# Overall avidity declines in TCR repertoires during latent CMV but not EBV infection

Barbara Couturaud, Bastien Doix, Laura Carretero-Iglesia, Mathilde Allard, Sylvain Pradervand, Michael Hebeisen, Nathalie Rufer

► To cite this version:

Barbara Couturaud, Bastien Doix, Laura Carretero-Iglesia, Mathilde Allard, Sylvain Pradervand, et al. Overall avidity declines in TCR repertoires during latent CMV but not EBV infection. Frontiers in Immunology, 2023, 14, pp.105202. 10.3389/fimmu.2023.1293090. hal-04991456

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EDITED BY

Asaf Poran,
BioNTech, Germany

REVIEWED BY

Mary A. Markiewicz,
University of Kansas Medical Center,
United States
Kilian Schober,
University Hospital Erlangen, Germany

*CORRESPONDENCE

Nathalie Rufer

<figure><img src="image_3.png" /></figure>

Nathalie.Rufer@unil.ch

†PRESENT ADDRESS

Barbara Couturaud,
genOway, Lyon, France
Bastien Doix,
EsoBiotec SA, Mont-Saint-Guibert, Belgium
Laura Carretero-Iglesia,
Ichnos Sciences, Epalinges, Switzerland
Mathilde Allard,
Nantes Université, Inserm UMR 1307, CNRS
UMR 6075, Universitéd'Angers, CRCI2NA,
Nantes, France

RECEIVED 12 September 2023

ACCEPTED 06 November 2023

PUBLISHED 20 November 2023

CITATION

Couturaud B, Doix B, Carretero-Iglesia L,
Allard M, Pradervand S, Hebeisen M
and Rufer N (2023) Overall avidity
declines in TCR repertoires during
latent CMV but not EBV infection.
*Front. Immunol.* 14:1293090.
doi: 10.3389/fimmu.2023.1293090

COPYRIGHT

© 2023 Couturaud, Doix, Carretero-Iglesia, Allard, Pradervand, Hebeisen and Rufer. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Overall avidity declines in TCR repertoires during latent CMV but not EBV infection

Barbara Couturaud<sup>1†</sup>, Bastien Doix<sup>1†</sup>, Laura Carretero-Iglesia<sup>1†</sup>, Mathilde Allard<sup>1†</sup>, Sylvain Pradervand<sup>1,2</sup>, Michael Hebeisen<sup>1</sup> and Nathalie Rufer<sup>1*</sup>

<sup>1</sup>Department of Oncology, Lausanne University Hospital and University of Lausanne, Epalinges, Switzerland, <sup>2</sup>Lausanne Genomic Technologies Facility (LGTF), University of Lausanne, Lausanne, Switzerland

**Introduction:** The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown.

**Methods:** To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαβ clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses.

**Results:** Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression.

**Conclusions:** These findings reveal an overall long-term avidity decline of CMV-but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus
infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

KEYWORDS

healthy donors, longitudinal study, latent herpesvirus infection, CD8 T cells, TCR clonotype, persistence, TCR off-rates, LILRB1

Introduction

Primary Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection is associated with a robust T cell response, followed by the establishment of a lifelong latency, while the T cell response persists long-term. In healthy individuals, herpesviruses are generally well controlled, through a fine balance between viral determinants and host immune surveillance. Yet, CMV and EBV cause considerable morbidity and mortality, particularly in immunocompromised individuals (1, 2). The maintenance of memory CD8 T cell responses during CMV and EBV latent infection have been shown to differ in terms of frequency, phenotype and function. Whereas the prevalence of EBV-specific memory CD8 T cells generally remains stable over time in healthy carriers (3, 4), CMV-specific T cells with certain specificities slowly accumulate as infection advances into latency until reaching high and stably maintained frequencies, in a process named "memory inflation" (5-8). Moreover, CD8 T cells specific for EBV mostly display a less differentiated memory phenotype than the CMV-specific T cell pool, that typically harbor a mature phenotype (9, 10) and share functional features (i.e. cytokine production and constitutive cytolytic activity) common among acute effector cells (9, 11-14). It is becoming increasingly clear that repetitive exposure to antigen is a key determinant of memory T cell inflation/expansion during CMV latent infection. Indeed, CMV is characterized by intermittent reactivation from latency, representing a low-level persistent infection, which subsequently impacts on the virus-specific CD8 T cell response (15). Conversely, EBV reactivation during latency is thought to occur only occasionally, with virus-specific T cells showing no sign of expansion/inflation comparable to CMV infection (16).

Extensive research has been undertaken to determine which main drivers are underlying the selection and long-term maintenance of the large populations of CMV-specific memory CD8 T cells (8, 17). Competition for antigen at the level of the antigen presenting cell (18), as well as between virus-specific T cells (19), have been shown to favor clonal dominance during memory inflation induced by murine CMV (MCMV) infection. Other studies have implicated a dominant role for the

immunoproteasome (20) and for the viral gene expression context (21) in defining T cell immunodominance against MCMV. More recently, latent MCMV infection of lymphatic endothelial cells, where sporadic reactivation events occur, has been shown to be sufficient to drive CD8 T cell memory inflation (22). Along the same lines, Welten and colleagues identified a subset of Tcf1+ MCMV-specific CD8 T cells which is able to maintain the peripheral cell pool of inflationary T cells (23). Another key observation is that memory CMV-specific CD8 T cell repertoires are often described as highly oligoclonal with an initial skewing following primary infection, resulting in a limited clonal diversity through the latency infection phase (5, 10, 24-26). Hence, it has been proposed that the strength of TCR-pMHC interactions (defined thereafter as TCR avidity) represents a major determinant of the TCR repertoire selection and dominance of human CMV-specific CD8 T cell clonotypes (10, 24-27). Investigations of TCR repertoire evolution during aging have suggested an accumulation of CMV-specific CD8 T cells of lower avidity or functional potential in elderly individuals (28-31). However, most of these studies only rely on cross-sectional analyses of different age groups and/or on short timespan evaluation. In a recent elegant study, high-avidity T cell clones were found to decline during the chronic phase of MCMV infection, leading to a switch in dominance towards low-avidity T cells (32). Nonetheless, there is a continued need to improve our knowledge about the intrinsic role of TCR-ligand avidity on T cell clonotype responses during the long-term course of human latent herpesvirus infection.

We had the unique opportunity to study the parameters driving TCRαβ clonal evolution between CMV and EBV chronic infection from six HLA-A2-positive healthy individuals in a longitudinal study, during an observational period of up to 18 years. Specifically, we assessed the structural TCR-pMHC avidity by monomeric TCR-ligand off-rate measurements (33, 34) combined to gene expression profiling and phenotypic and functional characterization of the clonal repertoire in CMV-specific (HLA-A2/pp65<sub>495-503</sub> and HLA-A2/IE-1<sub>316-324</sub>) versus EBV-specific (HLA-A2/BMFL1<sub>280-288</sub>) CD8 T cells over time. Our findings reveal a critical role played by TCR avidity-driven repertoire evolution in the long-term outcome (i.e. 15 to 18 years) of CMV-specific but not of EBV-specific CD8 T cell responses in healthy individuals. LILRB1 inhibitor checkpoint was also preferentially expressed in high-avidity CMV-specific T cell

<footnote>**Abbreviations:** NTAmer, NTA-His tag-containing multimer; pMHC, peptide-MHC; CMV, Cytomegalovirus; EBV, Epstein-Barr virus; TCR, T cell receptor.</footnote>
clonotypes and may potentially limit their life expectancy during persistent infection.

# Materials and methods

## Ethics statement

This study protocol was reviewed and approved according to the relevant regulatory standards from the Ethics Committee for Clinical Research of the University of Lausanne (Lausanne, Switzerland) in accordance with the Declaration of Helsinki. All healthy donors have provided written informed consent for the use of blood samples in medical research.

