundation Progreffe, the Fondation pour la Recherche Médicale, the Etoiles Montantes of Pays de la Loire and the Fondation du rein for personal support. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 825392. CG, IA and SB have patents that have been licensed to TxCell SA, a Sangamo company.

## REFERENCES

1. Möller G. Do suppressor T cells exist? *Scand J Immunol.* 1988;27(3):247-250.

2. Bézie S, Anegon I, Guillonneau C. Advances on CD8⁺ Treg cells and their potential in transplantation. *Transplantation*. 2018;102(9):1467-1478.

3. Bézie S, Meistermann D, Boucault L, et al. Ex vivo expanded human non-cytotoxic CD8⁺CD45RC<sup>low</sup>/− Tregs efficiently delay skin graft rejection and GVHD in humanized mice. *Front Immunol*. 2017;8:2014.

4. Guillonneau C, Hill M, Hubert F-X, et al. CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase. *J Clin Invest.* 2007;117(4):1096-1106.

5. Xystrakis E, Cavailles P, Dejean AS, et al. Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat. *J Immunol.* 2004;173(5):3140-3147.

6. Xystrakis E, Dejean AS, Bernard I, et al. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. *Blood*. 2004;104(10):3294-3301.

7. Picarda E, Bézie S, Venturi V, et al. MHC-derived allopeptide activates TCR-biased CD8⁺ Tregs and suppresses organ rejection. *J Clin Invest.* 2014;1(124):2497-2512.

8. Kim H-J, Barnitz RA, Kreslavsky T, et al. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. *Science*. 2015;350(6258):334-339.

9. Vuddamalay Y, Attia M, Vicente R, et al. Mouse and human CD8⁺ CD28(low) regulatory T lymphocytes differentiate in the thymus. *Immunology*. 2016;148(2):187-196.

10. Vieyra-Lobato MR, Vela-Ojeda J, Montiel-Cervantes L, López-Santiago R, Moreno-Lafont MC. Description of CD8⁺ regulatory T lymphocytes and their specific intervention in graft-versus-host and infectious diseases, autoimmunity, and cancer. *J Immunol Res.* 2018;2018:3758173.

11. Saligrama N, Zhao F, Sikora MJ, et al. Opposing T cell responses in experimental autoimmune encephalomyelitis. *Nature*. 2019;572(7770):481-487. https://doi.org/10.1038/s41586-019-1467-x

12. Najafian N, Chitnis T, Salama AD, et al. Regulatory functions of CD8⁺CD28⁻ T cells in an autoimmune disease model. *J Clin Invest.* 2003;112(7):1037-1048.

13. Yu P, Bamford RN, Waldmann TA. IL-15-dependent CD8⁺CD122⁺ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL-17 production by CD4⁺T cells: immunomodulation. *Eur J Immunol.* 2014;44(11):3330-3341.

14. Liu G-Z, Fang L-B, Hjelmström P, Gao X-G. Increased CD8⁺ central memory T cells in patients with multiple sclerosis. *Mult Scler J.* 2007;13(2):149-155.

15. Aristimuño C, de Andrés C, Bartolomé M, et al. IFNβ-1a therapy for multiple sclerosis expands regulatory CD8⁺T cells and decreases memory CD8⁺subset: a longitudinal 1-year study. *Clin Immunol.* 2010;134(2):148-157.

16. Negrini S, Fenoglio D, Parodi A, et al. Phenotypic alterations involved in CD8⁺Treg impairment in systemic sclerosis. *Front Immunol.* 2017;8:18.

17. Zhang L, Bertucci AM, Ramsey-Goldman R, Harsha-Strong ER, Burt RK, Datta SK. Major pathogenic steps in human lupus can be effectively suppressed by nucleosomal histone peptide epitope-induced regulatory immunity. *Clin Immunol.* 2013;149(3):365-378.

18. Zhang L, Bertucci AM, Ramsey-Goldman R, Burt RK, Datta SK. Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-producing CD8⁺Treg cells are associated with immunological remission of lupus. *J Immunol.* 2009;183(10):6346-6358.

19. Bernuzzi F, Fenoglio D, Battaglia F, et al. Phenotypical and functional alterations of CD8 regulatory T cells in primary biliary cirrhosis. *J Autoimmun.* 2010;35(3):176-180.

20. Boer MC, van Meijgaarden KE, Joosten SA, Ottenhoff T. CD8⁺ regulatory T cells, and not CD4⁺T cells, dominate suppressive phenotype and function after in vitro live *Mycobacterium bovis*-BCG activation of human cells. *PLoS ONE*. 2014;9(4):e94192.

21. Boer MC, Prins C, van Meijgaarden KE, van Dissel JT, Ottenhoff T, Joosten SA. *Mycobacterium bovis* BCG vaccination induces divergent proinflammatory or regulatory T cell responses in adults. *Clin Vaccine Immunol.* 2015;22(7):778-788.

22. Fenoglio D, Dentone C, Signori A, et al. CD8⁺CD28⁻CD127<sup>lo</sup>CD39⁺ regulatory T-cell expansion: a new possible pathogenic mechanism for HIV infection? *J Allergy Clin Immunol.* 2018;141(6):2220-2233.e4.

23. Popescu I, Macedo C, Abu-Elmagd K, et al. EBV-specific CD8⁺T cell reactivation in transplant patients results in expansion of CD8⁺type-1 regulatory T cells. *Am J Transplant.* 2007;7(5):1215-1223.

24. Chaput N, Louafi S, Bardier A, et al. Identification of CD8⁺CD25⁺Foxp3⁺ suppressive T cells in colorectal cancer tissue. *Gut.* 2009;58(4):520-529.
two antigenic prostate tumors (prostate-specific membrane antigen and prostate stem cell antigen) were able to destroy tumors expressing both antigens. However, these cells have no effect on cells with only one of the two antigens.²¹⁰ Conversely, it is possible to transduce T cells with an inhibitory CTLA-4- or PD-1-based iCAR to limit cytokine secretion, cytotoxicity, and proliferation.²¹² Overall, all this work provides an opportunity to specify selectivity and mimic the activity of modified T cells in order to reconcile processing power and safety.

## 8 | CONCLUSION

Altogether, we believe that CD8⁺ Tregs are powerful players in immune tolerance that should be developed further through careful and systematic analyses in rodent models and human diseases. Although there is always some way to go, all these recent advances and upheavals will bring us to a better understanding of the CD8⁺ Tregs and the next generation CD8⁺ Tregs usable for cell therapy in transplantation and autoimmune diseases.

## ACKNOWLEDGMENT

We thank the Labex IGO program supported by the National Research Agency via the investment of the future program ANR-11-LABX-0016-01 and the IHU-Cesti project program ANR-10-IBHU-005. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. We also thank the foundation Progreffe, the Fondation pour la Recherche Médicale, the Etoiles Montantes of Pays de la Loire and the Fondation du rein for personal support. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 825392. CG, IA and SB have patents that have been licensed to TxCell SA, a Sangamo company.

## REFERENCES

1. Möller G. Do suppressor T cells exist? *Scand J Immunol.* 1988;27(3):247-250.

2. Bézie S, Anegon I, Guillonneau C. Advances on CD8⁺ Treg cells and their potential in transplantation. *Transplantation*. 2018;102(9):1467-1478.

3. Bézie S, Meistermann D, Boucault L, et al. Ex vivo expanded human non-cytotoxic CD8⁺CD45RC<sup>low</sup>/− Tregs efficiently delay skin graft rejection and GVHD in humanized mice. *Front Immunol*. 2017;8:2014.

4. Guillonneau C, Hill M, Hubert F-X, et al. CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase. *J Clin Invest.* 2007;117(4):1096-1106.

5. Xystrakis E, Cavailles P, Dejean AS, et al. Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat. *J Immunol.* 2004;173(5):3140-3147.

6. Xystrakis E, Dejean AS, Bernard I, et al. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. *Blood*. 2004;104(10):3294-3301.

7. Picarda E, Bézie S, Venturi V, et al. MHC-derived allopeptide activates TCR-biased CD8⁺ Tregs and suppresses organ rejection. *J Clin Invest.* 2014;1(124):2497-2512.

8. Kim H-J, Barnitz RA, Kreslavsky T, et al. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. *Science*. 2015;350(6258):334-339.

9. Vuddamalay Y, Attia M, Vicente R, et al. Mouse and human CD8⁺ CD28(low) regulatory T lymphocytes differentiate in the thymus. *Immunology*. 2016;148(2):187-196.

10. Vieyra-Lobato MR, Vela-Ojeda J, Montiel-Cervantes L, López-Santiago R, Moreno-Lafont MC. Description of CD8⁺ regulatory T lymphocytes and their specific intervention in graft-versus-host and infectious diseases, autoimmunity, and cancer. *J Immunol Res.* 2018;2018:3758173.

11. Saligrama N, Zhao F, Sikora MJ, et al. Opposing T cell responses in experimental autoimmune encephalomyelitis. *Nature*. 2019;572(7770):481-487. https://doi.org/10.1038/s41586-019-1467-x

12. Najafian N, Chitnis T, Salama AD, et al. Regulatory functions of CD8⁺CD28⁻ T cells in an autoimmune disease model. *J Clin Invest.* 2003;112(7):1037-1048.

13. Yu P, Bamford RN, Waldmann TA. IL-15-dependent CD8⁺CD122⁺ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL-17 production by CD4⁺ T cells: immunomodulation. *Eur J Immunol.* 2014;44(11):3330-3341.

14. Liu G-Z, Fang L-B, Hjelmström P, Gao X-G. Increased CD8⁺ central memory T cells in patients with multiple sclerosis. *Mult Scler J.* 2007;13(2):149-155.

15. Aristimuño C, de Andrés C, Bartolomé M, et al. IFNβ-1a therapy for multiple sclerosis expands regulatory CD8⁺ T cells and decreases memory CD8⁺ subset: a longitudinal 1-year study. *Clin Immunol.* 2010;134(2):148-157.

