# Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors

Catherine Menier, Sylvain Meunier, Valeria Porcheddu, Laurène Romano, Evelyne Correia, Florence Busato, Jorg Tost, Bernard Maillère

<figure><img src="image_1.png" /></figure>

## To cite this version:

Catherine Menier, Sylvain Meunier, Valeria Porcheddu, Laurène Romano, Evelyne Correia, et al. Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors. European Journal of Immunology, 2024, 54 (4), 10.1002/eji.202350506. cea-04600272

HAL Id: cea-04600272

https://cea.hal.science/cea-04600272

Submitted on 4 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_1.png" /></figure>

Research Article

# Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors

Catherine Menier<fnref n="1" />, Sylvain Meunier<fnref n="1" />, Valeria Porcheddu<fnref n="1" />,
Laurène Romano<fnref n="1" />, Evelyne Correia<fnref n="1" />, Florence Busato<fnref n="2" />, Jorg Tost<fnref n="2" />
and Bernard Maillère<fnref n="1" />

Tolerance to self-proteins involves multiple mechanisms, including conventional CD4+
T-cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells
(nTregs). The significant incidence of autoantibodies specific for the blood coagulation
factor VIII (FVIII) in healthy donors illustrates that tolerance to self-proteins is not always
complete. In contrast to FVIII-specific Tconvs, FVIII-specific nTregs have never been
revealed and characterized. To determine the frequency of FVIII-specific Tregs in human
peripheral blood, we assessed the specificity of in vitro expanded Tregs by the mem-
brane expression of the CD137 activation marker. Amplified Tregs maintain high levels of
FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg-specific
demethylated region. The cells retained FOXP3 expression after long-term culture in vitro,
strongly suggesting that FVIII-specific Tregs are derived from the thymus. From eleven
healthy donors, we estimated the frequencies of FVIII-specific Tregs at 0.17 cells per mil-
lion, which is about 10-fold lower than the frequency of FVIII-specific CD4+ T cells we
previously published. Our results shed light on the mechanisms of FVIII tolerance by a
renewed approach that could be extended to other self- or non-self-antigens.

**Keywords:** Antigen-specific Treg • FVIII • Hemophilia A • Human regulatory T cells • Thymus-derived Treg

<figure><img src="image_2.png" /></figure>

Additional supporting information may be found online in the Supporting Information section
at the end of the article.

Introduction

Natural regulatory T cells (nTregs) constitute 5-10% of CD4+
T cells in healthy individuals and play a key role in maintain-
ing immune tolerance to self-antigens and homeostasis. In con-
trast to conventional CD4+ T cells (Tconvs), nTregs constitutively

express high levels of CD25 [1], none or low levels of CD127 [2],
and high expression of the master transcription factor FOXP3,
which confers immunosuppressive activity [3, 4]. Mutations in
the FOXP3 gene cause systemic autoimmune disorders in mice
and humans [5]. The majority of nTregs in the immune system
are generated in the thymus (tTregs). Their development appears
to be favored by high functional avidity T-cell receptor (TCR)
interaction with endogenous self-antigens, which is essential for
their immune regulatory capacity [6]. As shown in humans for

<footnote n="1">¹ CEA, INRAE, Département Médicaments et Technologies pour la Santé, SIMoS, Université de Paris-Saclay, Gif-sur-Yvette, France</footnote>

<footnote n="2">² Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France</footnote>

<footnote>Correspondence: Dr. Catherine Menier
e-mail: Catherine.Menier@cea.fr</footnote>
<figure><img src="image_3.png" /></figure>

Research Article

# Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors

Catherine Menier<fnref n="1" />, Sylvain Meunier<fnref n="1" />, Valeria Porcheddu<fnref n="1" />,
Laurène Romano<fnref n="1" />, Evelyne Correia<fnref n="1" />, Florence Busato<fnref n="2" />, Jorg Tost<fnref n="2" />
and Bernard Maillère<fnref n="1" />

Tolerance to self-proteins involves multiple mechanisms, including conventional CD4+ T-cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells (nTregs). The significant incidence of autoantibodies specific for the blood coagulation factor VIII (FVIII) in healthy donors illustrates that tolerance to self-proteins is not always complete. In contrast to FVIII-specific Tconvs, FVIII-specific nTregs have never been revealed and characterized. To determine the frequency of FVIII-specific Tregs in human peripheral blood, we assessed the specificity of in vitro expanded Tregs by the membrane expression of the CD137 activation marker. Amplified Tregs maintain high levels of FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg-specific demethylated region. The cells retained FOXP3 expression after long-term culture in vitro, strongly suggesting that FVIII-specific Tregs are derived from the thymus. From eleven healthy donors, we estimated the frequencies of FVIII-specific Tregs at 0.17 cells per million, which is about 10-fold lower than the frequency of FVIII-specific CD4+ T cells we previously published. Our results shed light on the mechanisms of FVIII tolerance by a renewed approach that could be extended to other self- or non-self-antigens.

**Keywords:** Antigen-specific Treg • FVIII • Hemophilia A • Human regulatory T cells • Thymus-derived Treg

<figure><img src="image_4.png" /></figure>

Additional supporting information may be found online in the Supporting Information section at the end of the article.

## Introduction

Natural regulatory T cells (nTregs) constitute 5–10% of CD4+ T cells in healthy individuals and play a key role in maintaining immune tolerance to self-antigens and homeostasis. In contrast to conventional CD4+ T cells (Tconvs), nTregs constitutively

express high levels of CD25 [1], none or low levels of CD127 [2], and high expression of the master transcription factor FOXP3, which confers immunosuppressive activity [3, 4]. Mutations in the FOXP3 gene cause systemic autoimmune disorders in mice and humans [5]. The majority of nTregs in the immune system are generated in the thymus (tTregs). Their development appears to be favored by high functional avidity T-cell receptor (TCR) interaction with endogenous self-antigens, which is essential for their immune regulatory capacity [6]. As shown in humans for

<footnote n="1">¹ CEA, INRAE, Département Médicaments et Technologies pour la Santé, SIMoS, Université de Paris-Saclay, Gif-sur-Yvette, France</footnote>

<footnote n="2">² Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France</footnote>

<footnote>Correspondence: Dr. Catherine Menier
e-mail: Catherine.Menier@cea.fr</footnote>
environmental antigens [7, 8], nTregs can be specific for foreign antigens. A part of nTregs can differentiate outside the thymus, notably in the gut, chronically inflamed tissues, or transplanted tissues, from CD4⁺CD25⁻FOXP3⁻ Tconv5 and they are called peripheral-generated Tregs (pTregs). Both tTreg and pTreg subsets are characterized by stable expression of FOXP3, which correlates with complete demethylation within the evolutionarily conserved region upstream of exon 1, named Treg-specific demethylated region (TSDR) [9]. Thereby, nTregs differ from activated CD4⁺ Tconv5 [10] and induced Tregs (iTregs) [11], which can be produced in vitro in the presence of high levels of IL-2 and TGF-β [12]. Unlike tTregs, pTregs, and iTregs can lose FOXP3 expression in long-term in vitro cultures [7, 9, 10].

Although nTregs exert their suppressive activity upon the engagement of their TCR with an antigen [13, 14], antigen-specific Tregs in humans have been poorly investigated and their characterization so far remains limited to some aeroantigens and few autoantigens [7, 8, 15, 16]. This is due to the low frequency of these particular subsets in human peripheral blood, which raises particular challenges for their ex vivo isolation and in vitro expansion. However, their study is undoubtedly essential to evaluate their involvement in regulating the immune response.

For instance, the immune tolerance mechanisms to the blood coagulation factor VIII (FVIII) are still unclear. In patients with hemophilia A (HA), missense mutations or extensive gene alterations can result in bleeding due to lack of FVIII production or production of a nonfunctional protein, which is prevented by infusions of plasma-derived or recombinant human FVIII (rFVIII). These gene deficiencies make the therapeutic FVIII a partially or fully non-self-protein for HA patients and thus promote the recruitment of FVIII-specific CD4⁺ Tconv5 [17, 18] while reducing the commitment of Tregs in the thymus. The appearance of neutralizing antibodies following FVIII treatments is more frequent in severe HA patients with none or low endogenous FVIII production, compared with patients with mild/moderate HA, in whom the endogenous, dysfunctional FVIII (cross-reacting material) [19] can still be recognized by the immune system and induce Tregs in the thymus. Nevertheless, tolerance to FVIII in healthy donors (HDs) also appears incomplete. Even though they express normal levels of functional endogenous FVIII, approximately 20% of the healthy population possess antibodies specific to FVIII [20]. Further, during their lifetime, individuals without altered hemostasis may develop acquired hemophilia A, a rare but severe autoimmune bleeding disorder caused by anti-FVIII autoantibody production [21].