## Healthy donors

Leukapheresis were collected at Interregional Blood Transfusion SRC from six HLA-A2-positive healthy individuals latently infected with the CMV and/or EBV viruses (BCL1, BCL2, BCL4, BCL6, BCL7 and BCL9) in 2002 (i.e. time-point Tn) as described previously (10). All donors had previously completed the Swiss National Medical questionary to verify that they fulfilled the criteria of apheresis donation. Blood samples from the same individuals were collected 15 to 18 years later (i.e. time-point Tn +15y and Tn+18y). No clinical data were available on their acute infection in the past, but all individuals remained in excellent health during this long-term follow-up. Peripheral blood mononuclear cells (PBMCs) centrifuged in Ficoll-Hypaque (Pharmacia) were cryopreserved in 10% DMSO and stored in liquid nitrogen until further use.

## TCRαβ clonotype repertoire sequencing

Thawed PBMCs were positively enriched using anti-CD8-coated magnetic microbeads (Miltenyi Biotec), stained in PBS, 0.2% BSA, and 5 mM EDTA with PE-labeled HLA-A\*0201 multimers loaded with native CMV/pp65<sub>495-503</sub> (NLVPMVATV) or EBV/BMFL1<sub>280-288</sub> (GLCTLVAML) peptide (Peptide and Tetramer Core Facility, CHUV/UNIL/LICR, Lausanne, Switzerland) at 4°C for 45 minutes, followed by cell surface marker APC-A750 anti-CD8 (Beckman Coulter) at 4°C for 30 minutes. Virus-specific CD8 T cells (CD8<sup>+</sup>multimer<sup>+</sup>) were sorted on a FACS Aria (BD Biosciences) flow cytometer as 300-1000 cells, before further cloning by limiting dilution and expansion in RPMI 1640 medium (Gibco) supplemented with 8% human serum, 150 U/ml human recombinant IL-2 (a gift from GlaxoSmithKline), 1 µg/ml PHA (Sodiag), and 1×10<sup>6</sup>/ml 30 Gy-irradiated allogeneic PBMCs as feeder cells. T cell clones (2x10<sup>4</sup> cells) were directly processed through direct cell lysis and cDNA synthesis. cDNA sample was then subjected to individual PCR using a set of previously validated forward primers specific for the different known TRBV or TRAV gene subfamilies and two reverse primers specific for the corresponding C-beta or C-alpha gene segments (10,

35). PCR products of interest were sequenced from the reverse primer (Fasteris SA). Clonotypes were defined as T cell clones sharing the same TRBV-CDR3 and TRAV-CDR3 amino acid sequences. TCR sequences were analyzed using SnapGene (v.4.1.9 GSL Biotech) and described according to the IMmunoGeneTics (IMGT) nomenclature (36).

## Direct ex vivo TRBV family and clonotype repertoire analyses

CD8-enriched T cells from PBMCs were initially stained at 4°C for 45 minutes with CMV/pp65-specific or EBV/BMFL1-specific multimers, followed by cell surface marker APC-A750, FITC or APC anti-CD8 (Beckman Coulter) and antibodies against the different identified TRBV families (Supplementary Table 1) at 4°C for 30 minutes. Samples were acquired on a LCRII cytometer (BD Biosciences) and analyzed using FlowJo 10.4.2 software (v.10.4.2, Tree Star). The frequency of CMV/pp65-specific TRBV clonotypes was further assessed for each *ex vivo* TRBV family identified by flow cytometry from donors BCL4 and BCL6. Briefly, virus-specific CD8 T cells, which stained positive for a given TRBV family were sorted, *in vitro* cloned and TRBV-CDR3 sequenced as described above.

## NTAmer-based dissociation kinetics

NTAmers were synthesized by the Peptide and Tetramer Core Facility, Ludwig Cancer Research, UNIL CHUV (Lausanne, Switzerland) as described previously (37). Dually labeled NTAmers are composed of streptavidin-phycoerythrin (SA-PE) complexed with biotinylated peptides and non-covalently bound to His-tagged HLA-A\*0201 monomers containing Cy5-labeled β2m (37), and were used for dissociation kinetic measurements as described previously (33, 34). Individual virus-specific CD8 T cell clones or bulk virus-specific CD8 T cell populations expanded following short-term *in vitro* stimulation with PHA and irradiated feeder cells were stained for 45 minutes at 4°C in PBS, 0.2% BSA and 5 mM EDTA with virus-specific NTAmers, in which the HLA-A\*0201 molecules were either loaded with native EBV/BMFL1<sub>280-288</sub> (GLCTLVAML), CMV/pp65<sub>495-503</sub> (NLVPMVATV) or CMV/IE-1<sub>316-324</sub> (VLEETSVML) peptides. NTAmers prepared with CD8 binding-deficient HLA-A\*0201 monomers (i.e. CD8-null NTA) bearing the D227K/T228A mutations in the HLA α3 domain (33) were used when indicated. To assess the differential dissociation kinetics relative to LILRB1 expression, CD8 T cells were first stained with SB600 anti-CD85j antibody (Invitrogen) for 20 minutes at room temperature before NTAmer staining. NTAmer staining was analyzed at 4°C on a SORP-LSR II cytometer (BD Biosciences). Following 30 seconds of baseline acquisition, imidazole (100 mM) was added allowing for the rapid dissociation of the SA-PE-NTA<sub>4</sub> scaffold and monomeric Cy5 fluorescence was measured during the following 5 to 10 minutes. Data were analyzed using the kinetic module of FlowJo software (v.9.7.6, Tree Star) and modeled (1-phase exponential decay) using Prism software (v.9, GraphPad).
Ex vivo global transcriptome profiling by
RNA sequencing analysis

CD8-enriched from PBMCs were stained with CMV/pp65-
specific multimers followed by cell surface marker APC-A750,
FITC or APC anti-CD8 (Beckman Coulter) and antibodies
against the different identified TRBV families (Supplementary
Table 1). CMV/pp65/TRBV-specific CD8 T cells were then sorted
on a FACSAria (BD Biosciences) flow cytometer in RNAlater
Stabilization Solution (Invitrogen). Total RNA from the ex vivo
sorted cells (between 300 and 8000 cells) was extracted using the
RNeasy Micro Kit (Qiagen) according to the manufacturer's
protocol. RNA-sequencing libraries were prepared using the
Clontech SMART Seq v4 Ultra Low RNA kit (Clontech
Laboratories, Inc.) and sequencing was performed on the Illumina
HiSeq 2500. Library preparation, sequencing and analyses were
performed by the Lausanne Genomic Technologies Facility (UNIL,
Lausanne, Switzerland) as described in detail in the Supplemental
Data section. Gene expression data have been deposited in the
NCBI Gene Expression Omnibus (GSE246111).

Ex vivo phenotype expression analysis

CD8-enriched T cells (Miltenyi Biotec) were isolated from thawed healthy donors PBMCs and stained in PBS, 0.2% BSA and 5 mM EDTA with AF700 anti-CD8 (Biolegend), APC-efluor780 anti-CD28 (eBioscience), ECD anti-CD45RA (Beckman Coulter), APC or SB600 anti-CD85j (LILRB1, Invitrogen) and BV421 anti-CD57 (BD Biosciences) for 30 minutes at room temperature. When required CD8<sup>pos</sup> T cells were further stained with appropriate TRBV antibodies to discriminate high- versus low-avidity CMV/pp65-specific TCRαβ clonotypes (Supplementary Table 1). Cells were stained with PE-labeled HLA-A*0201 multimers loaded with native CMV/pp65<sub>495-503</sub>, CMV/IE-1<sub>316-324</sub> or EBV/BMFL1<sub>280-288</sub> peptide for 45 minutes at 4°C. Vivid Aqua (Invitrogen) staining (20 minutes at 4°C in PBS) was used to discriminate live/dead cells. LSRII (BD Biosciences) and Cytoflex (Beckman Coulter) flow cytometers were used to acquire the data. The percentage of positive cells or the level of expression of each marker (geometric mean fluorescence intensity [gMFI]) were analyzed using FlowJo software (v.10.4.2, Tree Star). For the dimensional reduction analyses, samples at each time-point were downsized using the downsample V3 plugin (FlowJo, v.10.4.2) before concatenation and analysis with both the t-SNE built-in module and the FlowSOM plugin (38). FlowSOM plugin was run with the following settings: 5 metaclusters and a SOM grid of 8x8 with a seed of 3. The number of CMV/pp65- or CMV/IE-specific CD8 T cells from BCL6 shown at T<sub>n</sub> and T<sub>n+18y</sub> was adjusted by downsampling to be proportional to the multimer fractions found at each time-point.

