CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence

Report

CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence Graphical Abstract

Authors Scott M. Krummey, Anna B. Morris, Jesica R. Jacobs, ..., Brian D. Evavold, Haydn T. Kissick, Mandy L. Ford

Correspondence [email protected]

In Brief Krummey et al. show that viral CD8+ T cell memory has heterogeneous CD45 isoform expression. Low-affinity CD8+ T cells have high CD45RB expression and a CD27hiCD62Lhi phenotype relative to high-affinity CD45RBlo CD8+ T cells, which possess an effector-like phenotype. CD45RBhi cells survive better under homeostatic conditions in vivo.

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Viral-specific CD8+ T cell memory contains both CD45RBhi and CD45RBlo populations Low-affinity CD8+ T cell memory is CD45RBhi and possesses a CD27hiCD62Lhi phenotype CD45RBhi CD8+ T cell memory survive better than highaffinity CD45RBlo memory in vivo Human CD45RBhi and CD45RBlo CD8+ T cell memory populations have distinct phenotypes

Krummey et al., 2020, Cell Reports 30, 1282–1291 February 4, 2020 ª 2020 The Authors. https://doi.org/10.1016/j.celrep.2020.01.016

Cell Reports

Report CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence Scott M. Krummey,1,2,7,* Anna B. Morris,1 Jesica R. Jacobs,3 Donald J. McGuire,4 Satomi Ando,4 Katherine P. Tong,1 Weiwen Zhang,1 Jennifer Robertson,1 Sara A. Guasch,1 Koichi Araki,4,6 Christian P. Larsen,1 Brian D. Evavold,3 Haydn T. Kissick,5 and Mandy L. Ford1 1Emory

Transplant Center, Emory University School of Medicine, Atlanta, GA, USA of Pathology & Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 3Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA 4Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA, USA 5Department of Urology, Emory University School of Medicine, Atlanta, GA, USA 6Present address: Division of Infectious Diseases, Center for Inflammation and Tolerance, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA 7Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2020.01.016 2Department

SUMMARY

The identity of CD45 isoforms on the T cell surface changes following the activation of naive T cells and impacts intracellular signaling. In this study, we find that the anti-viral memory CD8+ T pool is unexpectedly comprised of both CD45RBhi and CD45RBlo populations. Relative to CD45RBlo memory T cells, CD45RBhi memory T cells have lower affinity and display greater clonal diversity, as well as a persistent CD27hi phenotype. The CD45RBhi memory population displays a homeostatic survival advantage in vivo relative to CD45RBlo memory, and long-lived high-affinity cells that persisted long term convert from CD45RBlo to CD45RBhi. Human CD45RO+ memory is comprised of both CD45RBhi and CD45RBlo populations with distinct phenotypes, and antigen-specific memory to two viruses is predominantly CD45RBhi. These data demonstrate that CD45RB status is distinct from the conventional central/effector T cell memory classification and has potential utility for monitoring and characterizing pathogen-specific CD8+ T cell responses. INTRODUCTION CD45 is a transmembrane phosphatase that plays a central role in T cell activation and signaling. Multiple isoforms of CD45 are simultaneously expressed on T cells due to alternative splicing of three exons corresponding to the extracellular domains A, B, and C or the exclusion of all three, denoted CD45RO (Hermiston et al., 2003). CD45 modulates T cell receptor (TCR) signaling by controlling the pool of active Lck, a Src family kinase required for TCR phosphorylation and signaling cascades. CD45 isoforms differ in their relative ability to activate Lck and localize proximally to the immunologic synapse, owing largely to inhibitory dimeriza-

tion that is more prevalent in CD45RO relative to larger isoforms (Xu and Weiss, 2002). Multiple groups have shown that CD45 isoforms with large extracellular domains more effectively mediate T cell activation, as measured by active Lck, calcium flux, and Nur77 expression (Xu and Weiss, 2002; Irles et al., 2003; Krummey et al., 2016). The current understanding of how CD45 isoform expression profiles correspond with T cell differentiation was largely defined by early studies. Following the identification of multiple CD45 species distinguished by molecular weight, the development of CD45-domain-specific monoclonal antibodies led to the finding that memory T cells express the low-molecular-weight isoform CD45RO (Michie et al., 1992; Merkenschlager et al., 1988; Merkenschlager and Beverley, 1989) and that high-molecular-weight isoforms corresponded with CD45RA+ or CD45RB+ populations in humans and mice, respectively (Rogers et al., 1992; Birkeland et al., 1989). Multiple groups established that naive T cells express high levels of CD45RA or CD45RB and that following stimulation in vitro, activated T cells become CD45RO+ and lose high-molecular-weight A- or B-domain-containing isoforms (Akbar et al., 1988; Serra et al., 1988). This seminal work formed a reciprocal and binary model of CD45 isoform expression on T cells, whereby naive T cells transition from CD45RO to CD45RO+ following activation and concurrently lose expression of high-molecular-weight A-, B-, and C-domain-containing isoforms (Trowbridge and Thomas, 1994; Hermiston et al., 2003). This description has carried through to more recent literature, in which memory T cells are described as CD45RO+ and presumed to be essentially devoid of high-molecular-weight isoforms on the cell surface (Sallusto et al., 1999; Lynch, 2004; Wu et al., 2008; Fecteau et al., 2001; Hermiston et al., 2003). However, despite a multitude of careful investigations into the regulation of CD45 in vitro, the role of CD45 isoforms on endogenous memory T cells has not been evaluated in vivo. Given the critical role for CD45 in facilitating T cell signaling, we sought to understand the role of CD45 isoforms on murine and human CD8+ T cell memory.