We previously demonstrated that FVIII-specific CD4⁺ T cells are present at a mean frequency of 1.7 per million total CD4⁺ T cells in the peripheral blood of HDs, thus escaping from thymic deletion [22]. The existence of FVIII-specific Tregs has been suggested in vitro and ex vivo by CD4⁺CD25⁺i nTreg depletion experiments [23, 24]. However, they have never been formally isolated, and their specificity has never been studied. Therefore, we aimed to detect and quantify nTregs specific for FVIII in human peripheral blood to shed light on the mechanisms underlying T-cell tolerance to FVIII.

# Results

## In vitro expanded Tregs display stable FOXP3 expression

To detect low-frequency nTregs specific for FVIII in human peripheral blood, we set up a new protocol to favor their expansion. First, we purified CD4⁺ T cells using magnetic beads, sorted polyclonal CD3⁺CD4⁺CD25⁺iCD127low/− Tregs by flow cytometry (Fig. 1A), and distributed them into multiple wells to limit the dilution of specific cells in each well. Then, Tregs were stimulated by four weekly round stimulations with autologous DCs previously loaded with rFVIII (Supporting information Fig. S1). As expected, purified Tregs seeded in culture expressed high levels of FOXP3 (Fig. 1B and Supporting information Fig. S2). Interestingly, we also observed a high level of FOXP3 expression for in vitro expanded Tregs (Fig. 1C and Supporting information Fig. S2). Accordingly, both day 0-purified and day 27-expanded Tregs displayed almost complete demethylation of FOXP3-TSDR (Fig. 2A). In addition, purified Tregs suppressed allogenic Tconv proliferation (Fig. 2B). Importantly, preliminary data showed that a day 27-expanded Treg cell line reduced allogenic Tconv proliferation and that this suppression was specific for FVIII (Supporting information Fig. S3). Altogether, these results strongly suggest that purified and expanded T cells displayed the characteristics of nTregs.

## FVIII-specific Tregs can be detected in vitro expansion

Following their in vitro culture, we investigated whether amplified Tregs included FVIII-specific Tregs. Each independent Treg cell line, that is, Tregs cultured in one well, was re-stimulated with rFVIII presented by autologous DCs and anti-CD28 antibody. Then we measured CD137 expression after a 24 h TCR activation on CD3⁺CD4⁺CD127low/−CD25⁺i Tregs (Supporting information Fig. S4) as CD137 is expressed at cell surface following TCR activation and at its maximal level after 24 h [25]. We considered that a Treg cell line was FVIII-specific if the ratio between the CD137 MFI of the stimulated to the unstimulated Treg cell line was ≥2. The percentage of CD137⁺ Tregs was obtained by subtracting the CD137⁺ background signal (non-stimulated Treg cell line). The lowest proportion of CD137⁺ cells was about 3%, and the highest was about 60% (Supporting information Fig. S5). The mean percentage of CD137⁺ Tregs for all the Treg cell lines from the eleven donors was 32.5. As exemplified for the same healthy individual (Fig. 3A), nonspecific Treg cell lines exhibit the same low level of CD137 expression comparing the rFVIII stimulated condition and the unstimulated condition. In contrast, high levels of CD137 expression were expressed by the FVIII-specific Treg cell line when stimulated by rFVIII, namely 32.5% of the cells expressing CD137 in the presence of rFVIII versus 4.3% in its absence. Notably, CD137⁻ Tregs expressed a low level of CD25, while the CD137⁺ Tregs expressed high levels of CD25 (pink dots)
environmental antigens [7, 8], nTregs can be specific for foreign antigens. A part of nTregs can differentiate outside the thymus, notably in the gut, chronically inflamed tissues, or transplanted tissues, from CD4⁺CD25⁻FOXP3⁻ Tconv5 and they are called peripheral-generated Tregs (pTregs). Both tTreg and pTreg subsets are characterized by stable expression of FOXP3, which correlates with complete demethylation within the evolutionarily conserved region upstream of exon 1, named Treg-specific demethylated region (TSDR) [9]. Thereby, nTregs differ from activated CD4⁺Tconv5 [10] and induced Tregs (iTregs) [11], which can be produced in vitro in the presence of high levels of IL-2 and TGF-β [12]. Unlike tTregs, pTregs, and iTregs can lose FOXP3 expression in long-term in vitro cultures [7, 9, 10].

Although nTregs exert their suppressive activity upon the engagement of their TCR with an antigen [13, 14], antigen-specific Tregs in humans have been poorly investigated and their characterization so far remains limited to some aeroantigens and few autoantigens [7, 8, 15, 16]. This is due to the low frequency of these particular subsets in human peripheral blood, which raises particular challenges for their ex vivo isolation and in vitro expansion. However, their study is undoubtedly essential to evaluate their involvement in regulating the immune response.

For instance, the immune tolerance mechanisms to the blood coagulation factor VIII (FVIII) are still unclear. In patients with hemophilia A (HA), missense mutations or extensive gene alterations can result in bleeding due to lack of FVIII production or production of a nonfunctional protein, which is prevented by infusions of plasma-derived or recombinant human FVIII (rFVIII). These gene deficiencies make the therapeutic FVIII a partially or fully non-self-protein for HA patients and thus promote the recruitment of FVIII-specific CD4⁺Tconv5 [17, 18] while reducing the commitment of Tregs in the thymus. The appearance of neutralizing antibodies following FVIII treatments is more frequent in severe HA patients with none or low endogenous FVIII production, compared with patients with mild/moderate HA, in whom the endogenous, dysfunctional FVIII (cross-reacting material) [19] can still be recognized by the immune system and induce Tregs in the thymus. Nevertheless, tolerance to FVIII in healthy donors (HDs) also appears incomplete. Even though they express normal levels of functional endogenous FVIII, approximately 20% of the healthy population possess antibodies specific to FVIII [20]. Further, during their lifetime, individuals without altered hemostasis may develop acquired hemophilia A, a rare but severe autoimmune bleeding disorder caused by anti-FVIII autoantibody production [21].

We previously demonstrated that FVIII-specific CD4⁺T cells are present at a mean frequency of 1.7 per million total CD4⁺T cells in the peripheral blood of HDs, thus escaping from thymic deletion [22]. The existence of FVIII-specific Tregs has been suggested in vitro and ex vivo by CD4⁺CD25⁺iTreg depletion experiments [23, 24]. However, they have never been formally isolated, and their specificity has never been studied. Therefore, we aimed to detect and quantify nTregs specific for FVIII in human peripheral blood to shed light on the mechanisms underlying T-cell tolerance to FVIII.

# Results

## In vitro expanded Tregs display stable FOXP3 expression

To detect low-frequency nTregs specific for FVIII in human peripheral blood, we set up a new protocol to favor their expansion. First, we purified CD4⁺T cells using magnetic beads, sorted polyclonal CD3⁺CD4⁺CD25⁺Tconv5/CD127⁻Tregs by flow cytometry (Fig. 1A), and distributed them into multiple wells to limit the dilution of specific cells in each well. Then, Tregs were stimulated by four weekly round stimulations with autologous DCs previously loaded with rFVIII (Supporting information Fig. S1). As expected, purified Tregs seeded in culture expressed high levels of FOXP3 (Fig. 1B and Supporting information Fig. S2). Interestingly, we also observed a high level of FOXP3 expression for in vitro expanded Tregs (Fig. 1C and Supporting information Fig. S2). Accordingly, both day 0-purified and day 27-expanded Tregs displayed almost complete demethylation of FOXP3-TSDR (Fig. 2A). In addition, purified Tregs suppressed allogenic Tconv proliferation (Fig. 2B). Importantly, preliminary data showed that a day 27-expanded Treg cell line reduced allogenic Tconv proliferation and that this suppression was specific for FVIII (Supporting information Fig. S3). Altogether, these results strongly suggest that purified and expanded T cells displayed the characteristics of nTregs.