In vitro LILRB1 blocking experiments

30-Gy-irradiated HLA-A\*0201-positive PBMCs were pulsed 1
hour at 37°C with the native CMV/pp65<sub>495-503</sub> peptide (10<sup>-8</sup>M),

washed, and incubated with CFSE-labelled (Invitrogen) CMV/
pp65-specific CD8 T cell clones of high or low avidity at an E/T
ratio of 1:1 in RPMI 1640 medium (Gibco) supplemented with 8%
human serum and 50 U/ml human recombinant IL-2
(GlaxoSmithKline) in the presence of monoclonal IgG<sub>2B</sub> mouse
anti-human LILRB1 (CD85j, ILT2) blocking antibody or an IgG<sub>2B</sub>
isotype control used at 5 µg/ml (R&D Systems). After 3 days, cells
were washed, stained in PBS with Near-IR Vivid (Invitrogen) at 4°C
for 30 minutes before acquisition on a LSRII (BD Biosciences)
cytometer. The percentage of divided cells, as well as the expansion
and replication indexes were determined using the proliferation
analysis tools available in FlowJo (v.10.4.2, Tree Star).

Statistical analyses

Data were analyzed using the GraphPad Prism software (v.9 or
v.10.0.1, www.graphpad.com), Statistical tests were used as
indicated throughout the manuscript.

Results

TCR clonotype dominance for CMV/pp65
but not EBV/BMFL1 varies over time

Previous reports have shown that CD8 T cell clonal repertoires
in response to chronic CMV or EBV infection are highly stable over
time (10, 39–42). Yet, the maximal timespan analyzed in those
studies was of five years, whereas CMV and EBV immune responses
persist for decades. To evaluate how lifelong persistent CMV and
EBV infections might impact on the TCRαβ clonal evolution, we
performed an in-depth clonotype repertoire analysis on virus-
specific CD8 T cells from six healthy donors latently infected with
CMV and/or EBV, during an observational period of 15 to 18 years
(Figure 1A). The proportion of CMV/pp65<sub>495-503</sub><sup>-</sup>, CMV/
immediate-early (IE)-1<sub>316-324</sub><sup>-</sup> and EBV/BMFL1<sub>280-288</sub>-specific
populations within total CD8 T cells over time was monitored by
ex vivo fluorescent multimer staining (Figure 1B; Supplementary
Figure 1A). No consistent pattern of frequency evolution was found
among CMV/pp65-specific T cells, as that of donor BCL6 increased
with time (from 1.06% to 4.08%), while the other three individuals
showed reduced frequencies across the two time-points with some
donor-related variations (between 0.14% to 0.97%). CMV/IE-1-
specific T cells were only detectable in donor BCL6, in agreement
with its previously described low prevalence in CMV-seropositive
HLA-A\*0201-expressing individuals (43). In contrast, EBV/
BMFL1-specific T cell populations were globally more stable
during the period of 15 years, with a maximum frequency
variation of 0.15% (Figure 1B; Supplementary Figure 1A).

Within CMV/pp65 and EBV-BMFL1 specificities, we assessed
the evolution of the TCRαβ clonal repertoires over time on ex vivo
CD8 T cells or large panels of single T cell clones derived by in vitro
limiting dilution cultures. The clonal composition was analyzed
based on TRBV-CDR3 and TRAV-CDR3 gene sequences (10, 35).
To validate the TCRαβ repertoires obtained from in vitro-generated
<figure><img src="image_4.png" /><figcaption>FIGURE 1</figcaption></figure>

Evolution of CMV/pp65- and EBV/BMFL1-specific CD8 TCRαβ clonotypes in longitudinal analyses. (A) Schematic representation of donor sampling at early (T<sub>n</sub>) and late (T<sub>n+15y</sub>, T<sub>n+18y</sub>) time-points during latent CMV and/or EBV infection. (B) Donor characteristics and frequencies of CMV (HLA-A*0201/pp65<sub>495-503</sub>)- and/or EBV (HLA-A*0201/BMFL1<sub>280-288</sub>)-specific T cells within total CD8 T populations over time. Of note, CMV (HLA*0201/IE-1<sub>316-324</sub>) specificity was only detectable in BCL6 and is shown at T<sub>n</sub>. (C) Frequencies of color-coded CMV/pp65-specific clonotypes per TRBV family for BCL4 and BCL6. Specific TRBV-CDR3 clonotypes were determined following ex vivo FACS-sorting of TRBV-positive CMV/pp65-specific T cells, in vitro single-cell cloning and TRBV-CDR3 sequencing. (D) Quantification of co-dominant CMV/pp65-specific TCRαβ clonotypes at T<sub>n</sub> and T<sub>n+15y</sub>, adjusted to the CMV/pp65-specific population size (multimer<sup>+</sup>CD8<sup>+</sup> T cells). Results are presented as percentages of color-coded CMV/pp65-specific T cell clonotypes (n = 1108 in vitro generated clones). (E) Delta-prevalence or variation in clone type prevalence from (D) (i.e. subtraction T<sub>n+15y</sub> - T<sub>n</sub> of each individual TCRαβ clone type frequency within total CD8 T cells) for the CMV/pp65 specificity. (F) Cumulative in vitro single cell cloning-based frequencies of preferential TRBV family usage within EBV/BMFL1-specific clonotypes at T<sub>n</sub>. (G) Quantification of co-dominant EBV/BMFL1-specific TCRαβ clonotypes at T<sub>n</sub> and T<sub>n+15y</sub>, adjusted to the EBV/BMFL1-specific population size (multimer<sup>+</sup>CD8<sup>+</sup> T cells). Results are presented as percentages of EBV/BMFL1-specific T cell clonotypes classified according to their color-coded TRBV family (n = 1114 in vitro generated clones). (D, G) Unique clonotypes are defined as "others" and depicted in white. For each donor, in vitro single-cell cloning of T<sub>n</sub> and T<sub>n+15y</sub> samples was performed alongside. (H) Variation in clone type prevalence from (G) (i.e. subtraction T<sub>n+15y</sub> - T<sub>n</sub> of each individual TCRαβ clone type frequency within total CD8 T cells) for the EBV/BMFL1 specificity. Data are presented according to the indicated healthy donors.

virus-specific T cell clones, we used a panel of TCRβ chain-targeted (i.e. TRBV) antibodies combined with epitope-specific multimers for the *ex vivo* staining of CMV/pp65- or EBV/BMFL1-specific CD8 T cells at the TRBV level (Supplementary Figure 1B). Robust correlations were obtained for both epitope specificities with similar frequencies of co-dominant TRBV families as determined by the two independent approaches (Supplementary Figure 1C) and in agreement with previous studies (10, 35, 44). In addition, TRBV-positive CMV/pp65-specific CD8 T cells were directly FACS-sorted, *in vitro* cloned and sequenced for *TRBV-CDR3*, revealing that over 95% of all TRBV family-recovered clones bore the corresponding *TRBV-CDR3* clone type (Figure 1C). These data indicate that *ex vivo* TRBV family staining is a powerful indicator of CMV/pp65-specific T cell clone type frequencies, as these are mostly composed of few well-defined co-dominant TCRαβ clonotypes bearing distinct TRBV families (Supplementary Table 2).

All CMV/pp65-specific clonotypes identified at the early time-
point were also found 15 years later (Figure 1D), revealing a notable
long-term persistence of the highly restricted clonal repertoires.
However, when adjusted to the population size, fluctuations were
still observed with certain clonotypes showing increased or
decreased prevalence over time (Figures 1D, E). These changes in
clonal dominance were also found when analyzing CMV/pp65-
specific populations at the direct ex vivo TRBV-chain family level
using a panel of TRBV-targeted antibodies (Supplementary
Figure 1D). This was more evident for donors BCL4, BCL6 and
BCL1 than for BCL9, presenting a relative stable TCR clone type
repertoire over the studied period. Despite presenting a diverse
clone type composition with 10 to 22 codominant clonotypes per
donor, EBV/BMFL1-specific T cell repertoires were highly biased in
their TRBV and TRAV family usage (Figure 1F; Supplementary
Table 3), as previously reported (10, 25, 45-47). Contrasting to
CMV/pp65-specific CD8 T cell repertoires, the EBV/BMFL1 clonal ones were highly stable over time, with only minor variations in the adjusted TCR clonotype prevalence between time-points and per healthy individual (Figures 1G, H).