1282 Cell Reports 30, 1282–1291, February 4, 2020 ª 2020 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

RESULTS CD45RBhi and CD45RBlo Memory Populations Are Generated Following LCMV Infection To investigate whether the CD45RB expression changed on endogenous memory CD8+ T cell populations during priming and differentiation into memory, we assessed T cells following acutely cleared viral infection with lymphocytic choriomeningitis virus (LCMV) Armstrong (Figure 1A). Activated CD44hi cells at 1 week post-infection contained a population of CD45RBlo cells (Figures 1A and 1B). Interestingly, in contrast to descriptions in the literature (Hermiston et al., 2003; Wu et al., 2008; Ogilvy et al., 2003; Fecteau et al., 2001), about half of the CD44hi effector and memory populations at 6 weeks post-infection maintained high CD45RB expression, similar to that seen in naive CD8+ T cells (Figures 1A and 1B). The frequency of CD45RBhi memory CD8+ T cells was slightly higher in the lymph nodes versus blood and spleen (Figure S1A). We found that CD44hiCD49dlo virtual memory cells, which undergo antigen-independent homeostatic proliferation, also maintain CD45RBhi status similar to naive CD8+ T cells (Figure S1B). In contrast to viral infection, fluorescence-activated cell sorting (FACS) of CD45RBhi and CD45RBlo memory cells that underwent homeostatic proliferation in a sub-lethally irradiated host largely maintain pre-transfer CD45RB expression levels (82% for CD45RBhi and 76% for CD45RBlo) (Figure S1C). To better characterize extracellular CD45 domain expression on LCMV memory, we assessed the expression of CD45RA, CD45RB, and CD45RC on antigen-specific CD8+ T cells following LCMV infection. Analysis of CD8+ T cells specific for H-2Db np396 revealed that the expression of CD45RA, CD45RB, and CD45RC each reached a nadir at approximately 10–14 days post-infection and remains remained relatively stable out to 42 days, with the frequency of CD45RAhi and CD45RBhi populations modestly increasing from day 14 to day 42 (Figures 1C and S1D). At 6 weeks post-infection, approximately 25%–40% of tetramer-positive populations were CD45RBhi (Figure 1D). CD45RBhi status corresponded with co-expression of CD45RA and CD45RC, whereas CD45RBlo memory was predominantly low or negative for CD45RA and CD45RC (Figure 1E). To assess the amount of CD45RO isoform, for which there is no available murine antibody, we quantified the frequency of transcripts with junctions between Ptprc exons 2–7 from sorted naive and CD45RBhi and CD45RBlo memory populations. We found that relative to naive CD8+ T cells, memory T cells expressed a greater proportion of the CD45RO transcript (Figure 1F). However, CD45RBlo memory T cells expressed relatively higher levels of the CD45RO transcript than CD45RBhi memory T cells (Figure 1F). In summary, we found that following LCMV infection, the endogenous CD45ROhi CD8+ T cell memory pool is comprised of CD45RBhi and CD45RBlo memory populations that express distinct profiles of CD45 isoforms. CD45RBhi Memory Cells Possess Lower Functional Avidity and Relative 2D Affinity Than CD45RBlo Memory Our previous work in a TCR transgenic model demonstrated that high-affinity priming of OT-I T cells leads to CD45RBlo memory, whereas low-affinity priming leads to CD45RBhi memory (Krum-

mey et al., 2016). We next assessed whether CD45RB status denotes differences in TCR affinity of endogenous CD8+ T cells specific for the viral antigen. We determined the functional avidity of CD8+ T cell memory to the H-2Db np396 epitope by using an interferon gamma (IFN-g) dose-response assay (Figure 1G). We found the half-maximal effective concentration (EC50) for CD45RBhi memory was approximately 2-fold higher than CD45RBlo memory (Figure 1H). Next, to measure the TCR affinity of CD45RBhi and CD45RBlo populations, we used the 2D micropipette adhesion assay, which provides a measure of the TCR:pMHC affinity independent of CD8 coreceptor binding (Huang et al., 2010). We used FACS with CD45RBhi and CD45RBlo memory populations and assessed the relative 2D affinity of these populations for H-2Db np396. We found that the mean 2D affinity of CD45RBlo memory cells was 2.8 3 10 4 mm4 (Figure 1I), similar to published values for known high-affinity interactions between CD8+ OT-I T cells and H-2Kb SIINFEKL (N4 OVA) (Krummey et al., 2016) and LCMV SMARTA CD4+ T cells for H-2Db GP61-85 (Sabatino et al., 2011). CD45RBhi memory cells, by contrast, had a mean value of 9.6 3 10 6 mm4 (Figure 1I), similar values obtained for low-affinity OT-I H-2Kb SIIVFEKL (V4 OVA) (Krummey et al., 2016) and autoreactive MOG35-55 CD4+ T cells (Sabatino et al., 2011; Hood et al., 2015). Thus, CD45RBhi and CD45RBlo memory cells have a significantly different sensitivity for the antigen, as measured by functional avidity and relative 2D affinity. CD45RBhi Memory Cells Have Greater Clonal Diversity Than CD45RBlo Memory Cells The strength of naive TCR priming affinity correlates with the magnitude of clonal burst following activation (Zehn et al., 2009). We assessed the TCR repertoire of CD45RBhi and CD45RBlo memory populations by sequencing the TCRb CDR3 region of memory cells specific for H-2Db np396. We found that the proportion of clones that are highly expanded (comprising 1%–10% of all clones) was greater in the CD45RBlo population than CD45RBhi (Figure 1J), whereas the proportion of less-expanded clones (comprising 0.01%–0.1% of all clones) was higher in CD45RBhi memory cells (Figure 1J). These results are consistent with greater clonal proliferation of higher affinity clones following activation in the CD45RBlo population relative to CD45RBhi. At the CDR3 sequence level, the diversity of the CD45RBhi population was significantly greater than the CD45RBlo population (Figure 1K). In total, these data demonstrate that the clonal diversity of the CD45RBhi memory population is greater than that of the CD45RBlo memory population. CD45RBhi and CD45RBlo Memory Populations Produce Similar Secondary Effector Populations Given the differences in affinity and avidity between CD45RBhi and CD45RBlo memory cells, we next wanted to assess the functional abilities of these populations in vivo. To assess the protective capacity of CD45RBhi and CD45RBlo memory cells, we adoptively transferred FACS-isolated populations into naive mice and challenged them with LCMV clone 13 (Figure 2A). We found that at day 5, viral loads were reduced in the spleen of both memory groups relative to the no-transfer control. Despite their lower affinity, CD45RBhi memory cells mediated similar

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Figure 1. CD8+ T Cell Memory Contains CD45RBhi and CD45RBlo Populations with Distinct TCR Affinity and Repertoire Diversity (A and B) FACS plots (A) and summary data (B) of CD45RB expression on CD44lo naive or CD44hi virus-specific populations following LCMV Armstrong infection. (C) Expression of CD45RA, CD45RB, and CD45RC on Db np396 at the indicated time points following infection with LCMV. See also Figure S1D. (D) Frequency of CD45RBhi cells within total Db np396 and Db gp276 populations at 6 weeks post-infection. (E) CD45RA and CD45RC expression among CD44lo naive or CD44hi CD8+ T cell populations at week 6 post-infection. (F) Frequency of the CD45RO transcript as a frequency of total Ptprc transcripts among naive and Db np396+ memory populations. (G) Representative frequency of IFN-g response following np396 peptide stimulation, normalized to maximum response, at week 6 post-infection. (H) EC50 from multiple mice analyzed as in (F). (I) Relative 2D micropipette adhesion assay values for Db np396 of FACS-isolated CD45RBhi and CD45RBlo memory CD8+ T cells at weeks 6–10 post-infection. (J) Clonal space homeostasis plots of CD45RBhi and CD45RBlo memory cells, depicting the proportion of T cell clones in three frequency ranges (1.0%–10%, 0.1%–0.01%, and 0.001%–0.0001%) within each memory population. Both the size of each clonal group and the radius reflect the proportion of the total. (K) Inverse Simpson’s diversity index for three populations of CD45RBhi and CD45RBlo memory cells. In (E), summary data depict 9 mice/group. For (J) and (K), each data point represents FACS-isolated populations of three pooled mice. Error bars represent mean ± SEM. Significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

levels of protection relative to high-affinity CD45RBlo memory cells (Figure 2B). Similar numbers of secondary effectors were recovered from the spleen (Figure 2C). Finally, both populations of secondary effectors had high expression of CD25 and CD127,

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which are associated with robust secondary effector cells (Figure 2D) (Masopust et al., 2006; Fraser et al., 2013). Thus, CD45RBhi and CD45RBlo memory cells are poised to become secondary effectors with similar functional capacity.