## FVIII-specific Tregs can be detected in vitro expansion

Following their in vitro culture, we investigated whether amplified Tregs included FVIII-specific Tregs. Each independent Treg cell line, that is, Tregs cultured in one well, was re-stimulated with rFVIII presented by autologous DCs and anti-CD28 antibody. Then we measured CD137 expression after a 24 h TCR activation on CD3⁺CD4⁺CD127⁻/CD25⁺Tregs (Supporting information Fig. S4) as CD137 is expressed at cell surface following TCR activation and at its maximal level after 24 h [25]. We considered that a Treg cell line was FVIII-specific if the ratio between the CD137 MFI of the stimulated to the unstimulated Treg cell line was ≥2. The percentage of CD137⁺Tregs was obtained by subtracting the CD137⁺background signal (non-stimulated Treg cell line). The lowest proportion of CD137⁺cells was about 3%, and the highest was about 60% (Supporting information Fig. S5). The mean percentage of CD137⁺Tregs for all the Treg cell lines from the eleven donors was 32.5. As exemplified for the same healthy individual (Fig. 3A), nonspecific Treg cell lines exhibit the same low level of CD137 expression comparing the rFVIII stimulated condition and the unstimulated condition. In contrast, high levels of CD137 expression were expressed by the FVIII-specific Treg cell line when stimulated by rFVIII, namely 32.5% of the cells expressing CD137 in the presence of rFVIII versus 4.3% in its absence. Notably, CD137⁻Tregs expressed a low level of CD25, while the CD137⁺Tregs expressed high levels of CD25 (pink dots)
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Gating strategy to sort Tregs and FOXP3 expression analysis by day 0- and day 27-Tregs. (A) Gating strategy used to identify and sort Tregs (day 0 of culture). A first gate was set on living lymphocytes through physical parameters (FSC-A vs. SSC-A) and on (FSC-H vs. FSC-A) to eliminate doublets, then on fluorescent parameters on CD3⁺CD4⁺ T cells and CD127low/−CD25hi to identify Tregs (number of separate experiments corresponding to distinct healthy donors *n* = 23). (B) Representative flow cytometry analysis of FOXP3 expression by freshly sorted (day 0-culture) Tregs, same donor as in A. (C) Representative flow cytometry analysis of FOXP3 expression by expanded Tregs (day 27-culture). The FOXP3 expression by day 0- and day 27-expanded Tregs was evaluated from six healthy donors using the same cytometer settings.</figcaption></figure>

(Fig. 3A). Both CD137⁻ Tregs and CD137⁺ Tregs, which are those specific for FVIII, expressed similar high levels of FOXP3 (Fig. 3B).

## FVIII-specific Tregs are present at low frequency in the peripheral blood of healthy individuals

Altogether, we could identify FVIII-specific Tregs in 10 out of 11 donors, with 43 FVIII-specific Treg cell lines among 197 seeded Treg lines, counting for an average of four specific Treg cell lines from 18 Treg cell lines per donor (Supporting information Table S1). We explain that only a part of the wells gave rise to FVIII-specific Tregs by the low frequency of Tregs circulating in the donors' blood. At low frequency, some wells are seeded with a few FVIII-specific Tregs, while others do not receive any specific Tregs, the cell distribution following a Poisson distribution [26]. By enumerating the FVIII-specific Treg cell lines, we could evaluate the frequency of FVIII-specific Tregs in the blood of HDs. Frequencies ranged from 0.03 to 0.44 cells, with a mean of 0.17 cells per million total CD4⁺ T cells among the 11 donors tested (Fig. 4), and approximately 3 FVIII-specific Tregs per million of total Tregs (Supporting information Table S1).

## Discussion

We reveal in this study the existence of FVIII-specific Tregs and quantify their frequency. Frequencies of FVIII-specific Tregs

ranged from 0.03 to 0.44 cells per million total CD4⁺ T cells. We estimated the frequency of FVIII-specific Tregs by a new approach relying on the weekly stimulation with rFVIII of CD3⁺CD4⁺CD127low/−CD25hi Tregs and assessment of their specificity by the upregulation of the CD137 activation marker in the presence of rFVIII. To our knowledge, only one previous study attempted to isolate FVIII-specific Tregs by stimulating CD4⁺CD25⁺ cells with CD4⁺ T-cell epitopes from the C2 domain followed by tetramer staining [27]. However, they failed to detect any specific Tregs, probably because of their low frequency, which increments the difficulty of their characterization ex vivo. Moreover, our approach gives the advantage of full-length protein applications and not single epitopes only.

In agreement with the mechanisms of thymic T-cell selection and differentiation, it is probable that the FVIII-specific Tregs we describe in this study have been committed to the Treg lineage within the thymus and therefore correspond to thymic Tregs [6, 28]. In fact, the Tregs specific for this self-protein did not lose Foxp3 expression in long-term in vitro cultures at variance to pTregs and iTregs [7, 9, 10]. Therefore, FVIII appears to be part of the category of endogenous antigens leading to both thymic escape and tTreg differentiation [28]. Although no study reports FVIII expression level in the thymus, the Human Protein Atlas reveals 3.1 FVIII transcripts per million in the thymus, which is associated with a presumably low protein level [29]. This limited thymic expression was shown to lead to partial intrathymic deletion and impaired generation of effector T cells but enhanced Treg generation [28].
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Gating strategy to sort Tregs and FOXP3 expression analysis by day 0- and day 27-Tregs. (A) Gating strategy used to identify and sort Tregs (day 0 of culture). A first gate was set on living lymphocytes through physical parameters (FSC-A vs. SSC-A) and on (FSC-H vs. FSC-A) to eliminate doublets, then on fluorescent parameters on CD3⁺CD4⁺ T cells and CD127low/CD25hi to identify Tregs (number of separate experiments corresponding to distinct healthy donors *n* = 23). (B) Representative flow cytometry analysis of FOXP3 expression by freshly sorted (day 0-culture) Tregs, same donor as in A. (C) Representative flow cytometry analysis of FOXP3 expression by expanded Tregs (day 27-culture). The FOXP3 expression by day 0- and day 27-expanded Tregs was evaluated from six healthy donors using the same cytometer settings.</figcaption></figure>

(Fig. 3A). Both CD137⁻ Tregs and CD137⁺ Tregs, which are those specific for FVIII, expressed similar high levels of FOXP3 (Fig. 3B).

## FVIII-specific Tregs are present at low frequency in the peripheral blood of healthy individuals

Altogether, we could identify FVIII-specific Tregs in 10 out of 11 donors, with 43 FVIII-specific Treg cell lines among 197 seeded Treg lines, counting for an average of four specific Treg cell lines from 18 Treg cell lines per donor (Supporting information Table S1). We explain that only a part of the wells gave rise to FVIII-specific Tregs by the low frequency of Tregs circulating in the donors' blood. At low frequency, some wells are seeded with a few FVIII-specific Tregs, while others do not receive any specific Tregs, the cell distribution following a Poisson distribution [26]. By enumerating the FVIII-specific Treg cell lines, we could evaluate the frequency of FVIII-specific Tregs in the blood of HDs. Frequencies ranged from 0.03 to 0.44 cells, with a mean of 0.17 cells per million total CD4⁺ T cells among the 11 donors tested (Fig. 4), and approximately 3 FVIII-specific Tregs per million of total Tregs (Supporting information Table S1).

## Discussion

We reveal in this study the existence of FVIII-specific Tregs and quantify their frequency. Frequencies of FVIII-specific Tregs

ranged from 0.03 to 0.44 cells per million total CD4⁺ T cells. We estimated the frequency of FVIII-specific Tregs by a new approach relying on the weekly stimulation with rFVIII of CD3⁺CD4⁺CD127low/CD25hi Tregs and assessment of their specificity by the upregulation of the CD137 activation marker in the presence of rFVIII. To our knowledge, only one previous study attempted to isolate FVIII-specific Tregs by stimulating CD4⁺CD25⁺ cells with CD4⁺ T-cell epitopes from the C2 domain followed by tetramer staining [27]. However, they failed to detect any specific Tregs, probably because of their low frequency, which increments the difficulty of their characterization ex vivo. Moreover, our approach gives the advantage of full-length protein applications and not single epitopes only.