16. Negrini S, Fenoglio D, Parodi A, et al. Phenotypic alterations involved in CD8⁺ Treg impairment in systemic sclerosis. *Front Immunol.* 2017;8:18.

17. Zhang L, Bertucci AM, Ramsey-Goldman R, Harsha-Strong ER, Burt RK, Datta SK. Major pathogenic steps in human lupus can be effectively suppressed by nucleosomal histone peptide epitope-induced regulatory immunity. *Clin Immunol.* 2013;149(3):365-378.

18. Zhang L, Bertucci AM, Ramsey-Goldman R, Burt RK, Datta SK. Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-producing CD8⁺ Treg cells are associated with immunological remission of lupus. *J Immunol.* 2009;183(10):6346-6358.

19. Bernuzzi F, Fenoglio D, Battaglia F, et al. Phenotypical and functional alterations of CD8 regulatory T cells in primary biliary cirrhosis. *J Autoimmun.* 2010;35(3):176-180.

20. Boer MC, van Meijgaarden KE, Joosten SA, Ottenhoff T. CD8⁺ regulatory T cells, and not CD4⁺ T cells, dominate suppressive phenotype and function after in vitro live *Mycobacterium bovis*-BCG activation of human cells. *PLoS ONE*. 2014;9(4):e94192.

21. Boer MC, Prins C, van Meijgaarden KE, van Dissel JT, Ottenhoff T, Joosten SA. *Mycobacterium bovis* BCG vaccination induces divergent proinflammatory or regulatory T cell responses in adults. *Clin Vaccine Immunol.* 2015;22(7):778-788.

22. Fenoglio D, Dentone C, Signori A, et al. CD8⁺CD28⁻CD127<sup>lo</sup>CD39⁺ regulatory T-cell expansion: a new possible pathogenic mechanism for HIV infection? *J Allergy Clin Immunol.* 2018;141(6):2220-2233.e4.

23. Popescu I, Macedo C, Abu-Elmagd K, et al. EBV-specific CD8⁺ T cell reactivation in transplant patients results in expansion of CD8⁺ type-1 regulatory T cells. *Am J Transplant.* 2007;7(5):1215-1223.

24. Chaput N, Louafi S, Bardier A, et al. Identification of CD8⁺CD25⁺Foxp3⁺ suppressive T cells in colorectal cancer tissue. *Gut.* 2009;58(4):520-529.
25. Kiniwa Y, Miyahara Y, Wang HY, et al. CD8⁺ Foxp3⁺ regulatory T cells mediate immunosuppression in prostate cancer. *Clin Cancer Res.* 2007;13(23):6947-6958.

26. Canale FP, Ramello MC, Núñez N, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8⁺ T cells. *Can Res.* 2018;78(1):115-128.

27. Boor P, Metselaar HJ, de Jonge S, Mancham S, van der Laan L, Kwekkeboom J. Human plasmacytoid dendritic cells induce CD8⁺ LAG-3⁺ Foxp3⁺ CTLA-4⁺ regulatory T cells that suppress allo-reactive memory T cells. *Eur J Immunol.* 2011;41(6):1663-1674.

28. Zheng J, Liu Y, Liu Y, et al. Human CD8⁺ regulatory T cells inhibit GVHD and preserve general immunity in humanized mice. *Sci Transl Med.* 2013;5(168):168ra9.

29. Barbon CM, Davies JK, Voskertchian A, et al. Alloanergization of human T cells results in expansion of alloantigen-specific CD8(+) CD28(−) suppressor cells. *Am J Transplant.* 2014;14(2):305-318.

30. Avivi I, Stroopinsky D, Rowe JM, Katz T. A subset of CD8⁺ T cells acquiring selective suppressive properties may play a role in GVHD management. *Transpl Immunol.* 2013;28(1):57-61.

31. Beres RJ, Haribhai D, Chadwick AC, Gonyo PJ, Williams CB, Drobyski WR. CD8⁺ Foxp3⁺ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity. *J Immunol.* 2012;189(1):464-474.

32. Robb RJ, Lineburg KE, Kuns RD, et al. Identification and expansion of highly suppressive CD8⁺FoxP3⁺ regulatory T cells after experimental allogeneic bone marrow transplantation. *Blood.* 2012;119(24):5898-5908.

33. Zimmerer JM, Ringwald BA, Elzein SM, et al. Antibody-suppressor CD8⁺T cells require CXCR5. *Transplantation.* 2019;103(9):1809-1820.

34. Zemmour D, Zilionis R, Kiner E, Klein AM, Mathis D, Benoist C. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. *Nat Immunol.* 2018;19(3): 291-301.

35. Li XL, Ménoret S, Bezie S, et al. Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance. *J Immunol.* 2010;185(2):823-833.

36. Bézie S, Picarda E, Tesson L, et al. Fibrinogen-like protein 2/fibroleukin induces long-term allograft survival in a rat model through regulatory B Cells. *PLoS ONE.* 2015;10(3):e0119686.

37. Bézie S, Picarda E, Ossart J, et al. IL-34 is a Treg-specific cytokine and mediates transplant tolerance. *J Clin Invest.* 2015;125(10):3952-3964.

38. Guillonneau C, Bézie S, Anegon I. Immunoregulatory properties of the cytokine IL-34. *Cell Mol Life Sci.* 2017;74(14):2569-2586.

39. Bézie S, Picarda E, Ossart J, Martinet B, Anegon I, Guillonneau C. Compensatory regulatory networks between CD8 T, B, and myeloid cells in organ transplantation tolerance. *J Immunol.* 2015;195(12):5805-5815.

40. Munn DH, Zhou M, Attwood JT, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. *Science.* 1998;281(5380):1191-1193.

41. Lindau R, Mehta RB, Lash GE, et al. Interleukin-34 is present at the fetal-maternal interface and induces immunoregulatory macrophages of a decidual phenotype in vitro. *Hum Reprod.* 2018;33(4):588-599.

42. Wang S, Sun F, Li M, et al. The appropriate frequency and function of decidual Tim-3⁺CTLA-4⁺CD8⁺ T cells are important in maintaining normal pregnancy. *Cell Death Dis.* 2019;10(6):407.

43. Tilburgs T, Strominger JL. CD8⁺ effector T cells at the fetal-maternal interface, balancing fetal tolerance and antiviral immunity. *Am J Reprod Immunol.* 2013;69(4):395-407.

44. Tilburgs T, Roelen DL, van der Mast BJ, et al. Differential distribution of CD4(+)-CD25(bright) and CD8(+)-CD28(−) T-cells in decidua and maternal blood during human pregnancy. *Placenta.* 2006;27(Suppl A):S47-S53.

45. Crespo ÁC, van der Zwan A, Ramalho-Santos J, Strominger JL, Tilburgs T. Cytotoxic potential of decidual NK cells and CD8+ T cells awakened by infections. *J Reprod Immunol.* 2017;119:85-90.

46. Tilburgs T, Crespo ÁC, van der Zwan A, et al. Human HLA-G⁺ extra-villous trophoblasts: immune-activating cells that interact with decidual leukocytes. *Proc Natl Acad Sci USA.* 2015;112(23):7219-7224.

47. Hu D, Ikizawa K, Lu L, Sanchirico ME, Shinohara ML, Cantor H. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. *Nat Immunol.* 2004;5(5):516-523.

48. Varthaman A, Khallou-Laschet J, Clement M, et al. Control of T cell reactivation by regulatory Qa-1-restricted CD8⁺ T cells. *J Immunol.* 2010;184(12):6585-6591.

49. Tang X, Maricic I, Purohit N, et al. Regulation of immunity by a novel population of Qa-1-restricted CD8αα⁺TCRαββα⁺T cells. *J Immunol.* 2006;177(11):7645-7655.

50. Jiang H, Canfield SM, Gallagher MP, et al. HLA-E-restricted regulatory CD8(+) T cells are involved in development and control of human autoimmune type 1 diabetes. *J Clin Invest.* 2010;120(10):3641-3650.

51. Li J, Goldstein I, Glickman-Nir E, Jiang H, Chess L. Induction of TCR Vbeta-specific CD8⁺ CTLs by TCR Vbeta-derived peptides bound to HLA-E. *J Immunol.* 2001;167(7):3800-3808.

52. Picarda E, Anegon I, Guillonneau C. T-cell receptor specificity of CD8(+) Tregs in allotransplantation. *Immunotherapy.* 2011;3(4 Suppl):35-37.

53. Beringer DX, Kleijwegt FS, Wiede F, et al. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex. *Nat Immunol.* 2015;16(11):1153-1161.

54. Dai Z, Zhang S, Xie Q, et al. Natural CD8⁺CD122⁺ T cells are more potent in suppression of allograft rejection than CD4⁺CD25⁺ regulatory T cells: CD8⁺CD122⁺ cells are supreme T suppressors. *Am J Transplant.* 2014;14(1):39-48.

55. Picarda E, Bézie S, Boucault L, et al. Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells. *JCI Insight.* 2017;2(3):e90088.

56. Heinrichs J, Li J, Nguyen H, et al. CD8(+) Tregs promote GVHD prevention and overcome the impaired GVL effect mediated by CD4(+) Tregs in mice. *Oncoimmunology.* 2016;5(6):e1146842.

57. Yang J, Brook MO, Carvalho-Gaspar M, et al. Allograft rejection mediated by memory T cells is resistant to regulation. *Proc Natl Acad Sci USA.* 2007;104(50):19954-19959.

58. Long X, Cheng Q, Liang H, et al. Memory CD4⁺ T cells are suppressed by CD8⁺ regulatory T cells in vitro and in vivo. *Am J Transl Res.* 2017;9(1):63-78.

59. Myers L, Croft M, Kwon BS, Mittler RS, Vella AT. Peptide-specific CD8 T regulatory cells use IFN-gamma to elaborate TGF-beta-based suppression. *J Immunol.* 2005;174(12):7625-7632.

60. Yamada Y, Sugita S, Horie S, Yamagami S, Mochizuki M. Mechanisms of immune suppression for CD8⁺ T cells by human corneal endothelial cells via membrane-bound TGFbeta. *Invest Ophthalmol Vis Sci.* 2010;51(5):2548-2557.