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Figure 2. CD45RBhi Have a Persistent Memory Phenotype and Similar Functional Capacity Relative to CD45RBlo Memory Cells (A) Schematic of viral rechallenge experiment in which CD45RBhi and CD45RBlo memory cells are adoptively transferred into naive congenic hosts and infected with 104 plaque-forming units (PFU) of LCMV clone 13. (B) Viral titers from spleen of clone-13-infected mice on day 5. (C) Absolute numbers of adoptively transferred CD45.2+ and Kb np396+ T cells on day 5. (D) Phenotype of CD45.2+ memory cells on day 5 post-infection. (E) Frequency of IFN-g-positive cells following 5 h with np396 peptide. See also Figure S2C.

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The predominant classification of CD8+ T cells is central memory (TCM), which is defined by high CD62L expression and relatively low expression of cytolytic proteins, such as granzyme B, and effector memory T cells (TEMs), which have low CD62L expression and higher granzyme B expression (Jameson and Masopust, 2009; Sallusto et al., 1999). In contrast to CD62Lhi TCM and CD62Llo TEM populations that express distinct levels of granzyme B, we found that CD45RBhi and CD45RBlo populations expressed similar levels of granzyme B (Figures S2A and S2B). We next assessed the relative ability of CD45RB populations to produce cytokines. Following brief ex vivo rechallenge with np396 peptide, a similar frequency of cells produced IFN-g. The slightly higher percentage of CD45RBlo cytokine producers relative to CD45RBhi reflects the greater abundance of np396specific CD45RBlo cells as measured by tetramer (Figure 2E). CD45RB expression did not change on memory CD8+ T cells during the 5-h peptide stimulation (Figure S3C). Finally, we assessed the ability of CD45RBhi and CD45RBlo memory cells to proliferate following rechallenge. We FACS-isolated memory CD44hiCD45RBhi and CD45RBlo cells and rechallenged them for 4 days ex vivo with the np396 peptide. Although differences in precursor frequency preclude a comparative analysis of the number of dividing cells, dividing cells in both populations underwent similar numbers of divisions and had similar proliferation indexes (Figure 2F). Thus, despite significant differences in TCR affinity, CD45RBhi and CD45RBlo memory cells have similar functional capabilities, as measured by cytolytic enzyme expression, cytokine production, and proliferative capacity. Memory CD45RBhi Cells Have a Long-Lived Memory Phenotype We next investigated whether CD45RBhi and CD45RBlo populations had distinct cellular phenotypes. We first investigated markers that have been associated with memory formation (Hendriks et al., 2000; Bose et al., 2013; Ha¨nninen et al., 2011; Hikono et al., 2007, and found that both CD45RB populations expressed high levels of Ly6C and CD11a (Figure S3A). In contrast, the CD45RBhi memory population expressed significantly higher levels of CD27 than CD45RBlo (Figure 2G). High CD27 expression has been associated with high-quality memory—with memory cell survival (Hendriks et al., 2000; van Gisbergen et al., 2011) and recall potential (Hikono et al., 2007)—so we next sought to investigate the expression of additional proteins associated with the phenotype of long-lived T cell memory. We found that relative to CD45RBlo memory cells, CD45RBhi memory cells were comprised of a higher frequency of CD27hiCD62Lhi and CD27hiCD127hi populations (Figure 2H). Expression of the senescence marker KLRG-1, however, was lower on CD45RBhi

than CD45RBlo memory cells. CD45RBlo memory cells expressed a higher frequency of the CD27loKLRG1hi population (Figures 2G and 2H). Following murine cytomegalovirus (MCMV) infection, CD45RBhi CD8+ memory T cells also expressed high levels of CD62L and CD27 relative to CD45RBlo CD8+memory T cells (Figure S3B). Thus, despite having a lower priming affinity, CD45RBhi cells have a phenotype consistent with established high-quality persistent memory CD8+ T cells. We performed RNA sequencing on FACS-isolated np396tetramer-positive memory populations at week 6 post-infection. We found 148 genes that were differentially expressed at least 1.5-fold between CD45RBhi and CD45RBlo memory cells (p < 0.05; Figure 2I). Genes that were upregulated in CD45RBhi memory cells include the markers of long-lived memory Sell (CD62L) and Il7r (CD127), as well as the transcription factor Satb1, which inhibits exhaustion in tumor-reactive T cells (Stephen et al., 2017), and the pro-survival oncogene Myc (Gnanaprakasam et al., 2017). In the CD45RBlo population, the multi-zinc-finger transcription factor Zeb2 was upregulated, which has been shown to control the balance of terminal differentiation by promoting KLRG-1hi effector CD8+ T cells and TEM (Omilusik et al., 2015). We performed gene set enrichment analysis (GSEA) on CD45RB populations relative to murine TCM and TEM populations. CD45RBlo memory cells were enriched for the TEM gene set relative to the TCM gene set, and the CD45RBhi memory cells was more enriched for TCM relative to the TEM gene set (Figure 2J). CD45RBlo memory cells were also enriched for genes from progressively differentiated memory populations and were most enriched for the most terminally differentiated population (Figure 2K). Thus, this analysis reveals that CD45RBlo memory CD8+ T cells are more well-differentiated effector memory cells and are more enriched for TEM genes, whereas CD45RBhi memory CD8+ T cells are more enriched for the TCM gene set. CD45RBhi Status Is Distinct from Homeostatic TCM Conversion and Confers a Long-Lived Memory Phenotype To assess the persistence of CD45RBhi memory relative to CD45RBlo, we first investigated the profile of CD45RB expression on long-term memory populations in vivo. We found that the frequency of CD45RBhi memory increased from an early memory time point (week 6) to a late time point (month 6) postinfection (Figure 3A). This increase was found in both the total CD44hi memory pool as well as the Db np396-tetramer-positive population (Figure 3B). The CD8+ memory pool is known to convert over time to a greater proportion of TCM due to greater homeostatic proliferation of TCM relative to TEM as well as differentiation of TEM into TCM (Sarkar et al., 2007; Wherry et al., 2003).