## TCR-pMHC dissociation rate is a stable intrinsic biomarker at the TCR clonotype level

We and others reported that the TCR-ligand off-rate (i.e. $k_{off}$) is a reliable parameter, independent of the activation state of the T cell (34, 48). Hence, we further explored its robustness by comparing $k_{off}$ from CMV/pp65-specific CD8 T cell clones expressing the same TCR$\alpha\beta$ clonotype, but isolated over 15 to 18 years, using reversible 2-color NTAmers (i.e. NTA) (33). Each specific clonotype showed a remarkable stability of TCR-pMHC off-rate measurements over time (Figure 2A; Supplementary Figure 2A), translating into strong positive correlation between $k_{off}$ rates obtained at $T_n$ and $T_{n+15y}$ from the four studied individuals (Figure 2B, left panel). Similar observations were made for the TCR$\alpha\beta$ clonotypes specific for EBV/BMFL1 (Figure 2B, right panel), or when using mutated NTAmers, that were deficient for CD8 binding to pMHC (i.e. CD8-null NTA) (Supplementary Figures 2B-D). This indicates that for a particular CMV/pp65- or EBV/BMFL1-specific TCR$\alpha\beta$ clonotype, the TCR-pMHC off-rate (i.e. NTA and CD8-null NTA) represents a stable intrinsic readout, unrelated to the time-point of blood sampling.

## Preferential long-term enrichment of low avidity CMV/pp65-specific T cell clonotypes

We next sought to determine whether the changing patterns of dominance observed between CMV/pp65-specific T cell clonotypes throughout the course of latent infection could be related to disparities in TCR-pMHC binding avidities. For each donor-specific clonal repertoire, NTA- and CD8-null NTA-based dissociation measurements allowed the identification of two distinct subgroups (Figures 2C, D). The first one was composed of CMV/pp65-specific T cell clonotypes of high binding avidity (i.e. slow off-rates/high $k_{off}$ and low CD8 binding dependency as shown by their capacity to bind CD8-null NTAmers), as depicted by clono 1 and 2 (BCL4), clono 5 and 6 (BCL6) and clono 8 (BCL1). Conversely, the second subgroup was characterized by TCRs of low binding avidity (i.e. fast off-rates/low $k_{off}$ and high CD8 binding dependency), such as clono 3 and 4 (BCL4), clono 7 (BCL6) and clono 9 (BCL1). Moreover, the relative prevalence of most high avidity clonotypes showed a decline over time, contrasting to the increase in relative frequency of the low avidity ones (Figure 2E). At the level of overall donor-specific repertoires, this led to a rise from $T_n$ to $T_{n+15y}$ in the ratios of low/high clonotype avidity (Figure 2F). The only exception was BCL9 with no major changes in the TCR repertoire avidity ratio over time, yet clono 12 with the lowest TCR binding avidity (Figures 2C, D) was the most prevalent clonotype,

representing nearly 50% of the CMV/pp65-specific repertoire (Figure 1D). Due to the limited number of available donors (n = 4) with such long timespans (i.e. 15 to 18 years), data do not reach levels of statistically significant differences, but suggest a preferential long-term enrichment of low avidity clonotypes (i.e. fast TCR-pMHC dissociation rates and greater dependency on CD8 coreceptor binding) within CMV/pp65-specific CD8 T cell repertoires.

## TCR binding avidity does not shape the clonotype evolution for EBV/BMFL1

We next investigated the impact of TCR-binding avidity on individually identified TCR$\alpha\beta$ clonotypes for EBV/BMFL1 using WT (i.e. NTA) and CD8 binding-deficient (i.e. CD8-null NTA) NTAmers. Similar to the CMV/pp65 clonal repertoires (Figures 2C, D), the TCR$\alpha\beta$ clonotypes covered a broad spectrum of TCR binding avidity (i.e. $k_{off}$ NTA) and CD8 binding capacity (Figure 3A; Supplementary Figures 3A, B). Moreover, TCR binding avidity and CD8 binding-dependency were closely related to distinct TRBV family usage (Figures 3B, C), but to a weaker extent to TRAV (Supplementary Figure 3C). Specifically, TRBV29 clonotypes displayed significantly faster TCR off-rates (i.e. lower $k_{off}$) and were largely CD8 binding-dependent compared to TRBV20 clonotypes. Both high and low avidity EBV/BMFL1-specific TCR clonotypes displayed a great stability in terms of relative frequency during the period of 15 years (Figure 3D). As a consequence, the overall ratios of low/high TCR clonotype avidity, for each donor, remained stable over time (Figure 3E). In conclusion, differing from the CMV/pp65 model, TCR-pMHC binding avidity does not appear to shape the clonotype evolution of EBV/BMFL1-specific CD8 T cells during the course of latent infection.

## Progressive long-term avidity declines of CMV/pp65- but not EBV/BMFL1-specific CD8 T cell repertoires

We hypothesized that the preferential enrichment at late time-points with T cell clonotypes expressing low TCR binding avidity for CMV/pp65 but not for EBV/BMFL1 would lead to an overall decline in TCR repertoire avidity over time. To address this question, we aimed at directly measuring global $k_{off}$-rates of polyclonal populations, similarly to a recent study (49). To do so, FACS-sorted virus epitope-specific CD8 T cells were rapidly expanded following short-term *in vitro* stimulation in order to reach sufficient cell numbers for the NT Amer assay, without introducing any major bias in the TRBV frequency distribution (Supplementary Figures 4A, B). Faster dissociation rates (i.e. lower $k_{off}$) were observed on CMV/pp65-specific bulk T cells at the later time-point (i.e. $T_{n+15y}$), revealing a significant decrease in the overall TCR binding avidity over time in three out of four donors (Figures 4A, B). Off-rates from BCL9 samples were highly heterogeneous, due to the presence of two distinct NT Amer
<figure><img src="image_5.png" /></figure>

FIGURE 2

Monomeric TCR-pMHC dissociation rates of CMV/pp65-specific TCRαβ clonotypes in longitudinal analyses. (A) TCR-pMHC dissociation rates ($k_{off}$) by wild-type NTAmers of each color-coded CMV-specific TCRαβ clonotypes from donor BCL6 at the indicated time-points. (B) Correlations of NTAmer-based TCR off-rates ($k_{off}$) between $T_n$ and $T_{n+15y}$ obtained from identical TCRαβ clonotypes for CMV/pp65 (left panel) or EBV/BMFL1 (right panel) specificity. Coefficient $R^2$ and p-values from simple linear regression analyses are indicated. (C, D) TCR-pMHC off-rates ($k_{off}$) by wild-type NTAmers (C; NTA) or mutated NTAmers (D; CD8null NTA) on a representative selection of T cell clones of each identified TCRαβ clone type and donor. CD8null NTA non-binder clones are represented in the grey boxes and are defined as CD8 binding-dependent clonotypes. The middle line represents the mean. (E) Relative prevalence of each CMV/pp65-specific TCRαβ clone type at $T_n$ and $T_{n+15y}$ categorized as high TCR binding avidity/CD8 binding-independent (left panel) or as low TCR binding avidity/CD8 binding-dependent (right panel). Clonotypes are color-coded as in Figure 1D. (F) Ratio of low (CD8 binding-dependent)/high (CD8 binding-independent) TCR binding avidities within the overall CMV/pp65-specific CD8 T cell repertoires at $T_n$ and $T_{n+15y}$ for each indicated donor. Values >1 (red arrow) indicate an enrichment of low over high avidity CMV/pp65-specific T cell clonotypes. Values <1 (blue arrow) indicate an enrichment of high over low avidity T cell clonotypes. (E, F) p-values by Wilcoxon non-parametric paired test ($p > 0.05$, not significant).
<figure><img src="image_6.png" /><figcaption>FIGURE 3</figcaption></figure>