61. Suzuki M, Jagger AL, Konya C, et al. CD8⁺CD45RA⁺CCR7⁺FOXP3⁺ T cells with immunosuppressive properties: a novel subset of inducible human regulatory T cells. *J Immunol.* 2012;189(5): 2118-2130.

62. Shalev I, Wong KM, Foerster K, et al. The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis. *Hepatology.* 2009;49(2):387-397.

63. Denning TL, Granger SW, Granger S, et al. Mouse TCRαβ⁺CD8αα intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses. *J Immunol.* 2007;178(7):4230-4239.

64. Liu Y, Xu S, Xiao F, et al. The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD
25. Kiniwa Y, Miyahara Y, Wang HY, et al. CD8⁺ Foxp3⁺ regulatory T cells mediate immunosuppression in prostate cancer. *Clin Cancer Res.* 2007;13(23):6947-6958.

26. Canale FP, Ramello MC, Núñez N, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8⁺ T cells. *Can Res.* 2018;78(1):115-128.

27. Boor P, Metselaar HJ, de Jonge S, Mancham S, van der Laan L, Kwekkeboom J. Human plasmacytoid dendritic cells induce CD8⁺ LAG-3⁺ Foxp3⁺ CTLA-4⁺ regulatory T cells that suppress allo-reactive memory T cells. *Eur J Immunol.* 2011;41(6):1663-1674.

28. Zheng J, Liu Y, Liu Y, et al. Human CD8⁺ regulatory T cells inhibit GVHD and preserve general immunity in humanized mice. *Sci Transl Med.* 2013;5(168):168ra9.

29. Barbon CM, Davies JK, Voskertchian A, et al. Alloanergization of human T cells results in expansion of alloantigen-specific CD8(+) CD28(−) suppressor cells. *Am J Transplant.* 2014;14(2):305-318.

30. Avivi I, Stroopinsky D, Rowe JM, Katz T. A subset of CD8⁺ T cells acquiring selective suppressive properties may play a role in GVHD management. *Transpl Immunol.* 2013;28(1):57-61.

31. Beres RJ, Haribhai D, Chadwick AC, Gonyo PJ, Williams CB, Drobyski WR. CD8⁺ Foxp3⁺ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity. *J Immunol.* 2012;189(1):464-474.

32. Robb RJ, Lineburg KE, Kuns RD, et al. Identification and expansion of highly suppressive CD8⁺FoxP3⁺ regulatory T cells after experimental allogeneic bone marrow transplantation. *Blood.* 2012;119(24):5898-5908.

33. Zimmerer JM, Ringwald BA, Elzein SM, et al. Antibody-suppressor CD8⁺T cells require CXCR5. *Transplantation.* 2019;103(9):1809-1820.

34. Zemmour D, Zilionis R, Kiner E, Klein AM, Mathis D, Benoist C. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. *Nat Immunol.* 2018;19(3): 291-301.

35. Li XL, Ménoret S, Bezie S, et al. Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance. *J Immunol.* 2010;185(2):823-833.

36. Bézie S, Picarda E, Tesson L, et al. Fibrinogen-like protein 2/fibroleukin induces long-term allograft survival in a rat model through regulatory B Cells. *PLoS ONE.* 2015;10(3):e0119686.

37. Bézie S, Picarda E, Ossart J, et al. IL-34 is a Treg-specific cytokine and mediates transplant tolerance. *J Clin Invest.* 2015;125(10):3952-3964.

38. Guillonneau C, Bézie S, Anegon I. Immunoregulatory properties of the cytokine IL-34. *Cell Mol Life Sci.* 2017;74(14):2569-2586.

39. Bézie S, Picarda E, Ossart J, Martinet B, Anegon I, Guillonneau C. Compensatory regulatory networks between CD8 T, B, and myeloid cells in organ transplantation tolerance. *J Immunol.* 2015;195(12):5805-5815.

40. Munn DH, Zhou M, Attwood JT, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. *Science.* 1998;281(5380):1191-1193.

41. Lindau R, Mehta RB, Lash GE, et al. Interleukin-34 is present at the fetal-maternal interface and induces immunoregulatory macrophages of a decidual phenotype in vitro. *Hum Reprod.* 2018;33(4):588-599.

42. Wang S, Sun F, Li M, et al. The appropriate frequency and function of decidual Tim-3⁺CTLA-4⁺CD8⁺ T cells are important in maintaining normal pregnancy. *Cell Death Dis.* 2019;10(6):407.

43. Tilburgs T, Strominger JL. CD8⁺ effector T cells at the fetal-maternal interface, balancing fetal tolerance and antiviral immunity. *Am J Reprod Immunol.* 2013;69(4):395-407.

44. Tilburgs T, Roelen DL, van der Mast BJ, et al. Differential distribution of CD4(+)-CD25(bright) and CD8(+)-CD28(−) T-cells in decidua and maternal blood during human pregnancy. *Placenta.* 2006;27(Suppl A):S47-S53.

45. Crespo ÁC, van der Zwan A, Ramalho-Santos J, Strominger JL, Tilburgs T. Cytotoxic potential of decidual NK cells and CD8+ T cells awakened by infections. *J Reprod Immunol.* 2017;119:85-90.

46. Tilburgs T, Crespo ÁC, van der Zwan A, et al. Human HLA-G⁺ extra-villous trophoblasts: immune-activating cells that interact with decidual leukocytes. *Proc Natl Acad Sci USA.* 2015;112(23):7219-7224.

47. Hu D, Ikizawa K, Lu L, Sanchirico ME, Shinohara ML, Cantor H. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. *Nat Immunol.* 2004;5(5):516-523.

48. Varthaman A, Khallou-Laschet J, Clement M, et al. Control of T cell reactivation by regulatory Qa-1-restricted CD8⁺ T cells. *J Immunol.* 2010;184(12):6585-6591.

49. Tang X, Maricic I, Purohit N, et al. Regulation of immunity by a novel population of Qa-1-restricted CD8αα⁺TCRαββα⁺T cells. *J Immunol.* 2006;177(11):7645-7655.

50. Jiang H, Canfield SM, Gallagher MP, et al. HLA-E-restricted regulatory CD8(+) T cells are involved in development and control of human autoimmune type 1 diabetes. *J Clin Invest.* 2010;120(10):3641-3650.

51. Li J, Goldstein I, Glickman-Nir E, Jiang H, Chess L. Induction of TCR Vbeta-specific CD8⁺ CTLs by TCR Vbeta-derived peptides bound to HLA-E. *J Immunol.* 2001;167(7):3800-3808.

52. Picarda E, Anegon I, Guillonneau C. T-cell receptor specificity of CD8(+) Tregs in allotransplantation. *Immunotherapy.* 2011;3(4 Suppl):35-37.

53. Beringer DX, Kleijwegt FS, Wiede F, et al. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex. *Nat Immunol.* 2015;16(11):1153-1161.

54. Dai Z, Zhang S, Xie Q, et al. Natural CD8⁺CD122⁺ T cells are more potent in suppression of allograft rejection than CD4⁺CD25⁺ regulatory T cells: CD8⁺CD122⁺ cells are supreme T suppressors. *Am J Transplant.* 2014;14(1):39-48.

55. Picarda E, Bézie S, Boucault L, et al. Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells. *JCI Insight.* 2017;2(3):e90088.

56. Heinrichs J, Li J, Nguyen H, et al. CD8(+) Tregs promote GVHD prevention and overcome the impaired GVL effect mediated by CD4(+) Tregs in mice. *Oncoimmunology.* 2016;5(6):e1146842.

57. Yang J, Brook MO, Carvalho-Gaspar M, et al. Allograft rejection mediated by memory T cells is resistant to regulation. *Proc Natl Acad Sci USA.* 2007;104(50):19954-19959.

58. Long X, Cheng Q, Liang H, et al. Memory CD4⁺ T cells are suppressed by CD8⁺ regulatory T cells in vitro and in vivo. *Am J Transl Res.* 2017;9(1):63-78.

59. Myers L, Croft M, Kwon BS, Mittler RS, Vella AT. Peptide-specific CD8 T regulatory cells use IFN-gamma to elaborate TGF-beta-based suppression. *J Immunol.* 2005;174(12):7625-7632.

60. Yamada Y, Sugita S, Horie S, Yamagami S, Mochizuki M. Mechanisms of immune suppression for CD8⁺ T cells by human corneal endothelial cells via membrane-bound TGFbeta. *Invest Ophthalmol Vis Sci.* 2010;51(5):2548-2557.

61. Suzuki M, Jagger AL, Konya C, et al. CD8⁺CD45RA⁺CCR7⁺FOXP3⁺ T cells with immunosuppressive properties: a novel subset of inducible human regulatory T cells. *J Immunol.* 2012;189(5): 2118-2130.

62. Shalev I, Wong KM, Foerster K, et al. The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis. *Hepatology.* 2009;49(2):387-397.

63. Denning TL, Granger SW, Granger S, et al. Mouse TCRαβ⁺CD8αα intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses. *J Immunol.* 2007;178(7):4230-4239.

64. Liu Y, Xu S, Xiao F, et al. The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD
through the MAPK pathway. *Biochem Biophys Res Commun.* 2010;396(2):555-561.

65. Paunicka K, Chen PW, Niederkorn JY. Role of IFN-γ in the establishment of anterior chamber-associated immune deviation (ACAID)-induced CD8+ T regulatory cells. *J Leukoc Biol.* 2012;91(3):475-483.

66. Olson BM, Jankowska-Gan E, Becker JT, Vignali D, Burlingham WJ, McNeel DG. Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade. *J Immunol.* 2012;189(12):5590-5601.

67. Balashov KE, Khoury SJ, Hafler DA, Weiner HL. Inhibition of T cell responses by activated human CD8+ T cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis. *J Clin Invest.* 1995;95(6):2711-2719.

68. Zhang J. Yin and yang interplay of IFN-gamma in inflammation and autoimmune disease. *J Clin Invest.* 2007;117(4):871-873.