(F) Proliferation of FACS-isolated CD45RBhi and CD45RBlo memory cells for 4 days ex vivo with Db np396 or Db gp276 peptide. (G) CD27 expression among naive CD44lo or memory CD45RBhi and CD45RBlo populations. (H) Expression profile of naive CD44lo or memory CD45RBhi and CD45RBlo populations. Right, statistical significance is indicated between CD45RBhi and CD45RBlo populations. See also Figure S3. (I) Volcano plot of RNA sequencing (RNA-seq) expression results between Db np396+ CD45RBhi and CD45RBlo memory populations. Colored dots depict genes with an expression change of greater than 1.5-fold. (J) Normalized enrichment score (NES) from gene set enrichment analysis of Db np396+ CD45RBhi and CD45RBlo memory populations versus central and effector memory CD8+ T cells. (K) NES from gene set enrichment analysis of Db np396+ CD45RBhi and CD45RBlo memory populations versus repetitively stimulated memory CD8+ T cells. Error bars represent mean ± SEM. Significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Figure 3. CD45RBhi Status Is Distinct from Homeostatic TCM Conversion and Confers a Long-Lived Memory Phenotype (A) Frequency of CD45RBhi CD8+ T cells at indicated time points up to 6 months post infection. (B) Example FACS plots and summary data depicting the frequency of CD44hi and Db np396+ CD8+ T cells that are CD45RBhi at 6 weeks and 6 months postinfection. (C) Frequency of CD62Lhi populations among CD45RBhi and CD45RBlo memory cells at week 6 or month 6 post-infection. (D) Ratio of CD62Lhi cells in (C). (E) Schematic depicting FACS isolation and adoptive transfer of CD45.2 CD45RBhi or CD45RBlo CD44hi memory into naive CD45.1 mice followed by parking for 3 weeks (25–27 days). (F) CD45RB expression of pre- and post-FACS-isolated populations as in (E) with summary data. (G) Ratio of the number of CD45RBlo/CD45RBhi CD45.2 memory cells recovered from spleen and lymph nodes on day 25 or 27 post-transfer. (H) Phenotype of memory populations recovered after parking. (I) Summary data of frequency of CD27hiCD62Lhi cells among CD45.2+ populations recovered. In (F), summary data depict 3 day-0-sorted samples and 9–10 mice/group at week 3. In (I), summary data depict 9–10 mice/group. Error bars represent mean ± SEM. Significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Figure 4. Human CD45ROhi Memory Cells Are Comprised of CD45RBhi and CD45RBlo Populations with Distinct Phenotypes (A) Example FACS gating and CD45RB expression of naive CD45ROlo and memory CD45ROhi CD8+ T cells from the peripheral blood of healthy donors. (B) Summary of the frequency of CD45RBhi cells among naive CD45ROlo and memory CD45ROhi CD8+ T cells from healthy donors. (C) Frequency of CD45RBhi cells among TCM and TEM cells from healthy donors. (D) Expression of CD27 and CD57 among CD45RBhi and CD45RBlo memory populations. (E) EBV BMLF1 tetramer staining and frequency of CD45RBhi cells among tetramer-specific CD8+ T cells in healthy donors. (F) CMV pp65 tetramer staining and frequencey of CD45RBhi cells among tetramer-specific CD8+ T cells in healthy donors. (G) Frequency of CD45RBhi among TCM, TEM, and TEMRA memory subsets for EBV BMLF1 antigen-specific and CMV pp65 antigen-specific cells. See also Figure S4. (H) Expression of CD27 and CD57 on viral-specific CD8+ T cells as in (E) and (F). In (D), summary data depict 10 healthy donors. In (H), summary data depict 8 EBV-immune and 10 CMV-immune individuals. Error bars represent mean ± SEM. Significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Because the CD45RBhi memory pool is enriched for CD62Lhi cells, we questioned whether this increase in the CD45RBhi frequency was due to the selective conversion of CD45RBhi cells to CD62Lhi TCM over time. We found that both CD45RBhi and CD45RBlo memory cells expressed higher CD62L at the late memory (6 months post-infection) time point relative to an early (6 weeks post-infection) memory time point (Figure 3C). Interestingly, the relative increase in CD62Lhi TCM was greater in the

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CD45RBlo population than the CD45RBhi population (Figure 3D), demonstrating that the increase in the CD45RBhi frequency of the memory pool is distinct from the conversion of memory T cells to TCM over time. To assess whether the change in CD45RBhi frequency was due to differential survival and/or phenotypic conversion of CD45RB status, we FACS isolated CD45.2 CD44hiCD45RBhi and CD45RBlo memory cells and adoptively transferred them

into CD45.1 congenically distinct hosts and parked them for 25–27 days in the absence of restimulating antigen (Figure 3E). Interestingly, CD45RBhi cells maintained CD45RBhi expression after parking, whereas a significant portion of CD45RBlo cells converted to CD45RBhi over this time (Figure 3F). The CD45RBlo memory cells, however, did not survive as well as CD45RBhi memory cells (Figure 3G). Thus, although less persistent than CD45RBhi memory cells, the CD45RBlo cells that do persist alter their CD45 isoform profile to become CD45RBhi. Consistent with the notion that the CD45RBhi status of memory confers a survival advantage, cells that converted from CD45RBlo to CD45RBhi, so-called ex-CD45RBlo cells, also had a more persistent phenotype, as defined by CD62LhiCD27hi status (Figures 3H and 3I). Human CD45RO+ Memory Cells Are Comprised of Phenotypically Distinct CD45RBhi and CD45RBlo Populations Human memory T cells are typically defined by a high expression of CD45RO and low expression of CD45RA (Hermiston et al., 2003; Sallusto et al., 1999). In the peripheral blood of healthy donors, we found that naive CD45ROlo T cells expressed high levels of CD45RB (Figures 4A and 4B). In contrast, the CD45ROhi memory population contained an equal proportion of CD45RBhi and CD45RBlo populations (Figures 4A and 4B). CD45RB status did not correspond to the TCM and TEM phenotypes (Figure 4C). We investigated the memory phenotype of the CD45RBhi and CD45RBlo populations by using the costimulatory receptor CD27 and CD57, a marker of terminally differentiated effectors (Brenchley et al., 2003; Chattopadhyay et al., 2009). We found that CD45RBhi memory cells expressed a greater proportion of CD27hiCD57lo memory cells than CD45RBlo memory cells, which were predominantly CD27loCD57hi (Figure 4D). Thus, human CD8+ T cell memory CD45RB status corresponds to distinct memory phenotypes. Finally, we assessed the CD45RB status of viral-specific memory T cells. We investigated a cohort of healthy donors with preexisting immunity to cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Using major histocompatibility complex class I (MHC class I) tetramers to the EBV BMLF1 or CMV pp65 antigens, we found that the antigen-specific memory to these viruses was predominantly CD45RBhi (Figures 4E and 4F). There was no difference in the frequency of CD45RBhi memory cells within TCM, TEM, and TEMRA subsets for either infection (Figures S4 and 4G). CD45RBhi EBV-specific memory displayed a relatively CD27hiCD57lo phenotype, whereas we found that, consistent with prior reports (Appay et al., 2002; Hertoghs et al., 2010), CMV-specific memory displayed a relatively lower frequency of CD27hiCD57lo cells (Figure 4H). Thus, these data demonstrate that the total human CD45ROhi memory pool is comprised of both CD45RBhi and CD45RBlo populations and that virus-specific CD8+ T cell memory to two well-controlled viruses is predominantly CD45RBhi. However, additional virus-specific factors dictate the CD27 expression phenotype of human CD8+ T cell memory.