Monomeric TCR-pMHC dissociation rates of EBV/BMFL1-specific TCRαβ clonotypes in longitudinal analyses. (A) TCR-pMHC off-rates ($k_{off}$) by wild-type NTAmers (NTA; top panels) or mutated NTAmers (CD8null NTA, bottom panels) on a representative selection of EBV/BMFL-1-specific T cell clones of each identified TCRαβ clonal type and donor, classified according to the TRBV family usage. CD8null NTA non-binder clones are represented in the gray boxes and are defined as CD8 binding-dependent clonotypes. The middle line represents the mean. (B) Compiled TCR-pMHC off-rates ($k_{off}$) wild-type NTA) data from ([A]), top panels], classified according to their preferential TRBV family usage and depicted as box (25th and 75th percentiles) and whisker (min to max) plots with the middle line indicating the median. p-values by Kruskal-Wallis test; ****p < 0.0001. (C) Compiled TCR-pMHC off-rates ($k_{off}$) CD8null NTA) data from ([A]), bottom panels] showing the proportion of CD8-independent TCRαβ clonotypes, classified according to their color-coded TRBV family. (D) Relative prevalence of each EBV/BMFL1-specific TCRαβ clonal type at $T_n$ and $T_{n+15y}$ categorized as high TCR binding avidity/CD8 binding-independent (left panel) or as low TCR binding avidity/CD8 binding-dependent (right panel). Clonotypes are color-coded as in Figure 1G. (E) Ratio of low (CD8 binding-dependent)/high (CD8 binding-independent) TCR binding avidities within the overall EBV/BMFL1-specific CD8 T cell repertoires at $T_n$ and $T_{n+15y}$ for each indicated donor. Values >1 (red arrow) indicate an enrichment of low over high avidity EBV/BMFL1-specific T cell clonotypes. Values <1 (blue arrow) indicate an enrichment of high over low avidity T cell clonotypes. (D, E) p-values by Wilcoxon non-parametric paired test ($p$ > 0.05, not significant).

staining-based dissociation sub-populations, preventing accurate dissociation fitting and computation. Nonetheless, when we compared the proportion of CMV-specific T cells representative of the slower dissociation curves between time-points (Figure 4A, see FACS-gated region), there was as well a significant decline in the percentage of these slow-dissociating cells over time (Figure 4B).

Compared to the CMV/pp65 model, the overall off-rates of EBV/BMFL1-specific *ex vivo* generated bulk T cell populations showed a higher stability in $k_{off}$-rates between $T_n$ and $T_{n+15y}$ with an absence of overall avidity loss (Figures 4C, D). Moreover, in BCL2 and BCL9, we assessed NTAmer-based $k_{off}$ on a large panel of *in vitro* generated EBV-specific CD8 T cell clones for each time-point without further clonal type characterization or selection, thus representing an unbiased TCRαβ clonal type repertoire, and confirmed no significant differences in their global TCR-pMHC off-rates over time (Figure 4E). Similar findings were made when we reconstituted the overall repertoire avidity of each EBV-positive donor by pooling single clonal type-derived $k_{off}$ measurements, obtained for most TCRαβ clonotypes, according to their

respective prevalence (Supplementary Figure 4C). Collectively, these observations indicate that the TCR repertoires evolved towards an overall avidity decline during CMV (i.e. pp65$_{495}$-503 epitope), but not EBV (i.e. BMFL1$_{280}$-288 epitope) latency.

## Preferential LILRB1 expression in CMV/pp65-specific T cell clonotypes of high avidity

To gain further insight into the mechanisms underlying the selection of low avidity T cell clonotypes during chronic CMV infection, we performed a global transcriptome profiling by RNA-Seq on *ex vivo* sorted CMV-pp65/TRBV-specific sub-populations at $T_n$ and $T_{n+15y}$ (Figure 5A; Supplementary Figure 5A). Hierarchical clustering highlighted the presence of 9 genes that were found enriched in clonotypes of high TCR binding avidity when compared with those of low avidity (Figure 5A). Among them, we identified *LILRB1* encoding for an inhibitory receptor (also known as CD85j
<figure><img src="image_7.png" /><figcaption>**B** BCL4
p = 0.004

BCL6
p = 0.006

BCL1
p = 0.002

Slow dissociating cells (%)

BCL9
p = 0.005

k<sub>off</sub> NTA (s<sup>-1</sup>)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**C** EBV (HLA-A2/BMFL1<sub>280-288</sub>)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**D** BCL7
p = 0.485

BCL2
p = 0.016

BCL9
p = 0.008

k<sub>off</sub> NTA (s<sup>-1</sup>)</figcaption></figure>

<figure><img src="image_10.png" /></figure>

FIGURE 4

Overall TCR-pMHC dissociation rates on bulk CMV/pp65- and EBV/BMFL1-specific CD8 T cell populations in longitudinal analyses. (A) Representative FACS-based wild-type NTamer dissociation curves obtained from short-term ex vivo expanded CMV/pp65-specific bulk T cell populations at T~n~ and T~n+15y~. Insets show the gating region used to estimate the proportion of T cells of slower off-rates in donor BCL9. (B) Data are representative of pooled dissociation rate (k~off~) values from 1 to 3 independent experiments. The mean value is indicated. For BCL9, percentage of slow dissociating CMV/pp65-specific bulk cells at T~n~ and T~n+15y~ based on the quantification of slow FACS-gated dissociation curves [see insets in (A)] is depicted. p-values by Mann-Whitney test (BCL4, BCL1, BCL9) and Kruskal-Wallis test (α = 0.05) (BCL6). (C) Representative FACS-based wild-type NTamer dissociation curves obtained from short-term ex vivo expanded EBV/BMFL1-specific bulk T cell populations at T~n~ and T~n+15y~. (D) Data are representative of pooled dissociation rate (k~off~) values from 1 to 2 independent experiments. The mean value is indicated. (E) TCR-pMHC off-rate (k~off~) values from the first 40 to 60 *in vitro* generated EBV/BMFL1-specific, TRBV-unselected T cell clones at T~n~ and T~n+15y~ (BCL2, BCL9). For each *in vitro* single-cell cloning, T~n~ and T~n+15y~ samples were performed alongside. (D, E) p-values by Mann-Whitney test with p > 0.05, not significant ([B] (panel BCL9) and (E)) Data are depicted as box (25th and 75th percentiles) and whisker (min to max) plots with the middle line indicating the median.

or ILT2/LIR-1), associated in CD8 T cells with aging and CMV
latent infection (50). LILRB1 was significantly upregulated in high
avidity CMV-specific clonotypes (Figure 5B). These data were
validated by the NanoString technology (Supplementary
Figure 5B). In addition, CMV/pp65-specific T cell clonotypes of
high avidity presented enhanced surface levels of LILRB1

(Figures 5C, D) as well as of CD57 (Figure 5E), a marker of CD8
T cell differentiation, when compared to the low avidity ones.
Hence, we observed, for the high avidity clonotypes, a positive
correlation between LILRB1 and CD57 expression (Figure 5F). In
agreement with previously documented reports (51–53), LILRB1
expression further displayed a progressive increase along CD8 T cell
<figure><img src="image_11.png" /><figcaption>FIGURE 5</figcaption></figure>