69. Konieczny BT, Dai Z, Elwood ET, et al. IFN-gamma is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. *J Immunol.* 1998;160(5):2059-2064.

70. Sawitzki B, Kingsley CI, Oliveira V, Karim M, Herber M, Wood KJ. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. *J Exp Med.* 2005;201(12):1925-1935.

71. Collison LW, Workman CJ, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. *Nature.* 2007;450(7169):566-569.

72. Zhang J, Zhang Y, Wang Q, et al. Interleukin-35 in immune-related diseases: protection or destruction. *Immunology.* 2019;157(1):13-20.

73. Krupnick AS, Lin X, Li W, et al. Central memory CD8+ T lymphocytes mediate lung allograft acceptance. *J Clin Invest.* 2014;124(3):1130-1143.

74. Takahashi T, Hsiao HM, Tanaka S, et al. PD-1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction. *Am J Transplant.* 2018;18(1):216-225.

75. Zimmerer JM, Pham TA, Wright CL, et al. Alloprimed CD8+ T cells regulate alloantibody and eliminate allogprimed B cells through perforin- and FasL-dependent mechanisms: CD8+ T cells kill IgG1-producing B cells. *Am J Transplant.* 2014;14(2):295-304.

76. Bjarnadottir U, Lemarquis AL, Halldorsdottir S, Freysdottir J, Ludviksson BR. The suppressive function of human CD8(+)iTregs is inhibited by IL-1β and TNFα. *Scand J Immunol.* 2014;80(5):313-322.

77. Choi BK, Asai T, Vinay DS, Kim YH, Kwon BS. 4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-dioxygenase-dependent mechanisms. *Cytokine.* 2006;34(5-6):233-242.

78. Wang Z. Role of IFN-g in induction of Foxp3 and conversion of CD4+CD25- T cells to CD4+ Tregs. *J Clin Invest.* 2006;116(9):2434-2441. https://doi.org/10.1172/JCI25826

79. Dai H, Wan N, Zhang S, Moore Y, Wan F, Dai Z. Cutting edge: programmed death-1 defines CD8+CD122+ T cells as regulatory versus memory T cells. *J Immunol.* 2010;185(2):803-807.

80. Chikuma S, Terawaki S, Hayashi T, et al. PD-1-mediated suppression of IL-2 production induces CD8+ T cell anergy in vivo. *J Immunol.* 2009;182(11):6682-6689.

81. van der Merwe M, Abdelsamed HA, Seth A, Ong T, Vogel P, Pillai AB. Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation. *J Immunol.* 2013;191(11):5764-5776.

82. Francisco LM, Salinas VH, Brown KE, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. *J Exp Med.* 2009;206(13):3015-3029.

83. Amarnath S, Costanzo CM, Mariotti J, et al. Cells and human myeloid dendritic cells promote tolerance via programmed death ligand-1. *PLoS Biol.* 2010;8(2):e1000302.

84. Machicote A, Belén S, Baz P, Billordo LA, Fainboim L. Human CD8+HLA-DR+ regulatory T cells, similarly to classical CD4+Foxp3+ cells, suppress immune responses via PD-1/PD-L1 axis. *Front Immunol.* 2018;9:2788.

85. Lu L, Kim H-J, Werneck M, Cantor H. Regulation of CD8+ regulatory T cells: interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease. *Proc Natl Acad Sci USA.* 2008;105(49):19420-19425.

86. Mu C, Zhang X, Wang L, et al. Enhanced suppression of polyclonal CD8(+)25(+) regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes. *J Leukoc Biol.* 2017;101(5):1221-1231.

87. Akane K, Kojima S, Mak TW, Shiku H, Suzuki H. CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity. *Proc Natl Acad Sci USA.* 2016;113(9):2460-2465.

88. Chen Z, Han Y, Gu Y, et al. CD11c(high)CD8+ regulatory T cell feedback inhibits CD4 T cell immune response via Fas ligand-Fas pathway. *J Immunol.* 2013;190(12):6145-6154.

89. Daniel V, Sadeghi M, Wang H, Opelz G. CD4+CD25+Foxp3+IFNγ+CD178+ human induced Treg (iTreg) contribute to suppression of alloresponses by apoptosis of responder cells. *Hum Immunol.* 2013;74(2):151-162.

90. Boissonnas A, Scholer-Dahirel A, Simon-Blancal V, et al. Foxp3+ T cells induce perforin-dependent dendritic cell death in tumor-draining lymph nodes. *Immunity.* 2010;32(2):266-278.

91. Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. *Immunity.* 2004;21(4):589-601.

92. Guipouy D, Gertner-Dardenne J, Pfajfer L, German Y, Belmonte N, Dupré L. Granulysin- and granzyme-dependent elimination of myeloid cells by therapeutic ova-specific type 1 regulatory T cells. *Int Immunol.* 2019;31(4):239-250.

93. Gondek DC, Lu L-F, Quezada SA, Sakaguchi S, Noelle RJ. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. *J Immunol.* 2005;174(4):1783-1786.

94. Cao X, Cai SF, Fehniger TA, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. *Immunity.* 2007;27(4):635-646.

95. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. *Cell.* 2017;168(6):960-976.

96. Perl A. Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases. *Nat Rev Rheumatol.* 2016;12(3):169-182.

97. Zeng H, Yang K, Cloer C, Neale G, Vogel P, Chi H. mTORC1 couples immune signals and metabolic programming to establish Treg-cell function. *Nature.* 2013;499(7459):485-490.

98. Battaglia M, Stabilini A, Roncarolo M-G. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. *Blood.* 2005;105(12):4743-4748.

99. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. *J Immunol.* 2007;178(1):320-329.

100. Michalek RD, Gerriets VA, Jacobs SR, et al. Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets. *J Immunol.* 2011;186(6):3299-3303.

101. Coe DJ, Kishore M, Marelli-Berg F. Metabolic regulation of regulatory T cell development and function. *Front Immunol.* 2014;5:590.

102. Sun J, Yang Y, Huo X, et al. Efficient therapeutic function and mechanisms of human polyclonal CD8+ CD103+ Foxp3+
through the MAPK pathway. *Biochem Biophys Res Commun.* 2010;396(2):555-561.

65. Paunicka K, Chen PW, Niederkorn JY. Role of IFN-γ in the establishment of anterior chamber-associated immune deviation (ACAID)-induced CD8⁺ T regulatory cells. *J Leukoc Biol.* 2012;91(3):475-483.

66. Olson BM, Jankowska-Gan E, Becker JT, Vignali D, Burlingham WJ, McNeel DG. Human prostate tumor antigen-specific CD8⁺ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade. *J Immunol.* 2012;189(12):5590-5601.

67. Balashov KE, Khoury SJ, Hafler DA, Weiner HL. Inhibition of T cell responses by activated human CD8⁺ T cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis. *J Clin Invest.* 1995;95(6):2711-2719.

68. Zhang J. Yin and yang interplay of IFN-gamma in inflammation and autoimmune disease. *J Clin Invest.* 2007;117(4):871-873.

69. Konieczny BT, Dai Z, Elwood ET, et al. IFN-gamma is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. *J Immunol.* 1998;160(5):2059-2064.

70. Sawitzki B, Kingsley CI, Oliveira V, Karim M, Herber M, Wood KJ. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. *J Exp Med.* 2005;201(12):1925-1935.

71. Collison LW, Workman CJ, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. *Nature.* 2007;450(7169):566-569.

72. Zhang J, Zhang Y, Wang Q, et al. Interleukin-35 in immune-related diseases: protection or destruction. *Immunology.* 2019;157(1):13-20.

73. Krupnick AS, Lin X, Li W, et al. Central memory CD8⁺ T lymphocytes mediate lung allograft acceptance. *J Clin Invest.* 2014;124(3):1130-1143.

74. Takahashi T, Hsiao HM, Tanaka S, et al. PD-1 expression on CD8⁺ T cells regulates their differentiation within lung allografts and is critical for tolerance induction. *Am J Transplant.* 2018;18(1):216-225.

75. Zimmerer JM, Pham TA, Wright CL, et al. Alloprimed CD8⁺ T cells regulate alloantibody and eliminate allog primed B cells through perforin- and FasL-dependent mechanisms: CD8⁺ T cells kill IgG1-producing B cells. *Am J Transplant.* 2014;14(2):295-304.

76. Bjarnadottir U, Lemarquis AL, Halldorsdottir S, Freysdottir J, Ludviksson BR. The suppressive function of human CD8(+)iTregs is inhibited by IL-1β and TNFα. *Scand J Immunol.* 2014;80(5):313-322.

77. Choi BK, Asai T, Vinay DS, Kim YH, Kwon BS. 4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-dioxygenase-dependent mechanisms. *Cytokine.* 2006;34(5-6):233-242.

78. Wang Z. Role of IFN-g in induction of Foxp3 and conversion of CD4⁺CD25⁻ T cells to CD4⁺ Tregs. *J Clin Invest.* 2006;116(9):2434-2441. https://doi.org/10.1172/JCI25826

79. Dai H, Wan N, Zhang S, Moore Y, Wan F, Dai Z. Cutting edge: programmed death-1 defines CD8⁺CD122⁺ T cells as regulatory versus memory T cells. *J Immunol.* 2010;185(2):803-807.

80. Chikuma S, Terawaki S, Hayashi T, et al. PD-1-mediated suppression of IL-2 production induces CD8⁺ T cell anergy in vivo. *J Immunol.* 2009;182(11):6682-6689.

81. van der Merwe M, Abdelsamed HA, Seth A, Ong T, Vogel P, Pillai AB. Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4⁺Foxp3⁺ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation. *J Immunol.* 2013;191(11):5764-5776.

82. Francisco LM, Salinas VH, Brown KE, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. *J Exp Med.* 2009;206(13):3015-3029.

83. Amarnath S, Costanzo CM, Mariotti J, et al. Cells and human myeloid dendritic cells promote tolerance via programmed death ligand-1. *PLoS Biol.* 2010;8(2):e1000302.