in mouse and human, but this work predated a detailed understanding of effector and memory T cell subsets (Trowbridge and Thomas, 1994). The findings in the present study revise the binary model of differentiation-coupled CD45 isoform expression in several ways: approximately half of virus-specific CD8+ T cells retained CD45RBhi status into memory; CD45RB status is coupled to the TCR affinity of memory T cells; and finally, over time, CD45 isoforms are altered within the memory pool under homeostatic conditions to favor a CD45RBhi status. As most studies of TCR affinity in vivo have relied on TCR transgenic T cells and altered peptide ligands, additional studies will be needed to confirm the utility of CD45RB status as a marker of TCR affinity of endogenous T cells in other experimental systems. Although CD45RB conferred a modest difference in functional avidity (approximately 2-fold) relative to the OVA APL (altered peptide ligand) system (N4 OVA versus V4 OVA, 680-fold) (Zehn et al., 2009), we believe that this is reflective of the difference between assessing the avidity of a single T cell for multiple ligands versus assessing a polyclonal population of T cells for a single ligand. Additionally, we found a significantly different relative 2D affinity measurement of 29-fold between CD45RBhi and CD45RBlo memory. Although this technique has been shown to sensitively discriminate between high- and low-affinity ligands for a transgenic TCR, we cannot definitively rule out the contribution of additional receptors on the cell surface in our experiments investigating polyclonal T cell populations. Although CD45RBlo memory cells were enriched for terminally differentiated memory population, a portion of CD45RBlo cells were able to survive long term and convert to CD45RBhi status. Future studies will have to elucidate the factors that control heterogeneity within this population. A number of studies have shown that higher avidity CD8+ T cell responses are desirable in vaccine responses to pathogens and cancer (Appay et al., 2008). However, the persistence of immunity generated following vaccination is a problem for a number of pathogens (Gu et al., 2017). Our findings suggest that maintaining a CD45RBhi status is important for maximizing the longevity of pathogen-specific immunity. This could be potentially accomplished by modulating the affinity of the interaction between TCR and pMHC during antigen recognition or blocking CD45RB with monoclonal antibodies, which has been shown to affect CD45 isoform expression (Fecteau et al., 2001). In addition, future studies should investigate the potential utility of CD45RB status as a biomarker that can be used to stratify the quality of CD8+ T cell memory responses to pathogens. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d

DISCUSSION Key discoveries involving CD45 function on T cells mirrored the development of monoclonal antibodies against CD45 isoforms

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KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animals B Human Subjects B Viruses

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METHOD DETAILS B Flow Cytometry of Murine Populations B Viral Rechallenge Experiments B Memory Parking Experiments B In vivo Homeostatic Proliferation B Relative 2D affinity assay B Ex vivo peptide stimulations B RNASeq B T Cell Receptor Repertoire Analysis B Flow Cytometry of Human T Cell Populations QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2020.01.016. ACKNOWLEDGMENTS The authors would like to thank the Emory+Children’s Flow Cytometry Core and the Emory Flow Cytometry Core for FACS sorting, the Yerkes Genomics Core for sequencing services and support, and the NIH Tetramer Core for tetramer production. This work was supported by NIAID/NIH awards AI104699, AI070307 (M.L.F.), and K99 AI146271 (S.M.K.) and funding from the James M. Cox Foundation (C.P.L.) and the Carlos and Marguerite Mason Trust (C.P.L.). The authors also thank members of the Emory Transplant Center and Matthew Z. Madden for helpful discussions. Experimental schematics were created with Biorender.com. AUTHOR CONTRIBUTIONS Conceptualization – S.M.K. and M.L.F.; Methodology – S.M.K., H.T.K., B.D.E., K.A., and M.L.F.; Investigation – S.M.K., J.R.J., S.A.G., K.P.T., A.B.M., W.Z., S.A., and J.R.; Formal Analysis – S.M.K., A.B.M., J.R.J., S.A., J.R., D.J.M., and H.T.K.; Writing – Original Draft, S.M.K.; Writing – Reviewing & Editing, S.M.K., K.A., and M.L.F.; Supervision – C.P.L., K.A., B.D.E., and M.L.F. DECLARATION OF INTERESTS The authors declare no competing interests. Received: April 16, 2019 Revised: October 18, 2019 Accepted: January 3, 2020 Published: February 4, 2020 REFERENCES Ahmed, R., Salmi, A., Butler, L.D., Chiller, J.M., and Oldstone, M.B. (1984). Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160, 521–540. Akbar, A.N., Terry, L., Timms, A., Beverley, P.C., and Janossy, G. (1988). Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol. 140, 2171–2178. Appay, V., Dunbar, P.R., Callan, M., Klenerman, P., Gillespie, G.M., Papagno, L., Ogg, G.S., King, A., Lechner, F., Spina, C.A., et al. (2002). Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 8, 379–385. Appay, V., Douek, D.C., and Price, D.A. (2008). CD8+ T cell efficacy in vaccination and disease. Nat. Med. 14, 623–628.

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Krummey, S.M., Martinez, R.J., Andargachew, R., Liu, D., Wagener, M., Kohlmeier, J.E., Evavold, B.D., Larsen, C.P., and Ford, M.L. (2016). Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity. J. Immunol. 196, 2838–2846. Livingston-Rosanoff, D., Daley-Bauer, L.P., Garcia, A., McCormick, A.L., Huang, J., and Mocarski, E.S. (2012). Antiviral T cell response triggers cytomegalovirus hepatitis in mice. J. Virol. 86, 12879–12890. Lynch, K.W. (2004). Consequences of regulated pre-mRNA splicing in the immune system. Nat. Rev. Immunol. 4, 931–940. Masopust, D., Ha, S.J., Vezys, V., and Ahmed, R. (2006). Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. J. Immunol. 177, 831–839.