Ex vivo LILRB1 expression on CMV/pp65-specific TCRαβ clonotypes of high versus low avidity. (A) Heatmap plot based on the 19 differentially expressed genes (FDR < 0.1) between high- and low-avidity CMV/pp65-specific clonotypes obtained following ex vivo FACS-sorting using multimer/pp65- and TRBV-specific staining combined to RNA sequencing from BCL4, BCL6 and BCL1 at Tn and Tn+15y. Red indicates over- and blue indicates under-expression relative to mean gene expression. (B) Direct comparison of LILRB1 mRNA expression (as normalized counts) between high- and low-avidity CMV/pp65-specific clonotypes (top panel, n = 8) and at Tn and Tn+15y (bottom panel, n = 8). (C) Representative FACS-based histograms of ex vivo LILRB1 expression on high- and low-avidity CMV/pp65-specific clonotypes from BCL4 and BCL6 at Tn and Tn+18y. For each single staining, LILRB1 expression on global CMV/pp65-negative CD8 T cells is depicted alongside (as gray curves). Note that for BCL6, TRBV6-5 staining includes two high-avidity clonotypes (clono 5 and clono 6), whereas TRBV12 is only specific for the low-avidity clono 7 (see Figure 1C). L, Low avidity; H, High avidity. (D, E) Frequencies of LILRB1 [(D), n = 6-9] and CD57 [(E), n = 6-9] surface expression on low- versus high-avidity CMV/pp65-specific clonotypes (top panels) and at Tn versus Tn+18y (bottom panels) from BCL4 and BCL6. Data are representative of 3 independent experiments. (B, D, E) Data are depicted as box (25th and 75th percentiles) and whisker (min to max) plots with the middle line indicating the median. p-values by Mann-Whitney unpaired test with *p < 0.05 and **p < 0.01, ns, non-significant. (F) Correlation between LILRB1 and CD57 surface expression on high-avidity CMV/pp65-specific T cell clonotypes from BCL4 and BCL6 (n = 6). Coefficient R² and p-values from simple linear regression analyses are indicated. (G) Frequencies of LILRB1 expression on CMV/pp65- versus CMV/pp65+ CD8 T cells or EBV/BMFL1- versus EBV/BMFL1+ CD8 T cells from 10 healthy individuals. The dotted line indicates the mean values of LILRB1 expression found within pp65+ or BMFL1+ CD8 T cells, respectively. p-values by Wilcoxon non-parametric paired test with **p < 0.01. (H) Frequency of LILRB1 expression on EBV/BMFL1-specific T cells according to their preferential TRBV family usage (n = 10).
differentiation with highest levels found in effector CMV/pp65-
specific T cells of high avidity (Supplementary Figure 5C). Finally,
the frequency of LILRB1 expression on ex vivo CMV/pp65-specific
T cells strongly exceeded that of EBV/BMFL1-specific T cells
(Figure 5G). The latter cells expressing LILRB1 only at low
frequencies, independently of their preferential TRBV family
usage (Figure 5H; Supplementary Figure 5D) and consistent with
a previous study (54).

LILRB1 expression moderately restricts cell proliferation of CMV/pp65-specific T cell clonotypes of high avidity

We next evaluated the biological significance of LILRB1 on cell proliferation (by CFSE) of CMV/pp65-specific T cell clonotypes from BCL4 upon direct LILRB1 blockade and ex vivo short term A2/pp65<sub>495-503</sub> peptide-specific stimulation. Treatment with a blocking monoclonal antibody to LILRB1 (51) only induced a modest shift of the single CFSE dilution peaks obtained for clono 2 but not clono 1 high avidity T cells (Supplementary Figure 6). We further conducted CFSE quantification analyses after LILRB1 blockade and pp65 stimulation using a large panel of high versus low avidity *in vitro*-generated CMV/pp65-specific T cell clones. Actually, T cell clones of high avidities expressed significantly greater levels of LILRB1 than low avidity ones (Figure 6A). LILRB1 levels in high-avidity T cell clones were also enhanced when compared to the ex vivo LILRB1 expression data (Figure 5), likely resulting from the binding interactions occurring between LILRB1 and MHC class-I molecules (55) during the *in vitro* cultures of virus-specific CD8 T cells. LILRB1 blockade moderately improved the proliferation capacity (i.e. frequency of divided cells, expansion index and replication index) of high avidity but not that of low avidity T cell clones (Figures 6B, C). Despite the technical limitations linked to this functional assay, our data are in line with previously reported findings (51).

Progressive *in vivo* loss of CMV/pp65 T cell clonotypes of high avidity is associated to enhanced LILRB1 expression

Since the NT Amer assay relies on flow-cytometry avidity
analyses, it can be easily adapted to combine direct ex vivo
phenotype and off-rate measurements alongside. Studies were
further performed on the two immune-dominant CMV/A2 CD8
T cell epitopes (i.e. CMV/pp65495-503 versus CMV/immediate-early
(IE)-1316-324) only detectable in donor BCL6 (Figure 1B). In this
healthy individual, CMV/pp65-specific T cells accumulated during
the 18 years of follow-up, whereas CMV/IE-1-specific T cells
showed a gradual decline in frequency over time, concomitant to
a decrease in LILRB1 expression (Figure 7A). These observations
were confirmed using dimensionally reduction t-SNE and
FlowSOM clustering analysis (38). At Tn, LILRB1 was expressed
in a large fraction of the CMV/IE-1-specific T cells, whereas this
distinct sub-population had almost vanished at Tn+18y (Figure 7B).

This was not the case for CMV/pp65-specific T cells with an overall
preserved LILRB1 expression over this time-frame (Figures 7A, B).

We next assessed TCR-pMHC dissociation rates at the ex vivo CMV/IE-specific T cell population level. This analysis revealed the presence of several dissociation curves, arbitrarily defined as fraction A (i.e. faster off-rates) and fraction B (i.e. slower off-rates) (Figure 7C). Fraction A represented the predominant subpopulation at Tn, which further prevailed during the course of latent infection, while retaining relatively stable off-rates (Figure 7D). In contrast, fraction B, mostly composed of T cells of highest binding avidities, had entirely disappeared at Tn+18y. These data indicate that in donor BCL6, CMV/IE-specific T cells not only contracted during latent infection, but this process was associated with the preferential loss of high avidity T cell repertoires, in line with the data found for the CMV/pp65 model (Figure 4). Finally, we conducted a NT Amer-based off-rate analysis combined to LILRB1 and TRBV co-staining. Strikingly, LILRB1 expression corresponded to the fraction of IE-1-specific T cells of higher TCR binding avidity/slower off-rates at Tn, which was no longer detectable at Tn+18y (Figure 7E, see arrow). Despite conserved LILRB1 expression over time within CMV/pp65-specific T cells (Figures 7A, B), we further observed the gradual appearance of a subpopulation of lower avidity/faster off-rates, characterized by reduced LILRB1 levels (Figure 7F, see arrow) and by the presence of TRBV12 clonotype of known low binding avidity (Figures 7G, H; Supplementary Figure 7). Collectively, these observations indicate a progressive in vivo loss of high avidity CMV-specific CD8 T cells with time, corresponding to T cells expressing increased LILRB1 levels.

Discussion

Here, we investigated whether TCR-ligand avidity can directly
drive the long-term maintenance of particular herpesvirus-specific
CD8 TCRαβ clonotypes and searched for factors that regulate
clonal repertoires in latent human CMV versus EBV infection,
over a follow-up time of 15-18 years. Our data revealed the
progressive *in vivo* loss of CMV/pp65- and CMV/IE-1-specific T
cell clonotypes of high binding avidity (i.e. slow TCR off-rates,
reduced CD8 binding dependency) during long-term antigen
exposure. This was not the case for EBV/BMFL1-specific CD8 T
cell repertoires, in which the clonal composition and distribution
(i.e. dominant versus sub-dominant, slow versus fast TCR off-rates,
CD8 binding-independent versus -dependent) are kept highly
stable for at least 15 years. Together, these findings indicate that
TCR-pMHC binding avidity is a determining factor driving the
clonal evolution of long-lasting CMV- but not EBV-specific
memory CD8 T cell responses in humans. In addition, LILRB1
checkpoint receptor was preferentially expressed in high-avidity
CMV-specific TCRαβ clonotypes and correlated in those cells to
their gradual frequency decline over time. The main weakness of the
study is the relative low number of available volunteers (n = 6), due
to the exceptionally long period of follow-up and the fact that
chronic CMV/pp65-specific T cell responses are typically composed
of highly restricted co-dominant clonotypes (n = 15). Consequently,
<figure><img src="image_12.png" /><figcaption>FIGURE 6
Blocking LILRB1 binding on *in vitro* cell proliferation of CMV/pp65-specific CD8 TCRαβ clonotypes. (A) Representative FACS-based histograms of LILRB1 expression on a representative selection of high- (top panel) and low- (bottom panel) avidity CMV/pp65-specific T cell clones at T~n~. Compiled data of LILRB1 expression levels (in percentage; right panel), depicted as violin plots (n = 27 to 61 T cell clones), p-values by Mann-Whitney test (two-tailed) with ***p < 0.001. (B) Representative overlays of CFSE histograms obtained from CFSE-labelled T cell clones of high avidity (top panels) or low avidity (bottom panels) in the presence of blocking anti-LILRB1 (red line) or an isotype control antibody (grey histogram) at day 4 after CMV/pp65-specific stimulation. (C) CFSE-based analyses (% of divided cells, expansion index and replication index) of high (top panels) or low (bottom panels) avidity CMV/pp65-specific CD8 T cell clones, treated with blocking anti-LILRB1 or an isotype control antibody. Data are depicted as violin plots (n = 10 to 22). p-values by Wilcoxon matched-pairs signed rank test with **p < 0.01 and ***p < 0.001; ns, non-significant.</figcaption></figure>

this somehow limits the strength of our statistical-based conclusions.