84. Machicote A, Belén S, Baz P, Billordo LA, Fainboim L. Human CD8⁺HLA-DR⁺ regulatory T cells, similarly to classical CD4⁺Foxp3⁺ cells, suppress immune responses via PD-1/PD-L1 axis. *Front Immunol.* 2018;9:2788.

85. Lu L, Kim H-J, Werneck M, Cantor H. Regulation of CD8+ regulatory T cells: interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease. *Proc Natl Acad Sci USA.* 2008;105(49):19420-19425.

86. Mu C, Zhang X, Wang L, et al. Enhanced suppression of polyclonal CD8(+)25(+) regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes. *J Leukoc Biol.* 2017;101(5):1221-1231.

87. Akane K, Kojima S, Mak TW, Shiku H, Suzuki H. CD8⁺CD122⁺CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity. *Proc Natl Acad Sci USA.* 2016;113(9):2460-2465.

88. Chen Z, Han Y, Gu Y, et al. CD11c(high)CD8⁺ regulatory T cell feedback inhibits CD4 T cell immune response via Fas ligand-Fas pathway. *J Immunol.* 2013;190(12):6145-6154.

89. Daniel V, Sadeghi M, Wang H, Opelz G. CD4⁺CD25⁺Foxp3⁺IFNγ⁺CD178⁺ human induced Treg (iTreg) contribute to suppression of allo responses by apoptosis of responder cells. *Hum Immunol.* 2013;74(2):151-162.

90. Boissonnas A, Scholer-Dahirel A, Simon-Blancal V, et al. Foxp3⁺ T cells induce perforin-dependent dendritic cell death in tumor-draining lymph nodes. *Immunity.* 2010;32(2):266-278.

91. Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. *Immunity.* 2004;21(4):589-601.

92. Guipouy D, Gertner-Dardenne J, Pfajfer L, German Y, Belmonte N, Dupré L. Granulysin- and granzyme-dependent elimination of myeloid cells by therapeutic ova-specific type 1 regulatory T cells. *Int Immunol.* 2019;31(4):239-250.

93. Gondek DC, Lu L-F, Quezada SA, Sakaguchi S, Noelle RJ. Cutting edge: contact-mediated suppression by CD4⁺CD25⁺ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. *J Immunol.* 2005;174(4):1783-1786.

94. Cao X, Cai SF, Fehniger TA, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. *Immunity.* 2007;27(4):635-646.

95. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. *Cell.* 2017;168(6):960-976.

96. Perl A. Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases. *Nat Rev Rheumatol.* 2016;12(3):169-182.

97. Zeng H, Yang K, Cloer C, Neale G, Vogel P, Chi H. mTORC1 couples immune signals and metabolic programming to establish Treg-cell function. *Nature.* 2013;499(7459):485-490.

98. Battaglia M, Stabilini A, Roncarolo M-G. Rapamycin selectively expands CD4⁺CD25⁺FoxP3⁺ regulatory T cells. *Blood.* 2005;105(12):4743-4748.

99. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A. Selective survival of naturally occurring human CD4⁺CD25⁺Foxp3⁺ regulatory T cells cultured with rapamycin. *J Immunol.* 2007;178(1):320-329.

100. Michalek RD, Gerriets VA, Jacobs SR, et al. Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4⁺ T cell subsets. *J Immunol.* 2011;186(6):3299-3303.

101. Coe DJ, Kishore M, Marelli-Berg F. Metabolic regulation of regulatory T cell development and function. *Front Immunol.* 2014;5:590.

102. Sun J, Yang Y, Huo X, et al. Efficient therapeutic function and mechanisms of human polyclonal CD8⁺CD103⁺Foxp3⁺
regulatory T cells on collagen-induced arthritis in mice. *J Immunol Res.* 2019;19(2019):1-12.

103. Churlaud G, Pitoiset F, Jebbawi F, et al. Human and mouse CD8(+) CD25(+)FOXP3(+) regulatory T cells at steady state and during interleukin-2 therapy. *Front Immunol.* 2015;6:171.

104. Aoyama A, Klarin D, Yamada Y, et al. Low-dose IL-2 for in vivo expansion of CD4+ and CD8+ regulatory T cells in nonhuman primates. *J Am Transplant.* 2012;12(9):2532-2537.

105. Peterson LB, Bell C, Howlett SK, et al. A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease. *J Autoimmun.* 2018;95:1-14.

106. Saadoun D, Rosenzwajg M, Joly F, et al. Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. *N Engl J Med.* 2011;365(22):2067-2077.

107. Rosenzwajg M, Churlaud G, Mallone R, et al. Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. *J Autoimmun.* 2015;58:48-58.

108. He J, Zhang X, Wei Y, et al. Low-dose interleukin-2 treatment selectively modulates CD4+ T cell subsets in patients with systemic lupus erythematosus. *Nat Med.* 2016;22(9):991-993.

109. Hirakawa M, Matos T, Liu H, et al. Low-dose IL-2 selectively activates subsets of CD4+ Tregs and NK cells. *JCI Insight.* 2016;1(18):e89278.

110. Apert C, Romagnoli P, van Meerwijk JPM. IL-2 and IL-15 dependent thymic development of Foxp3-expressing regulatory T lymphocytes. *Protein Cell.* 2018;9(4):322-332.

111. Waickman AT, Ligons DL, Hwang SuJin, et al. CD4 effector T cell differentiation is controlled by IL-15 that is expressed and presented in trans. *Cytokine.* 2017;99:266-274.

112. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-beta to convert naive CD4⁺CD25⁻ cells to CD25⁺Foxp3⁺ regulatory T cells and for expansion of these cells. *J Immunol.* 2007;178(4):2018-2027.

113. Liu H, Qiu F, Wang Y, et al. CD8⁺CD122⁺PD-1⁺ Tregs synergize with costimulatory blockade of CD40/CD154, but Not B7/CD28, to prolong murine allograft survival. *Front Immunol.* 2019;10:306.

114. Wang L-X, Li Y, Yang G, et al. CD122⁺CD8⁺ Treg suppress vaccine-induced antitumor immune responses in lymphodepleted mice. *Eur J Immunol.* 2010;40(5):1375-1385.

115. Mayer CT, Floess S, Baru AM, Lahl K, Huehn J, Sparwasser T. CD8⁺ Foxp3⁺ T cells share developmental and phenotypic features with classical CD4⁺ Foxp3⁺ regulatory T cells but lack potent suppressive activity. *Eur J Immunol.* 2011;41(3):716-725.

116. Vuddamalay Y, van Meerwijk J. CD28⁻ and CD28<sup>low</sup>CD8⁺ regulatory T cells: of mice and men. *Front Immunol.* 2017;8:31.

117. Liu Q, Zheng H, Chen X, et al. Human mesenchymal stromal cells enhance the immunomodulatory function of CD8(+)CD28(−) regulatory T cells. *Cell Mol Immunol.* 2015;12(6):708-718.

118. Lei H, Kuchenbecker L, Streitz M, et al. Human CD45RA(−) FoxP3(hi) memory-type regulatory T cells show distinct TCR repertoires with conventional T cells and play an important role in controlling early immune activation. *Am J Transplant.* 2015;15(10):2625-2635.
119. Thornton AM, Korty PE, Tran DQ, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3⁺ T regulatory cells. *J Immunol.* 2010;184(7):3433-3441.

120. Sebastian M, Lopez-Ocasio M, Metidji A, Rieder SA, Shevach EM, Thornton AM. Helios controls a limited subset of regulatory T cell functions. *J Immunol.* 2016;196(1):144-155.

121. Singh RP, La Cava A, Wong M, Ebling F, Hahn BH. CD8⁺ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression. *J Immunol.* 2007;178(12):7649-7657.

122. Huter EN, Punkosy GA, Glass DD, Cheng LI, Ward JM, Shevach EM. TGF-β-induced Foxp3⁺ regulatory T cells rescue scurfy mice. *Eur J Immunol.* 2008;38(7):1814-1821.

123. Lin W, Haribhai D, Relland LM, et al. Regulatory T cell development in the absence of functional Foxp3. *Nat Immunol.* 2007;8(4):359-368.

124. Cretney E, Xin A, Shi W, et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. *Nat Immunol.* 2011;12(4):304-311.

125. Oh H, Grinberg-Bleyer Y, Liao W, et al. An NF-κB transcription-factor-dependent lineage-specific transcriptional program promotes regulatory T cell identity and function. *Immunity.* 2017;47(3):450-465.e5.

126. Zhang T, Pierson RN, Azimzadeh AM. Update on CD40 and CD154 blockade in transplant models. *Immunotherapy.* 2015;7(8):899-911.

127. Guillot C, Guillonneau C, Mathieu P, et al. Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40lg results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection. *J Immunol.* 2002;168(4):1600-1609.

128. Tsai Y, Yang KD, Wen Y, Hung C, Chien J, Lin C. Allergen-specific immunotherapy enhances CD8⁺CD25⁺CD137⁺ regulatory T cells and decreases nasal nitric oxide. *Pediatr Allergy Immunol.* 2019;30:531-539.

129. Zhang S, Ke X, Zeng S, et al. Analysis of CD8⁺ Treg cells in patients with ovarian cancer: a possible mechanism for immune impairment. *Cell Mol Immunol.* 2015;12(5):580-591.

130. Chen M-L, Yan B-S, Kozoriz D, Weiner HL. Novel CD8⁺ Treg suppress EAE by TGF-beta- and IFN-gamma-dependent mechanisms. *Eur J Immunol.* 2009;39(12):3423-3435.

131. Izawa A, Yamaura K, Albin MJ, et al. A novel alloantigen-specific CD8⁺PD1⁺ regulatory T cell induced by ICOS-B7h blockade in vivo. *J Immunol.* 2007;179(2):786-796.

132. Notley CA, McCann FE, Inglis JJ, Williams RO. ANTI-CD3 therapy expands the numbers of CD4⁺ and CD8⁺ treg cells and induces sustained amelioration of collagen-induced arthritis. *Arthritis Rheum.* 2010;62(1):171-178.