Sabatino, J.J., Jr., Huang, J., Zhu, C., and Evavold, B.D. (2011). High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses. J. Exp. Med. 208, 81–90. Sallusto, F., Lenig, D., Fo¨rster, R., Lipp, M., and Lanzavecchia, A. (1999). Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712. Sarkar, S., Teichgra¨ber, V., Kalia, V., Polley, A., Masopust, D., Harrington, L.E., Ahmed, R., and Wherry, E.J. (2007). Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation. J. Immunol. 179, 6704–6714. Serra, H.M., Krowka, J.F., Ledbetter, J.A., and Pilarski, L.M. (1988). Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J. Immunol. 140, 1435–1441.

Merkenschlager, M., and Beverley, P.C. (1989). Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: human CD8 memory CTLp selectively express CD45RO (UCHL1). Int. Immunol. 1, 450–459.

Stephen, T.L., Payne, K.K., Chaurio, R.A., Allegrezza, M.J., Zhu, H., PerezSanz, J., Perales-Puchalt, A., Nguyen, J.M., Vara-Ailor, A.E., Eruslanov, E.B., et al. (2017). SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells. Immunity 46, 51–64.

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Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., and Mesirov, J.P. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550.

Michie, C.A., McLean, A., Alcock, C., and Beverley, P.C. (1992). Lifespan of human lymphocyte subsets defined by CD45 isoforms. Nature 360, 264–265. Milner, J.J., Toma, C., Yu, B., Zhang, K., Omilusik, K., Phan, A.T., Wang, D., Getzler, A.J., Nguyen, T., Crotty, S., et al. (2017). Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours. Nature 552, 253–257. Moon, J.J., Chu, H.H., Hataye, J., Paga´n, A.J., Pepper, M., McLachlan, J.B., Zell, T., and Jenkins, M.K. (2009). Tracking epitope-specific T cells. Nat. Protoc. 4, 565–581. Ogilvy, S., Louis-Dit-Sully, C., Cooper, J., Cassady, R.L., Alexander, D.R., and Holmes, N. (2003). Either of the CD45RB and CD45RO isoforms are effective in restoring T cell, but not B cell, development and function in CD45-null mice. J. Immunol. 171, 1792–1800. Omilusik, K.D., Best, J.A., Yu, B., Goossens, S., Weidemann, A., Nguyen, J.V., Seuntjens, E., Stryjewska, A., Zweier, C., Roychoudhuri, R., et al. (2015). Transcriptional repressor ZEB2 promotes terminal differentiation of CD8+ effector and memory T cell populations during infection. J. Exp. Med. 212, 2027–2039. Robins, H.S., Campregher, P.V., Srivastava, S.K., Wacher, A., Turtle, C.J., Kahsai, O., Riddell, S.R., Warren, E.H., and Carlson, C.S. (2009). Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. Blood 114, 4099–4107. Rogers, P.R., Pilapil, S., Hayakawa, K., Romain, P.L., and Parker, D.C. (1992). CD45 alternative exon expression in murine and human CD4+ T cell subsets. J. Immunol. 148, 4054–4065.

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Cell Reports 30, 1282–1291, February 4, 2020 1291

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies Anti-mouse CD3 PE Dazzle 594 monoclonal antibody

Biolegend

Clone 17A2 Cat# 100246; RRID: AB_2565883

Anti-mouse CD4 APC Fire 750 monoclonal antibody

Biolegend

Clone RM4-4 Cat# 116020; RRID: AB_2715956

Anti-mouse CD8 Brilliant Violet 785 monoclonal antibody

Biolegend

Clone 53-6.7 Cat# 100750; RRID: AB_2562610

Anti-mouse CD11a FITC monoclonal antibody

Biolegend

Anti-mouse CD11c Brilliant Violet 510 monoclonal antibody

Biolegend

Clone M17/4 Cat# 350604; RRID: AB_10662904 Clone S-HCL-3 Cat# 371514; RRID: AB_2650797

Anti-mouse CD19 Brilliant Violet 510 monoclonal antibody

Biolegend

Clone 6D5 Cat# 115546; RRID: AB_2562137

Anti-mouse CD25 FITC monoclonal antibody

Biolegend

Clone 3C7 Cat# 101908; RRID: AB_961212

Anti-mouse CD27 PE-Cy7 monoclonal antibody

Biolegend

Clone LG.3A10 Cat# 124215; RRID: AB_10645330

Anti-mouse CD44 Alexa Fluor 700 monoclonal antibody

Biolegend

Clone IM7 Cat# 103026; RRID: AB_493713

Anti-mouse CD45RA Brilliant Violet 421 monoclonal antibody

BD Optibuild

Anti-mouse CD45RB Pacific Blue monoclonal antibody

Biolegend

Clone 14.8 Cat# 740022 Clone C363-16A Cat# 103316; RRID: AB_2174405

Anti-mouse CD45RB FITC monoclonal antibody

Biolegend

Clone C363-16A Cat# 103306; RRID: AB_313013

Anti-mouse CD45RC PE monoclonal antibody

BD PharMingen

Clone DNL-1.9 Cat# 557357; RRID: AB_396661

Anti-mouse CD45.1 Brilliant Violet 605 monoclonal antibody

Biolegend

Clone A20 Cat# 110738; RRID: AB_2562565

Anti-mouse CD45.2 APC monoclonal antibody

Biolegend

Clone 104 Cat# 109813l; RRID: AB_389210

Anti-mouse CD62L FITC monoclonal antibody

BD PharMingen

Anti-mouse CD127 Brilliant Violet 605 monoclonal antibody

Biolegend

Clone MEL-14 Cat# 553150; RRID: AB_394665 Clone A7R34 Cat# 135041; RRID: AB_2572047

Anti-mouse KLRG-1 Brilliant Violet 711 monoclonal antibody

Biolegend

Clone 2F1/KLRG1 Cat# 138427; RRID: AB_2629721

Anti-mouse Ly6c Brilliant Violet 711 monoclonal antibody

Biolegend

Clone HK1.4 Cat# 128037; RRID: AB_2562630

Anti-mouse Granzyme B PE monoclonal antibody

Biolegend

Clone QA16A02 Cat# 372207; RRID: AB_2687031

Anti-mouse IFN-gamma APC monoclonal antibody

Biolegend

Clone XMG1.2 Cat# 505810; RRID: AB_315404

Anti-human CD4 Brilliant UV 395 monoclonal antibody

BD PharMingen

Clone SK3 Cat# 563550 (Continued on next page)