Differences in the nature of these β-herpesviruses during latent infection could in part explain the gradual contraction of high avidity CMV/pp65- and CMV/IE-1-specific T cell clonotypes observed over time, contrasting with the striking stability of EBV/ BMFL1-specific CD8 T cell clonal repertoires. In the context of EBV infection, some infected B cells become long-lived, namely the latently infected memory B cells (16). EBV reactivation and replication in B cells occurs only sporadically, leading to intermittent cycles of T cell rest and stimulation, in contrast to CMV, which is considered more as a smoldering latent/chronic infection (16, 56). In fact, there is growing evidence that CMV undergoes low-level viral replication, which potentially impacts the virus-specific CD8 T cell responses (15), with the induction of large populations of highly functional T cell responses, maintained lifelong (14, 57). The latent CMV reservoir in MCMV is largely confined to non-hematopoietic cells, such as lymphatic endothelial cells, and can selectively induce memory CD8 T cell inflation (22, 58). In addition, reactivation of CMV or EBV is commonly found in immunocompromised individuals after allogeneic hematopoietic stem cell transplantation (allo-HSCT). CMV has been described as the most frequently reactivated virus following allo-HSCT, occurring in 65-68% of the patients, contrasting to the 9-15% of patients developing EBV reactivation (59, 60). Altogether, these observations including ours further indicate differing mechanisms of viral latency and reactivation between these two herpesviruses. This is in line with a large, recent comprehensive profiling study of flu- versus CMV-specific T cells across multiple tissue sites of organ donors revealing that memory persistence and functional regulation to viral antigens is primarily shaped by virus tropism and specificity (61).

Schober and colleagues (62) proposed different theoretical models of TCR repertoire evolution during latent CMV infection, and to which degree this process is controlled by TCR-pMHC

binding/structural avidity. One model hypothesizes an initial accumulation of high-avidity virus-specific T cells during the early phase of latency, followed by the succession of clones of lower TCR binding avidity over the course of latent infection (62, 63). During this clonal evolution, T cells of higher TCR binding avidity may progressively be lost after replicative senescence due to critical telomere length shortening (64, 65). In the context of MCMV infection, the same authors recently demonstrated that high-avidity TCRs dominated T cell responses at early time-points after infection, but then underwent clonal succession, cellular differentiation and senescence during late time-points, leading to a switch in dominance toward low-avidity TCR T cells (32). Our findings in humans are also compatible with this model, as we showed the preferential selection and expansion of CMV/pp65-specific clonotypes of lower TCR binding avidity, compared to those that had contracted after 15-18 years. Consequently, an overall avidity decline over time was observed at the CMV/ epitope-specific population (i.e. HLA-A2/pp65<sub>495_503</sub> and HLA-A2/IE-1<sub>316_324</sub>). As shown here in humans, longer observation periods (i.e. >15-20 years) are required to reveal the contraction of high-avidity CMV/epitope-specific clones in the latency infection phase, as T cell repertoires can be maintained relatively stable during at least several years (40, 44). A recent report, using adoptive transfer of low versus high avidity MCMV-specific CD8 T cell subpopulations, revealed that during the early phase of latent infection, the inflationary T cell pool was comprised mainly of high avidity CD8 T cells, outcompeting lower avidity CD8 T cells (66). These discordant results could be explained by the use of high number of transferred cells and a relatively short period of follow-up. In summary, our extended longitudinal analysis further argues for an intrinsic impact of the TCR-pMHC avidity on repertoire evolution over the course of latent CMV infection (32, 62).

The remarkable stability in the clonal evolution of EBV-specific repertoires raises the question whether this represents a global characteristic of EBV-specific CD8 T cell responses or only of
<figure><img src="image_13.png" /><figcaption>FIGURE 7</figcaption></figure>

Relationship between LILRB1 expression, TCR binding avidity and *in vivo* persistence of CMV-specific CD8 T clonotypes over time. (A) Representative FACS analysis of CMV/pp65 (top panel) or CMV/IE-1 (bottom panel) multimer staining on CD8 T cells from donor BCL6. Evolution of CMV/pp65 and CMV/IE-1 multimer-positive fractions (middle panel) and LILRB1 expression within CMV/pp65 or CMV/IE-1-specific CD8 T cells (right panel) over time (2002 to 2020). (B) T-SNE analysis & FlowSOM visualization at T<sub>n</sub> and T<sub>n+18y</sub> of merged events from CMV/pp65- (n = 2348 cells) and CMV/IE-1- (n = 2898 cells) specific CD8 T cells from BCL6. On the FlowSOM grid plots, each cluster is sized proportionally to the fraction of multimer-positive CD8 T cells. The cluster's color represents LILRB1 mean expression from low (blue) to high (red). (C) Representative FACS-based NTamer dissociation curves obtained from *ex vivo* expanded CMV/IE-1-specific bulk populations at T<sub>n</sub>, T<sub>n+4y</sub> and T<sub>n+18y</sub>. The population of cells with fast (fraction A) or slow (fraction B) dissociation rates, respectively are indicated. (D) Quantification of the CMV/IE-1-specific CD8 T cells present within fraction A or B dissociating populations (n > 9 independent experiments, left panel). Dissociation rate (k<sub>off</sub>) values derived from the cell populations present within fraction A or fraction B at T<sub>n</sub>, T<sub>n+4y</sub> and T<sub>n+18y</sub> (right panel). (E, F) Combined NTamer dissociation measurements and LILRB1 costaining on *ex vivo* expanded CMV/pp65- (E) and CMV/IE-1- (F) specific bulk T cells at the indicated time-points (T<sub>n</sub>, T<sub>n+4y</sub> and T<sub>n+18y</sub>). Representative FACS-based wild-type NTamer dissociation curves obtained after gating on LILRB1<sup>high</sup> (top panels) versus LILRB1<sup>low</sup> (bottom panels) expression. Compiled TCR-pMHC dissociation rates of LILRB1<sup>high</sup> (in red) versus LILRB1<sup>low</sup> (in black) subpopulations over time (n = 6). Arrows indicate the dissociating subpopulations of high-avidity and LILRB1<sup>high</sup> expression (in red) within bulk CMV/IE-1-specific T cells at T<sub>n</sub> (i.e. contracting cells) or of low-avidity and LILRB1<sup>low</sup> expression (in black) within bulk CMV/pp65-specific T cells at T<sub>n+18y</sub> (i.e. expanding cells), respectively. (G, H) Combined NTamer dissociation measurements and TRBV costaining on *ex vivo* expanded CMV/pp65-specific bulk T cells at the indicated time-points (n = 2). Representative FACS-based wild-type NTamer dissociation curves obtained after gating on TRBV6-5 (high avidity; (G)) versus TRBV12 (low avidity; (H)) clonotypes.
responses against the EBV epitope studied here (i.e. HLA-A2/BMFL1<sub>280-288</sub>). Since HLA-A2/BMFL1<sub>280-288</sub> is an epitope from the early protein BMFL1 essentially expressed during the acute phase of infection, the stability of the observed clone type repertoires over extended periods of time could potentially be explained by the relative absence of epitope expression during the latent phase. Nonetheless, TCR clone type composition and distribution against HLA-A2/BMFL1<sub>280-288</sub> were also highly preserved during transient immunological perturbations after non-myeloablative chemotherapy (44) or following lung transplant under immunosuppression in the presence of EBV reactivation (46). Moreover, long-lasting dominance of EBV-specific TCRαβ clonotypes against two latent epitopes, with no changes in the T cell hierarchy for at least 18 years has been previously reported (67). Collectively, these observations, including our own study, are consistent with the concept that TCRαβ clone type responses against EBV, once established, show steady repertoires over these long periods.