In agreement with the mechanisms of thymic T-cell selection and differentiation, it is probable that the FVIII-specific Tregs we describe in this study have been committed to the Treg lineage within the thymus and therefore correspond to thymic Tregs [6, 28]. In fact, the Tregs specific for this self-protein did not lose Foxp3 expression in long-term in vitro cultures at variance to pTregs and iTregs [7, 9, 10]. Therefore, FVIII appears to be part of the category of endogenous antigens leading to both thymic escape and tTreg differentiation [28]. Although no study reports FVIII expression level in the thymus, the Human Protein Atlas reveals 3.1 FVIII transcripts per million in the thymus, which is associated with a presumably low protein level [29]. This limited thymic expression was shown to lead to partial intrathymic deletion and impaired generation of effector T cells but enhanced Treg generation [28].
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

**Figure 2.** Analysis of the suppressive capacity of flow cytometry-sorted Tregs and FOXP3 TSDR DNA demethylation of day 0- and day 27-Tregs. (A) Analysis of DNA demethylation at the FOXP3 TSDR in Tregs. DNA was extracted from Tregs purified by flow cytometry as described in Figure 1A (D0 Treg) and from cells recovered at the end of the culture (D27 Treg). CD4<sup>-</sup> cells and CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>-</sup>CD127<sup>+</sup> T cells purified by flow cytometry (CD4<sup>+</sup> Tconv) (the best control) were used as positive DNA methylation control. DNA methylation was evaluated at 10 CpG sites from TSDR of the FOXP3 gene for each cell subset, and results were expressed as a mean value. We analyzed the FOXP3 TSDR DNA methylation of CD4<sup>+</sup> Tconvs, CD4<sup>-</sup> cells, day 0 Tregs, and day 27 Tregs from three HD. Scatter plots indicate means. The Kruskal-Wallis test one-way ANOVA was performed to test whether the difference in the percent of FOXP3 TSDR methylation was statistically significant. ns: nonsignificant, \*p < 0.05, \*\*p < 0.005. (B) Representative analysis of the suppressive capacity of the purified polyclonal Tregs seeded in culture (n = 4). Suppression assays were performed using freshly purified CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>hi</sup>CD127<sup>lo/-</sup> Tregs, autologous DCs, and allogeneic Tconvs as described in Methods. Allogeneic Tconvs were labeled with CellTrace Violet (CTV) dye before stimulation. On day 3, cells were stained by mAbs to CD3, CD4, CD25, and CD127, and flow cytometry analysis was performed by gating on the CTV-labeled cells (Tconv) and excluding Treg autofluorescence signal. The dilution of CTV reveals distinct generations of proliferating cells. Tconv cell proliferation was evaluated using FlowJo's proliferation platform. The orange peak (brightest CTV fluorescence signal) represents the non-proliferating Tconv cells, while the pink peaks represent successive generations of live Tconv cells (CTV dye dilution). The percentage of suppression by Treg was calculated using the formula: % Suppression = 100 - (division index [Treg:Tconv]/division index Tconv alone) × 100.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Analysis of the suppressive capacity of flow cytometry-sorted Tregs and FOXP3 TSDR DNA demethylation of day 0- and day 27-Tregs. (A) Analysis of DNA demethylation at the FOXP3 TSDR in Tregs. DNA was extracted from Tregs purified by flow cytometry as described in Figure 1A (D0 Treg) and from cells recovered at the end of the culture (D27 Treg). CD4<sup>-</sup> cells and CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>-</sup>CD127<sup>+</sup> T cells purified by flow cytometry (CD4<sup>+</sup> Tconv) (the best control) were used as positive DNA methylation control. DNA methylation was evaluated at 10 CpG sites from TSDR of the FOXP3 gene for each cell subset, and results were expressed as a mean value. We analyzed the FOXP3 TSDR DNA methylation of CD4<sup>+</sup> Tconvs, CD4<sup>-</sup> cells, day 0 Tregs, and day 27 Tregs from three HD. Scatter plots indicate means. The Kruskal-Wallis test one-way ANOVA was performed to test whether the difference in the percent of FOXP3 TSDR methylation was statistically significant. ns: nonsignificant, \*p < 0.05, \*\*p < 0.005. (B) Representative analysis of the suppressive capacity of the purified polyclonal Tregs seeded in culture (n = 4). Suppression assays were performed using freshly purified CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>hi</sup>CD127<sup>lo/-</sup> Tregs, autologous DCs, and allogeneic Tconvs as described in Methods. Allogeneic Tconvs were labeled with CellTrace Violet (CTV) dye before stimulation. On day 3, cells were stained by mAbs to CD3, CD4, CD25, and CD127, and flow cytometry analysis was performed by gating on the CTV-labeled cells (Tconv) and excluding Treg autofluorescence signal. The dilution of CTV reveals distinct generations of proliferating cells. Tconv cell proliferation was evaluated using FlowJo's proliferation platform. The orange peak (brightest CTV fluorescence signal) represents the non-proliferating Tconv cells, while the pink peaks represent successive generations of live Tconv cells (CTV dye dilution). The percentage of suppression by Treg was calculated using the formula: % Suppression = 100 - (division index [Treg:Tconv]/division index Tconv alone) × 100.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Expression analysis of the CD137 activation marker and FOXP3 by expanded Tregs. (A) Example of CD137 staining on non-FVIII-specific (left panel) and FVIII-specific Treg cell lines (right panel) for one Treg cell line displaying 32.5% of CD137⁺ Tregs, this percentage corresponding to the mean of CD137⁺ Tregs for all the Treg cell lines of the ten donors. Top contour plots correspond to unstimulated Treg cell lines, and bottom contour plots correspond to those stimulated. CD137 expression was analyzed by combining two gates: the first on CD3⁺CD4⁺ T cells and the second on two CD127⁺⁻ Treg subsets, CD25⁺⁰ (green contour plots) and CD25⁺¹ (pink contour plots), respectively. (B) Day 28-analysis of FOXP3 expression on CD137⁺⁰⁻ Tregs (gate P5 pink) and CD137⁺ Tregs (gate P6 purple) on all live CD3⁺CD4⁺CD25⁺CD127⁻ Tregs (n = 2).</figcaption></figure>

In a previous study, we quantified the frequency of FVIII-specific CD4⁺ T cells in HDs and highlighted their escape from negative selection, probably due to the low level of expression of this self-protein in the thymus [22]. Although comparing frequencies calculated in two different studies and hence from distinct donors must be considered with caution, the mean frequency of FVIII-specific Tregs is 10-fold lower than that of FVIII-specific CD4⁺ T cells. Frequencies of FVIII-specific CD4⁺ T cells and Tregs

are therefore in a similar proportion in their respective T-cell subset, CD4⁺ [22] and CD4⁺CD25⁺¹CD127⁺⁰⁻. Half of the FVIII-specific CD4⁺ T cells exhibited a memory phenotype, suggesting their recruitment in vivo by the endogenous FVIII under physiological conditions in HDs. However, their frequencies were in the range of the naïve T cells and hence at low frequency for memory cells, suggesting they have been recruited but have poorly proliferated. The discovery of FVIII-specific Tregs in HDs suggests their role in counteracting the FVIII-specific CD4⁺ Tconv cell response. Accordingly, CD4⁺CD25⁺¹ T-cell depletion from peripheral blood mononuclear cells (PBMCs) was shown to increase FVIII-specific responses, suggesting their active role in suppressing T-cell response to FVIII, in both healthy donors [23] and hemophilia A patients [24].

While many antigen models have been designed in mice to analyze the generation and function of antigen-specific nTregs, few studies have been published on antigen-specific nTregs in humans [7, 15, 16, 30]. Two of these publications investigated the T-cell subsets sorted with autoantigens tetramers and highlighted the role of nTregs in the control of autoimmune diseases and to account for HLA association with autoimmune diseases [15, 16]. The CD137 activation marker was used to reveal high frequencies of Tregs specific for food and aeroantigens in allergic and healthy donors [7, 8]. Frequencies of nTregs specific for these exogenous antigens were 300–1700 fold higher than FVIII-specific Tregs. This difference might result from the foreign origin of these antigens, as the corresponding nTregs should have differentiated upon T-cell activation in the periphery into memory pTregs. Furthermore, the repeated inhalation or ingestion of these environmental

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Frequencies of FVIII-specific Tregs in 11 healthy individuals. (A) Frequencies were expressed as the number of FVIII-specific Tregs (i.e. CD137⁺ Tregs) per million CD4⁺ T cells. They were calculated by multiplying the number of FVIII-specific Tregs per million Treg by the percentage of Treg per 1 CD4⁺ T cell. Frequencies of FVIII-specific Tregs per million Treg were calculated using Poisson distribution according to the following formula: Frequency = -Ln [(number of CD137⁻ negative wells/total number of wells)/number of Tregs per well).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Expression analysis of the CD137 activation marker and FOXP3 by expanded Tregs. (A) Example of CD137 staining on non-FVIII-specific (left panel) and FVIII-specific Treg cell lines (right panel) for one Treg cell line displaying 32.5% of CD137⁺ Tregs, this percentage corresponding to the mean of CD137⁺ Tregs for all the Treg cell lines of the ten donors. Top contour plots correspond to unstimulated Treg cell lines, and bottom contour plots correspond to those stimulated. CD137 expression was analyzed by combining two gates: the first on CD3⁺CD4⁺ T cells and the second on two CD127⁺⁻ Treg subsets, CD25⁺⁰ (green contour plots) and CD25⁺¹ (pink contour plots), respectively. (B) Day 28-analysis of FOXP3 expression on CD137⁺⁰⁻ Tregs (gate P5 pink) and CD137⁺ Tregs (gate P6 purple) on all live CD3⁺CD4⁺CD25⁺CD127⁻ Tregs (n = 2).</figcaption></figure>