e1 Cell Reports 30, 1282–1291.e1–e5, February 4, 2020

Continued REAGENT or RESOURCE

SOURCE

Anti-human CD3 Brilliant Ultraviolet 737 monoclonal antibody

BD PharMingen

IDENTIFIER Clone UCHT1 Cat# 564307

Anti-human CD8 Brilliant Violet 711 monoclonal antibody

BD PharMingen

Clone RPA-T8 Cat# 563677

Anti-human CD14 Brilliant Violet 510 monoclonal antibody

BD PharMingen

Clone MP9 Cat# 301842

Anti-human CD19 Brilliant Violet 510 monoclonal antibody

Biolegend

Clone H1B19 Cat# 302242

Anti-human CD20 Brilliant Violet 510 monoclonal antibody

BD PharMingen

Clone 2H7 Cat# 563067

Anti-human CD27 PerCP-Cy5.5 monoclonal antibody

Biolegend

Clone O323 Cat# 302820

Anti-human CD45RA Alexa Fluor 700 monoclonal antibody

BD PharMingen

Clone HI100 Cat# 560673

Anti-human CD45RA APC-H7 monoclonal antibody

BD PharMingen

Clone HI100 Cat# 560674

Anti-human CD45RB PE monoclonal antibody

Biolegend

Clone MEM-55 Cat# 310204

Anti-human CD45RB Brilliant Violet 650 monoclonal antibody

BD PharMingen

Clone MT4 (6B6) Cat# 745395

Anti-human CD45RO Alexa Fluor 700 monoclonal antibody

BD PharMingen

Clone UCHL1 Cat# 561136

Anti-human CD57 FITC monoclonal antibody

BD PharMingen

Clone NK-1 Cat# 555619

Anti-human CD197 (CCR7) PE monoclonal antibody

BD PharMingen

Clone C1.7 Cat# 329522

Chemicals, Peptides, and Recombinant Proteins LCMV np396-404 peptide (FQPQNGQFI)

GenScript

Cat# RP20090

H-2D(b) / FQPGNGQFI (LCMV np396) APC Tetramer

NIH Tetramer Core

N/A

H-2D(b) / SGVENPGGYCL (LCMV gp276) APC Tetramer

NIH Tetramer Core

N/A

H-2D(b) / KAVYFNATM (LCMV gp33) APC Tetramer

NIH Tetramer Core

N/A

HLA-B7 / TPRVTGGGAM (CMV pp65) APC Tetramer

NIH Tetramer Core

N/A

HLA-A2 / NLVPMVATV (CMV pp65) APC Tetramer

NIH Tetramer Core

N/A

HLA-A2 / GLCTLVAML (EBV BMLF1) APC Tetramer

NIH Tetramer Core

N/A

GolgiStop Protein Transport Inhibitor

BD Biosciences

Cat# 554724

CellTrace CFSE Cell Proliferation Kit, for flow cytometry

Invitrogen

Cat# C34554

CellTrace Violet Cell Proliferation Kit, for flow cytometry

Invitrogen

Cat# C34557

LIVE/DEAD Fixable Aqua Dead Cell Stain Kit

Invitrogen

Cat# L34965

Fixation/Permeabilization Solution Kit

BD Biosciences

Cat# 554714

AllPrep DNA/RNA Micro Kit

QIAGEN

Cat# 80204

Critical Commercial Assays

Experimental Models: Organisms/Strains Mouse: C57BL/6NCrl

Charles River

Stock# 003831

Mouse: B6.SJL-Ptprca Pepcb/BoyCrCrl (C57BL/6 Ly5.2-Cr; CD45.1)

Charles River

Stock# 564

Lymphocytic chroriomeningitis virus (LCMV), Armstrong strain

Dr. Rafi Ahmed (Ahmed et al., 1984)

N/A

Lymphocytic choriomeningitis virus (LCMV), Clone 13 strain

Dr. Rafi Ahmed (Ahmed et al., 1984)

N/A (Continued on next page)

Cell Reports 30, 1282–1291.e1–e5, February 4, 2020 e2

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Murine cytomegalovirus (MCMV), K181 strain

Dr. Edward Mocarski (LivingstonRosanoff et al., 2012)

N/A

FlowJo v9

FlowJo, LLC

https://www.flowjo.com

Prism 7

GraphPad Software

https://www.graphpad.com

Anti-APC Microbeads

Miltenyi Biotec

Cat# 130-090-855

LS magnetic columns

Miltenyi Biotec

Cat# 130-042-401

CountBright Absolute Counting Beads

ThermoFisher Scientific

Cat# C36950

BD Quantibrite Beads

BD Biosciences

Cat# 340495

Software and Algorithms

Other

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Scott M. Krummey ([email protected]). This study did not generate new unique reagents. EXPERIMENTAL MODEL AND SUBJECT DETAILS Animals Male C57BL/6NCr and C57BL/6 Ly5.2-Cr mice were obtained from the National Cancer Institute Grantee Program (Charles River, Frederick, MD) at 6-8 weeks of age. All experiments were conducted in age-matched males randomly assigned to experimental groups. This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee at Emory University. All animals were housed in specific pathogen-free animal facilities at Emory University. Human Subjects Fresh peripheral blood mononuclear cells (PBMC) were isolated from normal healthy donors using protocols approved by the Emory University Institutional Review Board. Informed consent was obtained for all subjects in accordance IRB protocols. The pool of healthy donors used in this study was comprised of 31 individuals (17 females and 14 males) with a mean age 43.2 years old (range 19-63 years old). Subjects were randomized to each experiment. Blood was collected using BD Vacutainer CPT tubes (BD Diagnostics). In some experiments, cells were frozen in 10% DMSO and 50% FBS and stored in liquid nitrogen. Cells were thawed and rested overnight at 37 C in 96-well plates before stimulation. Cells were cultured in RPMI 1640 supplemented with 10% FBS (Mediatech, VA), 2.4 mM L-glutamine, and 10 mM 2-mercaptoethanol (Sigma). Viruses Mice were infected with 2x105 pfu LCMV Armstrong intraperitoneally (i.p.), 1x104 pfu LCMV Clone 13 via intravenous tail vein, or 2x106 pfu MCMV K181 strain i.p. where indicated. METHOD DETAILS Flow Cytometry of Murine Populations Spleens from infected or naive mice were processed to single cell suspension and stained using the following antibodies against the following antigens obtained from Biolegend: CD3ε, CD4, CD8, CD11a, CD11c, CD19, CD27, CD44, CD45RA, CD45RB, CD45RC, CD62L, CD127, KLRG-1, Ly6c, Granzyme B, IFN-g. H-2Db np396 (FQPQNGQFI) and H-2Db gp276 (SGVENPGGYCL) tetramers were provided by the NIH Tetramer Core and were stained at 100 mg/mL (1:200 from provided concentration). Surface stains were performed for 20-30 min at room temperature. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences) following manufacturer’s instructions. For surface phenotyping, memory T cell populations were identified by gating on singlets, CD19-CD4-, followed by CD8+CD44hi. In each phenotyping experiment, CD45RB gates were set as a naive mouse CD8+CD44lo T cells 90% CD45RBhi. Data were acquired on a BD LSR II or a BD Fortessa and analyzed using FlowJo software (Tree Star). Viral Rechallenge Experiments Spleen from an LCMV infected CD45.2 mouse was processed to single cell suspension and FACS sorted into CD8+CD44hi CD45RBhi and CD45RBlo memory populations to > 95% purity using a FACS Aria II. Populations were adjusted to include similar frequencies of