133. Ellis S, McGovern JL, van Maurik A, Howe D, Ehrenstein MR, Notley CA. Induced CD8⁺FoxP3⁺ Treg cells in rheumatoid arthritis are modulated by p38 phosphorylation and monocytes expressing membrane tumor necrosis factor α and CD86. *Arthritis Rheumatol.* 2014;66(10):2694-2705.

134. Bisikirska B, Colgan J, Luban J, Bluestone JA, Herold KC. TCR stimulation with modified anti-CD3 mAb expands CD8⁺ T cell population and induces CD8⁺CD25⁺ Tregs. *J Clin Invest.* 2005;115(10):2904-2913.

135. Ablamunits V, Bisikirska B, Herold KC. Acquisition of regulatory function by human CD8(+) T cells treated with anti-CD3 antibody requires TNF. *Eur J Immunol.* 2010;40(10):2891-2901.

136. Kennedy NA, Heap GA, Green HD, et al. Predictors of anti-TNF treatment failure in anti-TNF-naive patients with active luminal Crohn's disease: a prospective, multicentre, cohort study. *Lancet Gastroenterol Hepatol.* 2019;4(5):341-353.

137. Pierini A, Strober W, Moffett C, et al. TNF-α priming enhances CD4⁺FoxP3⁺ regulatory T-cell suppressive function in murine GVHD prevention and treatment. *Blood.* 2016;128(6):866-871.

138. Guillonneau C, Séveno C, Dugast A-S, et al. Anti-CD28 antibodies modify regulatory mechanisms and reinforce tolerance in CD40lg-treated heart allograft recipients. *J Immunol.* 2007;179(12):8164-8171.

139. Horwitz DA, Bickerton S, Koss M, Fahmy TM, La Cava A. Suppression of murine lupus by CD4⁺ and CD8⁺ Treg cells induced by T cell-targeted nanoparticles loaded with interleukin-2 and transforming growth factor β. *Arthritis Rheumatol.* 2019;71(4):632-640.

140. Zhang L, Morgan RA, Beane JD, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding
regulatory T cells on collagen-induced arthritis in mice. *J Immunol* Res. 2019;19(2019):1-12.

103. Churlaud G, Pitoiset F, Jebbawi F, et al. Human and mouse CD8(+) CD25(+)FOXP3(+) regulatory T cells at steady state and during interleukin-2 therapy. *Front Immunol*. 2015;6:171.

104. Aoyama A, Klarin D, Yamada Y, et al. Low-dose IL-2 for in vivo expansion of CD4+ and CD8+ regulatory T cells in nonhuman primates. *J Am Transplant*. 2012;12(9):2532-2537.

105. Peterson LB, Bell C, Howlett SK, et al. A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease. *J Autoimmun*. 2018;95:1-14.

106. Saadoun D, Rosenzwajg M, Joly F, et al. Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. *N Engl J Med*. 2011;365(22):2067-2077.

107. Rosenzwajg M, Churlaud G, Mallone R, et al. Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. *J Autoimmun*. 2015;58:48-58.

108. He J, Zhang X, Wei Y, et al. Low-dose interleukin-2 treatment selectively modulates CD4⁺T cell subsets in patients with systemic lupus erythematosus. *Nat Med*. 2016;22(9):991-993.

109. Hirakawa M, Matos T, Liu H, et al. Low-dose IL-2 selectively activates subsets of CD4⁺Tregs and NK cells. *JCI Insight*. 2016;1(18):e89278.

110. Apert C, Romagnoli P, van Meerwijk JPM. IL-2 and IL-15 dependent thymic development of Foxp3-expressing regulatory T lymphocytes. *Protein Cell*. 2018;9(4):322-332.

111. Waickman AT, Ligons DL, Hwang SuJin, et al. CD4 effector T cell differentiation is controlled by IL-15 that is expressed and presented in trans. *Cytokine*. 2017;99:266-274.

112. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-beta to convert naive CD4⁺CD25⁻ cells to CD25⁺Foxp3⁺ regulatory T cells and for expansion of these cells. *J Immunol*. 2007;178(4):2018-2027.

113. Liu H, Qiu F, Wang Y, et al. CD8⁺CD122⁺PD-1⁺ Tregs synergize with costimulatory blockade of CD40/CD154, but Not B7/CD28, to prolong murine allograft survival. *Front Immunol*. 2019;10:306.

114. Wang L-X, Li Y, Yang G, et al. CD122⁺CD8⁺ Treg suppress vaccine-induced antitumor immune responses in lymphodepleted mice. *Eur J Immunol*. 2010;40(5):1375-1385.

115. Mayer CT, Floess S, Baru AM, Lahl K, Huehn J, Sparwasser T. CD8⁺ Foxp3⁺ T cells share developmental and phenotypic features with classical CD4⁺Foxp3⁺ regulatory T cells but lack potent suppressive activity. *Eur J Immunol*. 2011;41(3):716-725.

116. Vuddamalay Y, van Meerwijk J. CD28⁻ and CD28<sup>low</sup>CD8⁺ regulatory T cells: of mice and men. *Front Immunol*. 2017;8:31.

117. Liu Q, Zheng H, Chen X, et al. Human mesenchymal stromal cells enhance the immunomodulatory function of CD8(+)CD28(−) regulatory T cells. *Cell Mol Immunol*. 2015;12(6):708-718.

118. Lei H, Kuchenbecker L, Streitz M, et al. Human CD45RA(−)FoxP3(hi) memory-type regulatory T cells show distinct TCR repertoires with conventional T cells and play an important role in controlling early immune activation. *Am J Transplant*. 2015;15(10):2625-2635.
119. Thornton AM, Korty PE, Tran DQ, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3⁺T regulatory cells. *J Immunol*. 2010;184(7):3433-3441.

120. Sebastian M, Lopez-Ocasio M, Metidji A, Rieder SA, Shevach EM, Thornton AM. Helios controls a limited subset of regulatory T cell functions. *J Immunol*. 2016;196(1):144-155.

121. Singh RP, La Cava A, Wong M, Ebling F, Hahn BH. CD8⁺T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression. *J Immunol*. 2007;178(12):7649-7657.

122. Huter EN, Punkosdy GA, Glass DD, Cheng LI, Ward JM, Shevach EM. TGF-β-induced Foxp3⁺ regulatory T cells rescue scurfy mice. *Eur J Immunol*. 2008;38(7):1814-1821.

123. Lin W, Haribhai D, Relland LM, et al. Regulatory T cell development in the absence of functional Foxp3. *Nat Immunol*. 2007;8(4):359-368.

124. Cretney E, Xin A, Shi W, et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. *Nat Immunol*. 2011;12(4):304-311.

125. Oh H, Grinberg-Bleyer Y, Liao W, et al. An NF-κB transcription-factor-dependent lineage-specific transcriptional program promotes regulatory T cell identity and function. *Immunity*. 2017;47(3):450-465.e5.

126. Zhang T, Pierson RN, Azimzadeh AM. Update on CD40 and CD154 blockade in transplant models. *Immunotherapy*. 2015;7(8):899-911.

127. Guillot C, Guillonneau C, Mathieu P, et al. Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40lg results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection. *J Immunol*. 2002;168(4):1600-1609.

128. Tsai Y, Yang KD, Wen Y, Hung C, Chien J, Lin C. Allergen-specific immunotherapy enhances CD8⁺CD25⁺CD137⁺ regulatory T cells and decreases nasal nitric oxide. *Pediatr Allergy Immunol*. 2019;30:531-539.

129. Zhang S, Ke X, Zeng S, et al. Analysis of CD8⁺Treg cells in patients with ovarian cancer: a possible mechanism for immune impairment. *Cell Mol Immunol*. 2015;12(5):580-591.

130. Chen M-L, Yan B-S, Kozoriz D, Weiner HL. Novel CD8⁺Treg suppress EAE by TGF-beta- and IFN-gamma-dependent mechanisms. *Eur J Immunol*. 2009;39(12):3423-3435.

131. Izawa A, Yamaura K, Albin MJ, et al. A novel alloantigen-specific CD8⁺PD1⁺ regulatory T cell induced by ICOS-B7h blockade in vivo. *J Immunol*. 2007;179(2):786-796.

132. Notley CA, McCann FE, Inglis JJ, Williams RO. ANTI-CD3 therapy expands the numbers of CD4⁺ and CD8⁺ treg cells and induces sustained amelioration of collagen-induced arthritis. *Arthritis Rheum*. 2010;62(1):171-178.

133. Ellis S, McGovern JL, van Maurik A, Howe D, Ehrenstein MR, Notley CA. Induced CD8⁺FoxP3⁺ Treg cells in rheumatoid arthritis are modulated by p38 phosphorylation and monocytes expressing membrane tumor necrosis factor α and CD86. *Arthritis Rheumatol*. 2014;66(10):2694-2705.

134. Bisikirska B, Colgan J, Luban J, Bluestone JA, Herold KC. TCR stimulation with modified anti-CD3 mAb expands CD8⁺T cell population and induces CD8⁺CD25⁺ Tregs. *J Clin Invest*. 2005;115(10):2904-2913.

135. Ablamunits V, Bisikirska B, Herold KC. Acquisition of regulatory function by human CD8(+)T cells treated with anti-CD3 antibody requires TNF. *Eur J Immunol*. 2010;40(10):2891-2901.

136. Kennedy NA, Heap GA, Green HD, et al. Predictors of anti-TNF treatment failure in anti-TNF-naive patients with active luminal Crohn's disease: a prospective, multicentre, cohort study. *Lancet Gastroenterol Hepatol*. 2019;4(5):341-353.

137. Pierini A, Strober W, Moffett C, et al. TNF-α priming enhances CD4⁺FoxP3⁺ regulatory T-cell suppressive function in murine GVHD prevention and treatment. *Blood*. 2016;128(6):866-871.

138. Guillonneau C, Séveno C, Dugast A-S, et al. Anti-CD28 antibodies modify regulatory mechanisms and reinforce tolerance in CD40lg-treated heart allograft recipients. *J Immunol*. 2007;179(12):8164-8171.