In a previous study, we quantified the frequency of FVIII-specific CD4⁺ T cells in HDs and highlighted their escape from negative selection, probably due to the low level of expression of this self-protein in the thymus [22]. Although comparing frequencies calculated in two different studies and hence from distinct donors must be considered with caution, the mean frequency of FVIII-specific Tregs is 10-fold lower than that of FVIII-specific CD4⁺ T cells. Frequencies of FVIII-specific CD4⁺ T cells and Tregs

are therefore in a similar proportion in their respective T-cell subset, CD4⁺ [22] and CD4⁺CD25⁺¹CD127⁺⁰⁻. Half of the FVIII-specific CD4⁺ T cells exhibited a memory phenotype, suggesting their recruitment in vivo by the endogenous FVIII under physiological conditions in HDs. However, their frequencies were in the range of the naïve T cells and hence at low frequency for memory cells, suggesting they have been recruited but have poorly proliferated. The discovery of FVIII-specific Tregs in HDs suggests their role in counteracting the FVIII-specific CD4⁺ Tconv cell response. Accordingly, CD4⁺CD25⁺¹ T-cell depletion from peripheral blood mononuclear cells (PBMCs) was shown to increase FVIII-specific responses, suggesting their active role in suppressing T-cell response to FVIII, in both healthy donors [23] and hemophilia A patients [24].

While many antigen models have been designed in mice to analyze the generation and function of antigen-specific nTregs, few studies have been published on antigen-specific nTregs in humans [7, 15, 16, 30]. Two of these publications investigated the T-cell subsets sorted with autoantigens tetramers and highlighted the role of nTregs in the control of autoimmune diseases and to account for HLA association with autoimmune diseases [15, 16]. The CD137 activation marker was used to reveal high frequencies of Tregs specific for food and aeroantigens in allergic and healthy donors [7, 8]. Frequencies of nTregs specific for these exogenous antigens were 300–1700 fold higher than FVIII-specific Tregs. This difference might result from the foreign origin of these antigens, as the corresponding nTregs should have differentiated upon T-cell activation in the periphery into memory pTregs. Furthermore, the repeated inhalation or ingestion of these environmental

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Frequencies of FVIII-specific Tregs in 11 healthy individuals. (A) Frequencies were expressed as the number of FVIII-specific Tregs (i.e. CD137⁺ Tregs) per million CD4⁺ T cells. They were calculated by multiplying the number of FVIII-specific Tregs per million Treg by the percentage of Treg per 1 CD4⁺ T cell. Frequencies of FVIII-specific Tregs per million Treg were calculated using Poisson distribution according to the following formula: Frequency = -Ln [(number of CD137⁻ negative wells/total number of wells)/number of Tregs per well).</figcaption></figure>
antigens is expected to maintain Treg survival through frequent
activation.

In conclusion, we revealed the existence of human FVIII-
specific nTregs circulating in the blood of healthy donors. To
date, no studies have been able to evaluate FVIII-specific Tregs
in hemophilia A patients, but their possible role in controlling
inhibitor onset has been suggested [31]. The remaining cross-
reacting material of FVIII resulting from missense mutations or
some other gene alterations influences the prevalence of neutral-
izing antibodies and might influence the differentiation of FVIII-
specific Tregs. Further studies in hemophilia A patients should
clarify the mechanisms of FVIII peripheral tolerance and the role
of FVIII-specific Tregs in reducing rFVIII immunogenicity.

Study limitations and future perspectives

As stated above, it would be interesting to evaluate FVIII-specific
Treg numbers generated from various groups of hemophilia A
patients, for example, those receiving initial FVIII infusions, mul-
tiply infused patients, or patients with inhibitors undergoing
immune tolerance induction via intensive FVIII replacement ther-
apy, or patients with different hemophilia A severities. However,
the volume of blood samples collected from hemophilia patients
might be too limited to recover a sufficient number of cells to
derive FVIII-specific Treg cells. Other future perspectives include
autoimmune diseases and malignancies.

**Materials and Methods**

Cell preparation

PBMCs were isolated from buffy coats of adult anonymous HDs who gave informed consent (Etablissement Français du Sang). Monocytes from PBMCs were isolated by positive magnetic selection using CD14 microbeads (Miltenyi Biotec). They were subsequently differentiated into immature DCs in 5-day culture with 1000 IU/mL of human IL-4 and 1000 IU/mL of human GM-CSF (R&D Systems). Immature DCs were differentiated into mature DCs by incubating overnight at 37°C 5% CO₂ with 1 µg/mL LPS (eBioscience) in the presence of the same amounts of IL-4 and GM-CSF. CD4⁺ cells were isolated from thawed CD14⁻ cells using magnetic CD4 microbeads (Miltenyi Biotec).

Treg cell sorting

Purified CD14<sup>-</sup> cells were stained in PBS1X supplemented with 2% human AB-serum for 20 min at +4°C in the dark using the following anti-human antibodies: CD3-VioGreen (REA613), CD4-APC-Vio770 (M-T466) (Miltenyi Biotec), CD25-PE-CF594 (M-A251), and CD127-BV786 (HIL-7R-M21) (BD Biosciences). CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>hi</sup>CD127<sup>lo/-</sup> Tregs were purified by fluorescence-

activated cell sorting (FACS) using a BD FACSAria III flow cytome-
ter and the FACSDiva software (BD Biosciences).

FOXP3 expression analysis

Tregs were stained using the anti-human FOXP3 staining set APC (eBioscience) containing the buffers and the PCH101 monoclonal antibody (mAb) conjugated to APC necessary to stain and identify human FOXP3⁺ cells. Briefly, we first stained cell surface CD3, CD4, CD25, and CD127 antigens with the monoclonal antibodies used for Treg cell sorting, and then we fixed, permeabilized, and stained intracellular FOXP3 according to the supplier's protocol (eBioscience). We subsequently analyze FOXP3 expression on the BD FACSAria III with the FACSDiva software.

FOXP3 TSDR methylation analysis

Tconvs or CD4⁻ cells and Tregs from male HDs were frozen as soon as purified. Genomic DNA was isolated using the QIAamp DNA Micro or Mini Kit (QIAGEN) depending on cell numbers and bisulfite-treated with the EpiTect Fast 96 DNA Bisulfite Kit (QIAGEN) according to the manufacturer's instructions. The FOXP3 TSDR region was amplified by PCR (forward primer: 5'-TGGGTTAAGTTTGTGTAGGATAGG; reverse primer: 5'-Biotin-TCCCTTTCTAACTAAATTTCTCAAAAAC) in the bisulfite-treated samples and analyzed on a Q24 pyrosequencer (QIAGEN) using PyroMark Q24 Advanced CpG Reagents (QIAGEN), as described previously [32].

Suppression assay

First, allogeneic Tconvs were stained with CellTrace Violet (CTV) dye. Then, CD3⁺CD4⁺CD25⁺hiCD127⁺lo/- Tregs freshly purified with the BD FACSAria III and allogeneic CTV⁺ Tconvs were stimulated separately by immobilized anti-CD3 and anti-CD28 mAbs on day - 1. On day 0, Tregs and allogeneic Tconvs (0.5:1 ratio) were seeded on mature DCs autologous to Tregs. On day 3, T cells were stained by mAbs to CD3, CD4, CD25, and CD127, and analysis was performed using the BD FACSAria III. Tconv proliferation was evaluated using FlowJo's proliferation platform (v7.6 software).

Enrichment and culture of FVIII-specific Tregs

FVIII-specific Treg-cell lines were generated by four once-per-
week stimulations of approximately 100,000 Tregs per well of
a 96-well round-bottom plate with autologous matured DCs
(30,000 per well). Mature DCs were obtained through incubation
overnight at 37°C with LPS in the presence of IL-4 and GM-CSF,
loaded with 0.2 µM recombinant FVIII (Kogenate), and then used
as antigen-presenting cells. Cells were cultured in 200 µL per well
of CTS OpTimizer T-cell expansion SFM (Gibco, Thermo-Fisher),
antigens is expected to maintain Treg survival through frequent
activation.