Memory inflation describes the longitudinal development of stable, expanded CD8 T cell memory pools with a distinct phenotype and functional profile, but without sharing features of immune exhaustion (14). Whereas memory CD8 T cell inflation is a hallmark of CMV infection in mouse models, this process is less clearly defined in human CMV infection (68). This is probably due to (i) the large inter-individual variability including notably the duration of infection, HLA restriction, size of latent virus pool, and recurrence of viral reactivation events as well as (ii) the paucity of long-term longitudinal studies (57). In this regard, data from a recent 27-year-long longitudinal study showed only limited effect of duration of CMV infection on adaptive immunity and frailty (69). With the exception of donor BCL6, we did not observe a significant increase in CMV/pp65-specific CD8 T cell frequencies over the observation period of 15-18 years. Moreover, this highly contrasted to the drastic over time decline of CMV/IE-1-specific T cells in BCL6, the only donor sharing multiple specificities. Nevertheless, we still observed a progressive loss of T cells of high binding avidity, with an enrichment of low avidity T cells, in both immunodominant epitopes. Accordingly, we did not observe an inverse relationship between population size and TCR-pMHC half-lives, as recently illustrated for MCMV-specific T cell responses (32). It is possible that our study performed on middle-aged individuals (45 +/- 10 years) was still set too early in the course of the latent phase to visualize CMV-specific T cell expansion, or that memory inflation remains hard to be studied even in well-designed longitudinal studies of healthy individuals, due to the above-mentioned confounding factors (57).

As other inhibitory receptors, LILRB1 (also known as CD85j or
ILT2/LIR-1) is frequently found on the surface of human CD8 T
cells (70). This is the case for highly differentiated antigen-
experienced T cells, often expressing CD57 and lacking CD28,
while displaying strong effector functions (51–53). The
proportion of LILRB1-positive T cells has been shown to increase
with age and in chronic CMV infection (50, 51). LILRB1 recognizes
with high affinity HLA-G and UL18, a human CMV-encoded MHC
class I homologue and with lower affinity classical MHC class I

molecules (71). Importantly, interactions between inhibitory
leukocyte Ig-like receptors, including LILRB1, and endogenous
and pathogenic ligands can regulate both innate and adaptive
immune responses, leading to tolerance and immune evasion (72).
Specifically, LILRB1 expressed on CD8 T cells can inhibit immune
cell activation and effector function (51–53, 73–75). Therefore, it
has been proposed that LILRB1 functions as an immunosuppressive
receptor and may represent an attractive target to enhance tumor
cell killing by effector CD8 T cells (52, 53, 72–74). Extending on
these observations, we found that LILRB1 was preferentially
expressed on CMV-specific TCRαβ clonotypes of high avidity
compared to the low avidity ones and may potentially contribute
to their gradual *in vivo* loss over time during latent CMV infection.
This contrasted to the low, steady LILRB1 expression observed in
EBV-specific CD8 TCRαβ clonotypes, unrelated to their TCR-
pMHC binding avidity. Our data are further in line with the
previous observations that latent CMV infection drives stronger T
cell differentiation and clonal expansion than the EBV one,
resulting in clonal diversity loss (5–10). Interestingly, Schober and
colleagues (32) recently showed that MCMV-specific T cell clones
that declined during the chronic infection phase were also those of
higher TCR avidity and underwent cellular senescence without
displaying signs of classical exhaustion.

Robust techniques allowing for the large-scale *ex vivo*
assessment of TCR-pMHC binding kinetics at the surface of live
T cells have proven technically challenging until recently (76).
Using fluorescent reversible NTAmers, we previously showed that
the k<sub>off</sub> parameter represents a powerful biomarker by which the
functional potency of antigen-specific CD8 T cell responses can be
directly evaluated (33, 34) and graded to better characterize their
impact on the efficacy of therapeutic vaccines (77). Here, we used
NTAmers on well-identified virus-specific TCRαβ clonotypes or at
the polyclonal CD8 T cell population level and observed for the first
time a differential impact of TCR-pMHC binding kinetics on the
long-term TCR clonal evolution during CMV (i.e. pp65 and IE-1)
versus EBV/BMFL1 latent infection. Moreover, comparable TCR-
ligand off-rate (k<sub>off</sub>) values were found for all studied representatives
of a given CMV- or EBV-specific clone type over time (T<sub>n</sub> versus
T<sub>n+15y</sub>), indicating that TCR-pMHC dissociation rate represents a
stable intrinsic biometric at the TCRαβ clone type level. Our
findings nicely fit with another recent report performed on
MCMV-specific TCR repertoires during the course of infection
using TCR-pMHC k<sub>off</sub>-rate measurements with reversible
Streptamers (32). Moreover, the NT Amer technology is based on
reversible peptide-MHC multimers and real-time flow cytometry,
allowing to directly combine TCR-pMHC off-rate analysis to
LILRB1 and specific TRBV staining. Using this upgraded
approach, we demonstrated a direct relationship between CMV
epitope-specific TCR binding avidity and LILRB1 expression.
Specifically, our results suggest a selective expansion of low-
avidity LILRB1<sup>low</sup> T cells, outcompeting high-avidity LILRB1<sup>high</sup>
T cells. Alternatively, high-avidity LILRB1<sup>high</sup> T cells may directly
be depleted over time as shown for the CMV/IE-1 epitope. Either
way, TCR avidity-based repertoire evolution may further be
described as a "clonal succession model", in which T cells of
relative higher avidity are continuously being replaced over time by
the next generation of lower avidity T cells, in a constant and
dynamic recruitment process.

Conclusive remarks

Our study reinforces the key role driven by TCR-ligand binding
avidity in tailoring CMV- but not EBV-specific clonal evolution
during long periods of viral latency. We further propose that
LILRB1 may act as an inhibitory checkpoint receptor, by
potentially limiting the expansion of high avidity T cell
clonotypes over the course of latent CMV infection. In the
context of CMV infection, repetitive exposure to antigen is a key
determinant for memory expansion/inflation, and therefore
regulatory mechanisms that can repress CD8 T cell expansion are
likely beneficial (15). Such mechanisms include regulatory T cells
and anti-inflammatory cytokine production, which might otherwise
lead to the extensive proliferation of highly antigen-sensitive T cells,
possibly overwhelming the global T cell pool (57). Supporting this
notion, LILRB1 may therefore provide another mechanism by
which memory expansion of given CMV-specific TCRαβ
clonotypes might be tightly regulated during lifelong latent/
reactivating CMV infection, while preserving the global functional
T cell repertoire.

Data availability statement

The data presented in this study are deposited in the GEO
repository, accession number GSE246111.

Ethics statement

The studies involving humans were approved by the Ethics
Committee for Clinical Research of the University of Lausanne
(Lausanne, Switzerland). The studies were conducted in accordance
with the local legislation and institutional requirements. The
participants provided their written informed consent to
participate in this study.

Author contributions

BC: Conceptualization, Data curation, Formal Analysis, Methodology, Writing – original draft. BD: Formal Analysis, Methodology, Writing – review & editing. LC-I: Formal Analysis, Methodology, Writing – review & editing. MA: Formal Analysis, Methodology, Writing – review & editing. SP: Formal Analysis, Methodology, Writing – review & editing. MH:

Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing – review & editing. NR: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This study
was sponsored and supported by the Department of Oncology
(University of Lausanne), the MEDIC Foundation (Switzerland)
and the Promedica Stiftung (Chur, Switzerland).

Acknowledgments

We are grateful to the blood donors for their dedicated long-
term collaboration in this study. We are thankful for Prof. Daniel
Speiser for comments and critical reading of the manuscript. We
also acknowledge Mariana Bustamante Eduardo, Nicole
Montandon and Natasa Jovanovic for excellent technical and
secretarial help, and Anne Wilson and the flow cytometry facility
of Lausanne for operational support.

Conflict of interest

The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2023
.1293090/full#supplementary-material
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