139. Horwitz DA, Bickerton S, Koss M, Fahmy TM, La Cava A. Suppression of murine lupus by CD4⁺ and CD8⁺ Treg cells induced by T cell-targeted nanoparticles loaded with interleukin-2 and transforming growth factor β. *Arthritis Rheumatol*. 2019;71(4):632-640.

140. Zhang L, Morgan RA, Beane JD, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding
interleukin-12 for the immunotherapy of metastatic melanoma. *Clin Cancer Res.* 2015;21(10):2278-2288.

141. Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4⁺CD25⁺FOXP3⁺ regulatory T cells in renal transplant recipients. *Transplantation*. 2006;82(4):550-557.

142. Zeiser R, Leveson-Gower DB, Zambricki EA, et al. Differential impact of mammalian target of rapamycin inhibition on CD4⁺CD25⁺Foxp3⁺ regulatory T cells compared with conventional CD4⁺ T cells. *Blood*. 2008;111(1):453-462.

143. Pidala J, Kim J, Jim H, et al. A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation. *Haematologica*. 2012;97(12):1882-1889.

144. Ramírez E, Morales JM, Lora D, et al. Peripheral blood regulatory T cells in long-term kidney transplant recipients. *Transpl Proc*. 2009;41(6):2360-2362.

145. Mathew JM, H-Voss J, LeFever A, et al. A phase I clinical trial with ex vivo expanded recipient regulatory T cells in living donor kidney transplants. *Sci Rep*. 2018;8(1):7428.

146. Fraser H, Safinia N, Grageda N, et al. A rapamycin-based GMP-compatible process for the isolation and expansion of regulatory T cells for clinical trials. *Mol Ther Methods Clin Dev*. 2018;8:198-209.

147. Marín Morales JM, Münch N, Peter K, et al. Automated clinical grade expansion of regulatory T cells in a fully closed system. *Front Immunol*. 2019;10:38.

148. Safinia N, Vaikunthanathan T, Fraser H, et al. Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation. *Oncotarget*. 2016;7(7):7563-7577.

149. Putnam AL, Safinia N, Medvec A, et al. Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation: clinical grade alloantigen-reactive Tregs. *Am J Transplant*. 2013;13(11):3010-3020.

150. Fuchs A, Gliwiński M, Grageda N, et al. Minimum information about T regulatory cells: a step toward reproducibility and standardization. *Front Immunol*. 2017;8:1844.

151. Zhang Q, Lu W, Liang C-L, et al. Chimeric antigen receptor (CAR) Treg: a promising approach to inducing immunological tolerance. *Front Immunol*. 2018;9:2359.

152. Wieczorek G, Asemissen A, Model F, et al. Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. *Can Res*. 2009;69(2):599-608.

153. Allan SE, Crome SQ, Crellin NK, et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. *Int Immunol*. 2007;19(4):345-354.

154. Fribourg M, Anderson L, Fischman C, et al. T-cell exhaustion correlates with improved outcomes in kidney transplant recipients. *Kidney Int*. 2019;96:436-449.

155. Fuertes Marraco SA, Neubert NJ, Verdeil G, Speiser DE. Inhibitory receptors beyond T cell exhaustion. *Front Immunol*. 2015;6:310.

156. Yu Y, Zitzner JR, Houlihan J, et al. Common gamma chain cytokines promote rapid in vitro expansion of allo-specific human CD8⁺ suppressor T cells. *PLoS ONE*. 2011;6(12):e28948.

157. Sega EI, Leveson-Gower DB, Florek M, Schneidawind D, Luong RH, Negrin RS. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation. *PLoS ONE*. 2014;9(1):e86551.

158. Kennedy-Nasser AA, Ku S, Castillo-Caro P, et al. Ultra low-dose IL-2 for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory T cells without diminishing antiviral and antileukemic activity. *Clin Cancer Res*. 2014;20(8):2215-2225.

159. Brehm MA, Kenney LL, Wiles MV, et al. Lack of acute xenogeneic graft- versus -host disease, but retention of T-cell function

following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression. *FASEB J*. 2019;33(3):3137-3151.

160. Betts BC, Pidala J, Kim J, et al. IL-2 promotes early Treg reconstitution after allogeneic hematopoietic cell transplantation. *Haematologica*. 2017;102(5):948-957.

161. Scotta C, Esposito M, Fazekasova H, et al. Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4⁺CD25⁺FOXP3⁺ T regulatory cell subpopulations. *Haematologica*. 2013;98(8):1291-1299.

162. Gołąb K, Grose R, Placencia V, et al. Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype. *Oncotarget*. 2018;9(11):9728-9740.

163. Brunstein CG, Miller JS, McKenna DH, et al. Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. *Blood*. 2016;127(8):1044-1051.

164. Dijke IE, Hoepli RE, Ellis T, et al. Discarded human thymus is a novel source of stable and long-lived therapeutic regulatory T cells. *Am J Transplant*. 2016;16(1):58-71.

165. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. *Cell*. 2006;126(4):663-676.

166. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. *Cell*. 2007;131(5):861-872.

167. Timmermans F, Velghe I, Vanwalleghem L, et al. Generation of T cells from human embryonic stem cell-derived hematopoietic zones. *J Immunol*. 2009;182(11):6879-6888.

168. Vizcardo R, Masuda K, Yamada D, et al. Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8⁺ T cells. *Cell Stem Cell*. 2013;12(1):31-36.

169. Wakao H, Yoshikiyo K, Koshimizu U, et al. Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation. *Cell Stem Cell*. 2013;12(5):546-558.

170. Maeda T, Nagano S, Ichise H, et al. Regeneration of CD8αβ T cells from T-cell-derived iPSC imparts potent tumor antigen-specific cytotoxicity. *Can Res*. 2016;76(23):6839-6850.

171. Kawamoto H, Masuda K, Nagano S, Maeda T. Cloning and expansion of antigen-specific T cells using iPS cell technology: development of "off-the-shelf" T cells for the use in allogeneic transfusion settings. *Int J Hematol*. 2018;107(3):271-277.

172. Haque R, Lei F, Xiong X, et al. Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity. *J Immunol*. 2012;189(3):1228-1236.

173. Haque M, Song J, Fino K, et al. Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity. *Sci Rep*. 2016;6(1):20588.

174. Haque M, Lei F, Xiong X, et al. Stem cell-derived tissue-associated regulatory T cells suppress the activity of pathogenic cells in autoimmune diabetes. *JCI Insight*. 2019;4(7):126471.

175. Vo LT, Kinney MA, Liu X, et al. Regulation of embryonic haematopoietic multipotency by EZH1. *Nature*. 2018;553(7689):506-510.

176. Saudemont A, Jespers L, Clay T. Current status of gene engineering cell therapeutics. *Front Immunol*. 2018;9:153.

177. Kim YC, Zhang A-H, Su Y, et al. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses. *Blood*. 2015;125(7):1107-1115.

178. MacDonald KG, Hoepli RE, Huang Q, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. *J Clin Invest*. 2016;126(4):1413-1424.

179. Brusko TM, Koya RC, Zhu S, et al. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer. *PLoS ONE*. 2010;5(7):e11726.
interleukin-12 for the immunotherapy of metastatic melanoma. *Clin Cancer Res.* 2015;21(10):2278-2288.

141. Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4⁺CD25⁺FOXP3⁺ regulatory T cells in renal transplant recipients. *Transplantation*. 2006;82(4):550-557.

142. Zeiser R, Leveson-Gower DB, Zambricki EA, et al. Differential impact of mammalian target of rapamycin inhibition on CD4⁺CD25⁺Foxp3⁺ regulatory T cells compared with conventional CD4⁺ T cells. *Blood*. 2008;111(1):453-462.

143. Pidala J, Kim J, Jim H, et al. A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation. *Haematologica*. 2012;97(12):1882-1889.

144. Ramírez E, Morales JM, Lora D, et al. Peripheral blood regulatory T cells in long-term kidney transplant recipients. *Transpl Proc*. 2009;41(6):2360-2362.

145. Mathew JM, H-Voss J, LeFever A, et al. A phase I clinical trial with ex vivo expanded recipient regulatory T cells in living donor kidney transplants. *Sci Rep*. 2018;8(1):7428.

146. Fraser H, Safinia N, Grageda N, et al. A rapamycin-based GMP-compatible process for the isolation and expansion of regulatory T cells for clinical trials. *Mol Ther Methods Clin Dev*. 2018;8:198-209.

147. Marín Morales JM, Münch N, Peter K, et al. Automated clinical grade expansion of regulatory T cells in a fully closed system. *Front Immunol*. 2019;10:38.

148. Safinia N, Vaikunthanathan T, Fraser H, et al. Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation. *Oncotarget*. 2016;7(7):7563-7577.

149. Putnam AL, Safinia N, Medvec A, et al. Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation: clinical grade alloantigen-reactive Tregs. *Am J Transplant*. 2013;13(11):3010-3020.

150. Fuchs A, Gliwiński M, Grageda N, et al. Minimum information about T regulatory cells: a step toward reproducibility and standardization. *Front Immunol*. 2017;8:1844.

151. Zhang Q, Lu W, Liang C-L, et al. Chimeric antigen receptor (CAR) Treg: a promising approach to inducing immunological tolerance. *Front Immunol*. 2018;9:2359.

152. Wieczorek G, Asemissen A, Model F, et al. Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. *Can Res*. 2009;69(2):599-608.

153. Allan SE, Crome SQ, Crellin NK, et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. *Int Immunol*. 2007;19(4):345-354.

154. Fribourg M, Anderson L, Fischman C, et al. T-cell exhaustion correlates with improved outcomes in kidney transplant recipients. *Kidney Int*. 2019;96:436-449.

155. Fuertes Marraco SA, Neubert NJ, Verdeil G, Speiser DE. Inhibitory receptors beyond T cell exhaustion. *Front Immunol*. 2015;6:310.

156. Yu Y, Zitzner JR, Houlihan J, et al. Common gamma chain cytokines promote rapid in vitro expansion of allo-specific human CD8⁺ suppressor T cells. *PLoS ONE*. 2011;6(12):e28948.