In conclusion, we revealed the existence of human FVIII-
specific nTregs circulating in the blood of healthy donors. To
date, no studies have been able to evaluate FVIII-specific Tregs
in hemophilia A patients, but their possible role in controlling
inhibitor onset has been suggested [31]. The remaining cross-
reacting material of FVIII resulting from missense mutations or
some other gene alterations influences the prevalence of neutral-
izing antibodies and might influence the differentiation of FVIII-
specific Tregs. Further studies in hemophilia A patients should
clarify the mechanisms of FVIII peripheral tolerance and the role
of FVIII-specific Tregs in reducing rFVIII immunogenicity.

Study limitations and future perspectives

As stated above, it would be interesting to evaluate FVIII-specific Treg numbers generated from various groups of hemophilia A patients, for example, those receiving initial FVIII infusions, multiply infused patients, or patients with inhibitors undergoing immune tolerance induction via intensive FVIII replacement therapy, or patients with different hemophilia A severities. However, the volume of blood samples collected from hemophilia patients might be too limited to recover a sufficient number of cells to derive FVIII-specific Treg cells. Other future perspectives include autoimmune diseases and malignancies.

**Materials and Methods**

Cell preparation

PBMCs were isolated from buffy coats of adult anonymous HDs who gave informed consent (Etablissement Français du Sang). Monocytes from PBMCs were isolated by positive magnetic selection using CD14 microbeads (Miltenyi Biotec). They were subsequently differentiated into immature DCs in 5-day culture with 1000 IU/mL of human IL-4 and 1000 IU/mL of human GM-CSF (R&D Systems). Immature DCs were differentiated into mature DCs by incubating overnight at 37°C 5% CO₂ with 1 µg/mL LPS (eBioscience) in the presence of the same amounts of IL-4 and GM-CSF. CD4⁺ cells were isolated from thawed CD14⁻ cells using magnetic CD4 microbeads (Miltenyi Biotec).

Treg cell sorting

Purified CD14<sup>-</sup> cells were stained in PBS1X supplemented with 2% human AB-serum for 20 min at +4°C in the dark using the following anti-human antibodies: CD3-VioGreen (REA613), CD4-APC-Vio770 (M-T466) (Miltenyi Biotec), CD25-PE-CF594 (M-A251), and CD127-BV786 (HIL-7R-M21) (BD Biosciences). CD3<sup>+</sup>CD4<sup>+</sup>CD25<sup>hi</sup>CD127<sup>lo/-</sup> Tregs were purified by fluorescence-

activated cell sorting (FACS) using a BD FACSAria III flow cytometer and the FACSDiva software (BD Biosciences).

FOXP3 expression analysis

Tregs were stained using the anti-human FOXP3 staining set APC (eBioscience) containing the buffers and the PCH101 monoclonal antibody (mAb) conjugated to APC necessary to stain and identify human FOXP3⁺ cells. Briefly, we first stained cell surface CD3, CD4, CD25, and CD127 antigens with the monoclonal antibodies used for Treg cell sorting, and then we fixed, permeabilized, and stained intracellular FOXP3 according to the supplier's protocol (eBioscience). We subsequently analyze FOXP3 expression on the BD FACSAria III with the FACSDiva software.

FOXP3 TSDR methylation analysis

Tconvs or CD4⁻ cells and Tregs from male HDs were frozen as soon as purified. Genomic DNA was isolated using the QIAamp DNA Micro or Mini Kit (QIAGEN) depending on cell numbers and bisulfite-treated with the EpiTect Fast 96 DNA Bisulfite Kit (QIAGEN) according to the manufacturer's instructions. The FOXP3 TSDR region was amplified by PCR (forward primer: 5'-TGGGTTAAGTTTGTGTAGGATAGG; reverse primer: 5'-Biotin-TCCCTTTCTAACTAAATTTCTCAAAAAC) in the bisulfite-treated samples and analyzed on a Q24 pyrosequencer (QIAGEN) using PyroMark Q24 Advanced CpG Reagents (QIAGEN), as described previously [32].

Suppression assay

First, allogeneic Tconvs were stained with CellTrace Violet (CTV) dye. Then, CD3⁺CD4⁺CD25hiCD127lo/- Tregs freshly purified with the BD FACSAria III and allogeneic CTV⁺ Tconvs were stimulated separately by immobilized anti-CD3 and anti-CD28 mAbs on day -1. On day 0, Tregs and allogeneic Tconvs (0.5:1 ratio) were seeded on mature DCs autologous to Tregs. On day 3, T cells were stained by mAbs to CD3, CD4, CD25, and CD127, and analysis was performed using the BD FACSAria III. Tconv proliferation was evaluated using FlowJo's proliferation platform (v7.6 software).

Enrichment and culture of FVIII-specific Tregs

FVIII-specific Treg-cell lines were generated by four once-per-week stimulations of approximately 100,000 Tregs per well of a 96-well round-bottom plate with autologous matured DCs (30,000 per well). Mature DCs were obtained through incubation overnight at 37°C with LPS in the presence of IL-4 and GM-CSF, loaded with 0.2 µM recombinant FVIII (Kogenate), and then used as antigen-presenting cells. Cells were cultured in 200 µL per well of CTS OpTimizer T-cell expansion SFM (Gibco, Thermo-Fisher),
supplemented with 2% human AB-serum, Glutamax (Gibco), 100 IU/mL Penicillin, 100 µg/mL Streptomycin, 100 IU/mL human IL-2 (premium grade; Miltenyi Biotec), and 10 ng/mL human IL-15 (R&D Systems). The CTS OpTimizer T-cell expansion SFM is a serum-free, xeno-free medium supporting the growth of human T cells. Medium was replenished every 3-4 days and Tregs were split as needed.

## Assessment of Treg cell specificity

Tregs were left to rest for three days in OpTimizer 2% human AB-serum. On day 27, each Treg cell line was stimulated with autologous DCs previously loaded with rFVIII or with unloaded autologous DCs in the presence of 1 µg/mL CD28 functional-grade pure Ab (15E8 clone) (Miltenyi Biotec) in 96-well flat-bottom plates. On day 28, Tregs were stained with the same antibodies used for FACS-sorting, and recombinant human CD137-PE antibody (REA765) (Miltenyi Biotec). Samples were acquired on a BD FACSaria III flow cytometer and analyzed using FACSDiva software.

**Acknowledgements:** The authors thank Pedro Paz and Jeanette Lo (Bayer, San Francisco, CA) for the kind gift of Kogenate. This work was supported by the Commissariat à l'Energie Atomique et aux Energies Alternatives.

**Conflict of interest:** The authors declare no commercial or financial conflict of interest.

**Author Contributions:** Catherine Menier designed research, performed research, analyzed data, and wrote the paper; Valeria Porcheddu participated in designing the specific suppression test and correcting the manuscript; Sylvain Meunier, Laurène Romano, and Evelyne Correia performed some experiments; Florence Busato performed the experiments to analyze the methylation of *FOXP3* TSDR; Jorg Tost designed the methodology to analyze the methylation of *FOXP3* TSDR and Bernard Maillère supervised the project and wrote the paper.

**Data availability statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Peer review:** The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202350506

## References

1. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 recep-

tor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. *J Immunol.* 1995. 155: 1151-1164.

2. Liu, W., Putnam, A. L., Xu-yu, Z., Szot, G. L., Lee, M. R., Zhu, S., Gottlieb, P. A. et al., CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. *J. Exp. Med.* 2006. 203: 1701-1711.

3. Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell development by the transcription factor Foxp3. *Science*. 2003. 299: 1057-1061.

4. Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. *Nat Immunol.* 2003. 4: 330-336.

5. Sakaguchi, S., Mikami, N., Wing, J. B., Tanaka, A., Ichiyama, K. and Ohkura, N., Regulatory T cells and human disease. *Annu Rev Immunol.* 2020. 38: 541-566.

6. Kieback, E., Hilgenberg, E., Stervbo, U., Lampropoulou, V., Shen, P., Bunse, M., Jaimes, Y. et al., Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity. *Immunity*. 2016. 44: 1114-1126.

7. Bacher, P., Kniemeyer, O., Schönbrunn, A., Sawitzki, B., Assenmacher, M., Rietschel, E., Steinbach, A. et al., Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. *Mucosal Immunol.* 2014. 7: 916-928.

8. Bacher, P., Heinrich, F., Stervbo, U., Nienen, M., Vahldieck, M., Iwert, C., Vogt, K. et al., Regulatory T cell specificity directs tolerance versus allergy against aeroantigens in humans. *Cell*. 2016. 167: 1067-1078.e16.

9. Ohkura, N. and Sakaguchi, S., Transcriptional and epigenetic basis of Treg cell development and function: its genetic anomalies or variations in autoimmune diseases. *Cell Res.* 2020. 30: 465-474.

10. Baron, U., Floess, S., Wieczorek, G., Baumann, K., Grützkau, A., Dong, J., Thiel, A. et al., DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells. *Eur. J. Immunol.* 2007. 37: 2378-2389.

11. Polansky, J. K., Kretschmer, K., Freyer, J., Floess, S., Garbe, A., Baron, U., Olek, S. et al., DNA methylation controls Foxp3 gene expression. *Eur. J. Immunol.* 2008. 38: 1654-1663.

12. Chen, W., Jin, W., Hardegen, N., Lei, K., Li, L., Marinos, N., McGrady, G. et al., Conversion of peripheral CD4+CD25+ naïve T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. *J. Exp. Med.* 2003. 198: 1875-1886.

13. Levine, A. G., Arvey, A., Jin, W. and Rudensky, A. Y., Continuous requirement for the TCR in regulatory T cell function. *Nat Immunol.* 2014. 15: 1070-1078.

14. Vahl, J. C., Drees, C., Heger, K., Heink, S., Fischer, J. C., Nedjic, J., Ohkura, N. et al., Continuous T cell receptor signals maintain a functional regulatory T cell pool. *Immunity*. 2014. 41: 722-736.

15. Ooi, J. D., Petersen, J., Tan, Y. H., Huynh, M., Willett, Z. J., Ramarathinam, S. H., Eggenhuizen, P. J. et al., Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. *Nature*. 2017. 545: 243-247.

16. Wen, X., Yang, J., James, E., Chow, I.-T., Reijonen, H. and Kwok, W. W., Increased islet antigen-specific regulatory and effector CD4+ T cells in healthy individuals with the type 1 diabetes-protective haplotype. *Sci. Immunol.* 2020. 5: eaax8767.

17. the PATH (Personalized Alternative Therapies for Hemophilia) Study Investigators, Pandey, G. S., Yanover, C., Miller-Jenkins, L. M., Garfield, S., Cole, S. A., Curran, J. E. et al., Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A. *Nat Med.* 2013. 19: 1318-1324.

18. Jacquemin, M. and Saint-Remy, J.-M., T cell response to FVIII. *Cell. Immunol.* 2016. 301: 8-11.
supplemented with 2% human AB-serum, Glutamax (Gibco), 100 IU/mL Penicillin, 100 µg/mL Streptomycin, 100 IU/mL human IL-2 (premium grade; Miltenyi Biotec), and 10 ng/mL human IL-15 (R&D Systems). The CTS OpTimizer T-cell expansion SFM is a serum-free, xeno-free medium supporting the growth of human T cells. Medium was replenished every 3-4 days and Tregs were split as needed.

## Assessment of Treg cell specificity

Tregs were left to rest for three days in OpTimizer 2% human AB-serum. On day 27, each Treg cell line was stimulated with autologous DCs previously loaded with rFVIII or with unloaded autologous DCs in the presence of 1 µg/mL CD28 functional-grade pure Ab (15E8 clone) (Miltenyi Biotec) in 96-well flat-bottom plates. On day 28, Tregs were stained with the same antibodies used for FACS-sorting, and recombinant human CD137-PE antibody (REA765) (Miltenyi Biotec). Samples were acquired on a BD FACSaria III flow cytometer and analyzed using FACSDiva software.

**Acknowledgements:** The authors thank Pedro Paz and Jeanette Lo (Bayer, San Francisco, CA) for the kind gift of Kogenate. This work was supported by the Commissariat à l'Energie Atomique et aux Energies Alternatives.

**Conflict of interest:** The authors declare no commercial or financial conflict of interest.

**Author Contributions:** Catherine Menier designed research, performed research, analyzed data, and wrote the paper; Valeria Porcheddu participated in designing the specific suppression test and correcting the manuscript; Sylvain Meunier, Laurène Romano, and Evelyne Correia performed some experiments; Florence Busato performed the experiments to analyze the methylation of *FOXP3* TSDR; Jorg Tost designed the methodology to analyze the methylation of *FOXP3* TSDR and Bernard Maillère supervised the project and wrote the paper.

**Data availability statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Peer review:** The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202350506

## References

1. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 recep-

tor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. *J Immunol.* 1995. 155: 1151-1164.

2. Liu, W., Putnam, A. L., Xu-yu, Z., Szot, G. L., Lee, M. R., Zhu, S., Gottlieb, P. A. et al., CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. *J. Exp. Med.* 2006. 203: 1701-1711.

3. Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell development by the transcription factor Foxp3. *Science*. 2003. 299: 1057-1061.

4. Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. *Nat Immunol.* 2003. 4: 330-336.

5. Sakaguchi, S., Mikami, N., Wing, J. B., Tanaka, A., Ichiyama, K. and Ohkura, N., Regulatory T cells and human disease. *Annu Rev Immunol.* 2020. 38: 541-566.

6. Kieback, E., Hilgenberg, E., Stervbo, U., Lampropoulou, V., Shen, P., Bunse, M., Jaimes, Y. et al., Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity. *Immunity*. 2016. 44: 1114-1126.

7. Bacher, P., Kniemeyer, O., Schönbrunn, A., Sawitzki, B., Assenmacher, M., Rietschel, E., Steinbach, A. et al., Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. *Mucosal Immunol.* 2014. 7: 916-928.

8. Bacher, P., Heinrich, F., Stervbo, U., Nienen, M., Vahldieck, M., Iwert, C., Vogt, K. et al., Regulatory T cell specificity directs tolerance versus allergy against aeroantigens in humans. *Cell*. 2016. 167: 1067-1078.e16.

9. Ohkura, N. and Sakaguchi, S., Transcriptional and epigenetic basis of Treg cell development and function: its genetic anomalies or variations in autoimmune diseases. *Cell Res.* 2020. 30: 465-474.

10. Baron, U., Floess, S., Wieczorek, G., Baumann, K., Grützkau, A., Dong, J., Thiel, A. et al., DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells. *Eur. J. Immunol.* 2007. 37: 2378-2389.

11. Polansky, J. K., Kretschmer, K., Freyer, J., Floess, S., Garbe, A., Baron, U., Olek, S. et al., DNA methylation controls Foxp3 gene expression. *Eur. J. Immunol.* 2008. 38: 1654-1663.

12. Chen, W., Jin, W., Hardegen, N., Lei, K., Li, L., Marinos, N., McGrady, G. et al., Conversion of peripheral CD4+CD25+ naïve T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. *J. Exp. Med.* 2003. 198: 1875-1886.

13. Levine, A. G., Arvey, A., Jin, W. and Rudensky, A. Y., Continuous requirement for the TCR in regulatory T cell function. *Nat Immunol.* 2014. 15: 1070-1078.

14. Vahl, J. C., Drees, C., Heger, K., Heink, S., Fischer, J. C., Nedjic, J., Ohkura, N. et al., Continuous T cell receptor signals maintain a functional regulatory T cell pool. *Immunity*. 2014. 41: 722-736.

15. Ooi, J. D., Petersen, J., Tan, Y. H., Huynh, M., Willett, Z. J., Ramarathinam, S. H., Eggenhuizen, P. J. et al., Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. *Nature*. 2017. 545: 243-247.

16. Wen, X., Yang, J., James, E., Chow, I.-T., Reijonen, H. and Kwok, W. W., Increased islet antigen-specific regulatory and effector CD4+ T cells in healthy individuals with the type 1 diabetes-protective haplotype. *Sci. Immunol.* 2020. 5: eaax8767.

17. the PATH (Personalized Alternative Therapies for Hemophilia) Study Investigators, Pandey, G. S., Yanover, C., Miller-Jenkins, L. M., Garfield, S., Cole, S. A., Curran, J. E. et al., Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A. *Nat Med.* 2013. 19: 1318-1324.

18. Jacquemin, M. and Saint-Remy, J.-M., T cell response to FVIII. *Cell. Immunol.* 2016. 301: 8-11.
19 Meunier, S., de Bourayne, M., Hamze, M., Azam, A., Correia, E., Menier, C. and Maillère, B., Specificity of the T cell response to protein biopharmaceuticals. *Front. Immunol.* 2020. 11: 1550.