157. Sega EI, Leveson-Gower DB, Florek M, Schneidawind D, Luong RH, Negrin RS. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation. *PLoS ONE*. 2014;9(1):e86551.

158. Kennedy-Nasser AA, Ku S, Castillo-Caro P, et al. Ultra low-dose IL-2 for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory T cells without diminishing antiviral and antileukemic activity. *Clin Cancer Res*. 2014;20(8):2215-2225.

159. Brehm MA, Kenney LL, Wiles MV, et al. Lack of acute xenogeneic graft- versus -host disease, but retention of T-cell function

following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression. *FASEB J*. 2019;33(3):3137-3151.

160. Betts BC, Pidala J, Kim J, et al. IL-2 promotes early Treg reconstitution after allogeneic hematopoietic cell transplantation. *Haematologica*. 2017;102(5):948-957.

161. Scotta C, Esposito M, Fazekasova H, et al. Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4⁺CD25⁺FOXP3⁺ T regulatory cell subpopulations. *Haematologica*. 2013;98(8):1291-1299.

162. Gołąb K, Grose R, Placencia V, et al. Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype. *Oncotarget*. 2018;9(11):9728-9740.

163. Brunstein CG, Miller JS, McKenna DH, et al. Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. *Blood*. 2016;127(8):1044-1051.

164. Dijke IE, Hoepli RE, Ellis T, et al. Discarded human thymus is a novel source of stable and long-lived therapeutic regulatory T cells. *Am J Transplant*. 2016;16(1):58-71.

165. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. *Cell*. 2006;126(4):663-676.

166. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. *Cell*. 2007;131(5):861-872.

167. Timmermans F, Velghe I, Vanwalleghem L, et al. Generation of T cells from human embryonic stem cell-derived hematopoietic zones. *J Immunol*. 2009;182(11):6879-6888.

168. Vizcardo R, Masuda K, Yamada D, et al. Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8⁺ T cells. *Cell Stem Cell*. 2013;12(1):31-36.

169. Wakao H, Yoshikiyo K, Koshimizu U, et al. Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation. *Cell Stem Cell*. 2013;12(5):546-558.

170. Maeda T, Nagano S, Ichise H, et al. Regeneration of CD8αβ T cells from T-cell-derived iPSC imparts potent tumor antigen-specific cytotoxicity. *Can Res*. 2016;76(23):6839-6850.

171. Kawamoto H, Masuda K, Nagano S, Maeda T. Cloning and expansion of antigen-specific T cells using iPS cell technology: development of "off-the-shelf" T cells for the use in allogeneic transfusion settings. *Int J Hematol*. 2018;107(3):271-277.

172. Haque R, Lei F, Xiong X, et al. Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity. *J Immunol*. 2012;189(3):1228-1236.

173. Haque M, Song J, Fino K, et al. Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity. *Sci Rep*. 2016;6(1):20588.

174. Haque M, Lei F, Xiong X, et al. Stem cell-derived tissue-associated regulatory T cells suppress the activity of pathogenic cells in autoimmune diabetes. *JCI Insight*. 2019;4(7):126471.

175. Vo LT, Kinney MA, Liu X, et al. Regulation of embryonic haematopoietic multipotency by EZH1. *Nature*. 2018;553(7689):506-510.

176. Saudemont A, Jespers L, Clay T. Current status of gene engineering cell therapeutics. *Front Immunol*. 2018;9:153.

177. Kim YC, Zhang A-H, Su Y, et al. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses. *Blood*. 2015;125(7):1107-1115.

178. MacDonald KG, Hoepli RE, Huang Q, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. *J Clin Invest*. 2016;126(4):1413-1424.

179. Brusko TM, Koya RC, Zhu S, et al. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer. *PLoS ONE*. 2010;5(7):e11726.
180. Hull CM, Nickolay LE, Estorninho M, et al. Generation of human islet-specific regulatory T cells by TCR gene transfer. *J Autoimmun.* 2017;79:63-73.

181. Wright GP, Notley CA, Xue S-A, et al. Adoptive therapy with redi-rected primary regulatory T cells results in antigen-specific suppres-sion of arthritis. *Proc Natl Acad Sci USA.* 2009;106(45):19078-19083.

182. Rivière I, Sadelain M. Chimeric antigen receptors: a cell and gene therapy perspective. *Mol Ther.* 2017;25(5):1117-1124.

183. Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. *Proc Natl Acad Sci USA.* 1993;90(2):720-724.

184. Savoldo B, Ramos CA, Liu E, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. *J Clin Invest.* 2011;121(5):1822-1826.

185. Zhao Z, Condomines M, van der Stegen S, et al. Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. *Cancer Cell.* 2015;28(4):415-428.

186. Van Schandevyl S, Kerre T. Chimeric antigen receptor T-cell therapy: design improvements and therapeutic strategies in cancer treatment. *Acta Clin Belg.* 2018;13:1-7.

187. Gauthier J, Yakoub-Agha I. Chimeric antigen-receptor T-cell therapy for hematological malignancies and solid tumors: clinical data to date, current limitations and perspectives. *Curr Res Transl Med.* 2017;65(3):93-102.

188. Jain MD, Davila ML. Concise review: emerging principles from the clinical application of chimeric antigen receptor T cell therapies for B cell malignancies: principles of CAR T therapy. *Stem Cells.* 2018;36(1):36-44.

189. Elinav E, Waks T, Eshhar Z. Redirection of regulatory T cells with predetermined specificity for the treatment of experimental colitis in mice. *Gastroenterology.* 2008;134(7):2014-2024.

190. Blat D, Zigmond E, Alteber Z, Waks T, Eshhar Z. Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells. *Mol Ther.* 2014;22(5):1018-1028.

191. Trzonkowski P, Bieniaszewska M, Juścińska J, et al. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4⁺CD25⁺CD127⁻ T regulatory cells. *Clin Immunol.* 2009;133(1):22-26.

192. Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. *Blood.* 2011;117(14):3921-3928.

193. Brunstein CG, Miller JS, Cao Q, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. *Blood.* 2011;117(3):1061-1070.

194. Brunstein CG, Blazar BR, Miller JS, et al. Adoptive transfer of umbilical cord blood-derived regulatory T cells and early viral reactivation. *Biol Blood Marrow Transplant.* 2013;19(8):1271-1273.

195. Boardman DA, Philippeos C, Fruhwirth GO, et al. Expression of a chimeric antigen receptor specific for donor HLA class I enhances the potency of human regulatory T cells in preventing human skin transplant rejection. *Am J Transplant.* 2017;17(4):931-943.

196. Noyan F, Zimmermann K, Hardtke-Wolenski M, et al. Prevention of allograft rejection by use of regulatory T cells with an MHC-specific chimeric antigen receptor. *Am J Transplant.* 2017;17(4):917-930.

197. Pierini A, Iliopoulou BP, Peiris H, et al. T cells expressing chimeric antigen receptor promote immune tolerance. *JCI Insight.* 2017;2(20):92865.

198. Okada M, Kanamori M, Someya K, Nakatsukasa H, Yoshimura A. Stabilization of Foxp3 expression by CRISPR-dCas9-based

epigenome editing in mouse primary T cells. *Epigenetics Chromatin.* 2017;10:24.

199. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY. Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. *Nat Immunol.* 2019;20(2):232-242.

200. Miska J, Lee-Chang C, Rashidi A, et al. HIF-1α is a metabolic switch between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression of Tregs in glioblastoma. *Cell Rep.* 2019;27(1):226-237.e4.

201. Xu Y, Melo-Cardenas J, Zhang Y, et al. The E3 ligase Hrd1 stabilizes Tregs by antagonizing inflammatory cytokine-induced ER stress response. *JCI Insight.* 2019;4(5):121887.

202. Bordignon C, Bonini C, Verzeletti S, et al. Transfer of the HSV-tk Gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. The San Raffaele Hospital, Milan, Italy. *Hum Gene Ther.* 1995;6(6):813-819.

203. Tiberghien P, Cahn J-Y, Brion A, et al. Use of donor T-lymphocytes expressing herpes-simplex thymidine kinase in allogeneic bone marrow transplantation: a phase I-II study. Laboratoire d'Histocompatibilité et Thérapeutique Immuno-Moléculaire, Besançon, France. *Hum Gene Ther.* 1997;8(5):615-624.

204. Bonifant CL, Jackson HJ, Brentjens RJ, Curran KJ. Toxicity and management in CAR T-cell therapy. *Mol Ther Oncolytics.* 2016;3:16011.

205. Tey S-K, Dotti G, Rooney CM, Heslop HE, Brenner MK. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. *Biol Blood Marrow Transplant.* 2007;13(8):913-924.

206. Zhou X, Di Stasi A, Tey S-K, et al. Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene. *Blood.* 2014;123(25):3895-3905.

207. Straathof KC. An inducible caspase 9 safety switch for T-cell therapy. *Blood.* 2005;105(11):4247-4254.

208. Minagawa K, Al-Obaidi M, Di Stasi A. Generation of suicide gene-modified chimeric antigen receptor-redirected T-cells for cancer immunotherapy. *Methods Mol Biol.* 2019;1895:57-73.

209. Wilkie S, van Schalkwyk M, Hobbs S, et al. Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling. *J Clin Immunol.* 2012;32(5):1059-1070.

210. Kloss CC, Condomines M, Cartellieri M, Bachmann M, Sadelain M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. *Nat Biotechnol.* 2013;31(1):71-75.

211. Roybal K, Rupp L, Morsut L, et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. *Cell.* 2016;164(4):770-779.

212. Fedorov VD, Themeli M, Sadelain M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. *Sci Transl Med.* 2013;5(215):215ra172.

213. Rosenblum MD, Way SS, Abbas AK. Regulatory T cell memory. *Nat Rev Immunol.* 2016;16(2):90-101.

**How to cite this article:** Flippe L, Bézie S, Anegon I, Guillonneau C. Future prospects for CD8⁺ regulatory T cells in immune tolerance. *Immunol Rev.* 2019;292:209-224. https://doi.org/10.1111/imr.12812