20 Whelan, S. F. J., Hofbauer, C. J., Horling, F. M., Allacher, P., Wolfsegger, M. J., Oldenburg, J., Male, C. et al., Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients. *Blood*. 2013. 121: 1039–1048.

21 Franchini, M. and Mannucci, P. M., Acquired haemophilia A: a 2013 update. *Thromb Haemost.* 2013. 110: 1114–1120.

22 Meunier, S., Menier, C., Marcon, E., Lacroix-Desmazes, S. and Maillère, B., CD4 T cells specific for factor VIII are present at high frequency in healthy donors and comprise naïve and memory cells. *Blood Adv.* 2017. 1: 1842–1847.

23 Kamaté, C., Lenting, P. J., Van Den Berg, H. M. and Mutis, T., Depletion of CD4⁺/CD25 high regulatory T cells may enhance or uncover factor VIII-specific T-cell responses in healthy individuals. *J Thromb Haemost.* 2007. 5: 611–613.

24 Ettinger, R. A., James, E. A., Kwok, W. W., Thompson, A. R. and Pratt, K. P., HLA-DR-restricted T-cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P. *Haemophilia*. 2010. 16: 44–55.

25 Litjens, N. H. R., Boer, K. and Betjes, M. G. H., Identification of circulating human antigen-reactive CD4⁺ FOXP3⁺ natural regulatory T cells. *J.I.* 2012. 188: 1083–1090.

26 Delluc, S., Ravot, G. and Maillère, B. Quantitative analysis of the CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors. *FASEB J.* 2011. 25: 2040–2048.

27 James, E. A., Kwok, W. W., Ettinger, R. A., Thompson, A. R. and Pratt, K. P., T-cell responses over time in a mild hemophilia A inhibitor subject: epi-tope identification and transient immunogenicity of the corresponding self-peptide. *J Thromb Haemost.* 2007. 5: 2399–2407.

28 Malhotra, D., Linehan, J. L., Dileepan, T., Lee, Y. J., Purtha, W. E., Lu, J. V., Nelson, R. W. et al., Tolerance is established in polyclonal CD4⁺ T cells by distinct mechanisms, according to self-peptide expression patterns. *Nat Immunol.* 2016. 17: 187–195.

29 The Tabula Sapiens Consortium. The Tabula Sapiens a multiple organ single cell transcriptomic atlas of humans. *Science*. 2022. 376: eabl4896.

30 De Groot, A. S., Moise, L., McMurry J. A., Wambre, E., Van Overtvelt, L., Moingeon, P., Scott, DW. et al., Activation of natural regulatory T cells by IgG Fc-derived peptide “Tregitopes”. *Blood*. 2008. 112: 3303–3311.

31 Varthaman, A. and Lacroix-Desmazes, S., Pathogenic immune response to therapeutic factor VIII: exacerbated response or failed induction of tolerance? *Haematologica*. 2019. 104: 236–244.

32 Tost, J. and Gut, I. G., DNA methylation analysis by pyrosequencing. *Nat Protoc.* 2007. 2: 2265–2275.

**Abbreviations:** CTV: CellTrace Violet dye · FACS: fluorescence-activated cell sorting · HA: hemophilia A · HDs: healthy donors · nTregs: natural regulatory T cells · PBMCs: Peripheral blood mononuclear cells · pTregs: peripheral-generated Tregs · rFVIII: recombinant factor VIII · Tconvs: conventional CD4⁺ T cells · TCR: T-cell receptor · Tregs: regulatory T cells · TSDR: Treg-specific demethylated region · tTregs: thymus-derived Tregs

**Full correspondence:** Dr. Catherine Menier, CEA Paris-Saclay, DRF / DMTS / SIMoS, 91190 Gif-sur-Yvette
e-mail: Catherine.Menier@cea.fr

**Current address:** Sylvain Meunier, INSERM U955 - Institut Mondor de Recherche Biomédicale (IMRB) Equipe Yves Lévy - CHU Henri-Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France; Laurène Romano, Ermium Therapeutics - Pépinière Paris Santé Cochin, 29, rue du Faubourg Saint Jacques, 75014 Paris, France

Received: 27/3/2023

Revised: 5/12/2023

Accepted: 5/12/2023

Accepted article online: 23/2/2024
19 Meunier, S., de Bourayne, M., Hamze, M., Azam, A., Correia, E., Menier, C. and Maillère, B., Specificity of the T cell response to protein biopharmaceuticals. *Front. Immunol.* 2020. 11: 1550.

20 Whelan, S. F. J., Hofbauer, C. J., Horling, F. M., Allacher, P., Wolfsegger, M. J., Oldenburg, J., Male, C. et al., Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients. *Blood*. 2013. 121: 1039–1048.

21 Franchini, M. and Mannucci, P. M., Acquired haemophilia A: a 2013 update. *Thromb Haemost.* 2013. 110: 1114–1120.

22 Meunier, S., Menier, C., Marcon, E., Lacroix-Desmazes, S. and Maillère, B., CD4 T cells specific for factor VIII are present at high frequency in healthy donors and comprise naïve and memory cells. *Blood Adv.* 2017. 1: 1842–1847.

23 Kamaté, C., Lenting, P. J., Van Den Berg, H. M. and Mutis, T., Depletion of CD4⁺/CD25 high regulatory T cells may enhance or uncover factor VIII-specific T-cell responses in healthy individuals. *J Thromb Haemost.* 2007. 5: 611–613.

24 Ettinger, R. A., James, E. A., Kwok, W. W., Thompson, A. R. and Pratt, K. P., HLA-DR-restricted T-cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P. *Haemophilia*. 2010. 16: 44–55.

25 Litjens, N. H. R., Boer, K. and Betjes, M. G. H., Identification of circulating human antigen-reactive CD4⁺ FOXP3⁺ natural regulatory T cells. *J.I.* 2012. 188: 1083–1090.

26 Delluc, S., Ravot, G. and Maillère, B. Quantitative analysis of the CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors. *FASEB J.* 2011. 25: 2040–2048.

27 James, E. A., Kwok, W. W., Ettinger, R. A., Thompson, A. R. and Pratt, K. P., T-cell responses over time in a mild hemophilia A inhibitor subject: epi-tope identification and transient immunogenicity of the corresponding self-peptide. *J Thromb Haemost.* 2007. 5: 2399–2407.

28 Malhotra, D., Linehan, J. L., Dileepan, T., Lee, Y. J., Purtha, W. E., Lu, J. V., Nelson, R. W. et al., Tolerance is established in polyclonal CD4⁺ T cells by distinct mechanisms, according to self-peptide expression patterns. *Nat Immunol.* 2016. 17: 187–195.

29 The Tabula Sapiens Consortium. The Tabula Sapiens a multiple organ single cell transcriptomic atlas of humans. *Science*. 2022. 376: eabl4896.

30 De Groot, A. S., Moise, L., McMurry J. A., Wambre, E., Van Overtvelt, L., Moingeon, P., Scott, DW. et al., Activation of natural regulatory T cells by IgG Fc-derived peptide “Tregitopes”. *Blood*. 2008. 112: 3303–3311.

31 Varthaman, A. and Lacroix-Desmazes, S., Pathogenic immune response to therapeutic factor VIII: exacerbated response or failed induction of tolerance? *Haematologica*. 2019. 104: 236–244.

32 Tost, J. and Gut, I. G., DNA methylation analysis by pyrosequencing. *Nat Protoc.* 2007. 2: 2265–2275.

**Abbreviations:** CTV: CellTrace Violet dye · FACS: fluorescence-activated cell sorting · HA: hemophilia A · HDs: healthy donors · nTregs: natural regulatory T cells · PBMCs: Peripheral blood mononuclear cells · pTregs: peripheral-generated Tregs · rFVIII: recombinant factor VIII · Tconvs: conventional CD4⁺ T cells · TCR: T-cell receptor · Tregs: regulatory T cells · TSDR: Treg-specific demethylated region · tTregs: thymus-derived Tregs

**Full correspondence:** Dr. Catherine Menier, CEA Paris-Saclay, DRF / DMTS / SIMoS, 91190 Gif-sur-Yvette
e-mail: Catherine.Menier@cea.fr

**Current address:** Sylvain Meunier, INSERM U955 - Institut Mondor de Recherche Biomédicale (IMRB) Equipe Yves Lévy - CHU Henri-Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France; Laurène Romano, Ermium Therapeutics - Pépinière Paris Santé Cochin, 29, rue du Faubourg Saint Jacques, 75014 Paris, France

Received: 27/3/2023

Revised: 5/12/2023

Accepted: 5/12/2023

Accepted article online: 23/2/2024