e3 Cell Reports 30, 1282–1291.e1–e5, February 4, 2020

MHC Class I tetramer+ cells (Kb np396, Db gp276, and Db gp33 (KAVYFNATM) in each CD45RBhi and CD45RBlo population, and adoptively transferred i.v. to the tail vein of naive CD45.1 mice. Mice were then infected with 1x104 pfu of LCMV Clone 13 i.v. in the tail vein. On day 5 post-infection, the spleen was harvested and a portion was weighed and preserved in 1 mL RPMI with 2% FBS. The remainder of the spleen was processed to single cell suspension, stained with CD45.2 APC and enriched using antiAPC magnetic beads as described previously (Krummey et al., 2016; Moon et al., 2009). Single cell suspensions were incubated with 3 mL CD45.2 APC for 15 min at 4 C, then washed with 15 mL FACS buffer (PBS with 0.5% BSA), followed by incubation with 20 mL anti-APC microbeads (Miltenyi) for 10 min at 4 C and wash with 15 mL FACS buffer. Cell suspensions were separated on magnetic LS columns (Miltenyi) according to manufacturer’s instructions. Flow-through and column-bound fractions were stained with surface markers to identify transferred populations as CD45.1-CD45.2+CD8+CD44hi. Absolute cell numbers were obtained using AccuCheck beads (Invitrogen). Viral titer of the spleen samples was performed by plaque assay (Ahmed et al., 1984). Serially diluted homogenized spleen samples were incubated on Vero E6 cell monolayers in 6-well tissue culture plates for 60 min at 37 C. The cells were then overlayed with 4 mL 0.5% agarose in Media 199. After 4 days incubation at 37 C, the cells were overlayed with 2 mL 0.5% agarose in Media 199 with 0.02% neutral red, and the number of plaques were counted the following day. Memory Parking Experiments Spleen from an LCMV infected CD45.2 mouse was processed to single cell suspension and FACS sorted into CD8+CD44hi CD45RBhi and CD45RBlo memory populations to > 95% purity using a FACS Aria II. Equal numbers of cells (0.8-1.5x105) were adoptively transferred i.v. to the tail vein of naive CD45.1 mice. On week 3 post transfer (day 25 or 27), the spleen and popliteal, inguinal, axial, brachial and mesenteric lymph nodes were harvested and pooled together in a single cell suspension. Cells were stained with CD45.2 APC and enriched using anti-APC magnetic beads as described above (Krummey et al., 2016; Moon et al., 2009). Transferred populations were identified as CD45.1-CD45.2+CD8+CD44hi. Absolute cell numbers were obtained using AccuCheck beads (Invitrogen). In vivo Homeostatic Proliferation CD45.1 mice were irradiated with 50 cGy as hosts. Spleen from an LCMV infected CD45.2 mouse was processed to single cell suspension and FACS sorted into CD8+CD44hi CD45RBhi and CD45RBlo memory populations to > 95% purity using a FACS Aria II. Cells were stained with CFSE 20 mM (Invitrogen) for 3 min and washed with 15 mL complete R10. Equal numbers of cells (0.8-1.5x105) were adoptively transferred i.v. to the tail vein of irradiated CD45.1 mice. On day 7 post transfer the spleen was harvested processed to a single cell suspension. Cells were stained with CD45.2 APC and enriched using anti-APC magnetic beads as described above. Transferred populations were identified as CD45.1-CD45.2+CD8+CD44hi. Relative 2D affinity assay Human RBCs were isolated in accordance with the Institutional Review Board at Emory University coated with Biotin-X-NHS (EMD) and 0.5 mg/ml streptavidin (ThermoFisher) and 1-2 mg of H-2Db np396 bioinylated monomers with mouse b-2 microglobulin (NIH Tetramer Core). Monomers cannot bind CD8 due to substitution of the mouse H-2Db a3 domain with the human HLA-A2 a3 domain. The concentration of monomer bound to RBCs was quantified with anti-H-2Db antibody (eBioscience) and QuantiBrite Beads (BD Biosciences). Splenic CD3ε+CD19-CD8+CD44hi cells from week 6-10 LCMV infected mice were sorted into CD45RBhi and CD45RBlo populations using a FACS Aria II (BD), and the TCR expression was quantified with anti-TCRb (H57-597; BD Biosciences) and QuantiBrite beads. The micropipette adhesion frequency assay was performed as previously described (Huang et al., 2010; Sabatino et al., 2011). In brief, a pMHC-coated RBC and T cell were placed on opposing micropipettes and brought into contact by micromanipulation for a controlled contact area (Ac) and time (t). The T cell was retracted at the end of the contact period, and the presence of adhesion (indicating TCR:pMHC binding) was observed by elongation of the RBC membrane. This TCR-RBC contact was repeated 30 times and the adhesion frequency (Pa) was calculated. The relative 2D affinity (AcKm) of each cell that had a Pa of greater than 10% was calculated using the Pa at equilibrium (where t / N) using the following equation: AcKa = ln[1-Pa(N)]/(mrml), where mr and ml reflect the receptor (TCR) and ligand (pMHC) densities, respectively. Ex vivo peptide stimulations For cytokine production, spleens from LCMV infected mice were processed to single cell suspension and 1x106 cells were stimulated for 4 hours at 37 C with saturating 10 mM np396 peptide (FQPQNGQFI, GenScript). After 30 min, 10 mg/ml GolgiPlug (BD Biosciences) was added. For proliferation experiments, splenocytes from LCMV infected mice were FACS sorted into CD45RBhi and CD45RBlo memory populations as described above. 1x105 sorted CD45.2 cells were stained with Cell Trace Violet (Invitrogen) according to manufacturer’s instructions, and added to 9x105 naive CD45.1 splenocytes in a flat bottom 96-well plate with 10 mM np396 peptide (GenScript). After 4 days, cells were stained with Live/Dead Aqua (Invitrogen) and surface markers to identify CD45.2 cells. RNASeq Week 6 post-infection cells from the spleen and mesenteric lymph nodes were sorted into CD8+CD44hiCD14-CD19CD11c-CD4-Dbnp396+ CD45RBhi and CD45RBlo memory populations to > 99% purity. RNA and DNA extracted using the QIAGEN Allprep DNA/RNA Micro kit. RNA sequencing was performed using the Illumina HiSeq platform. Sequences were sorted and indexed with Samtools, counted using featurecount, and differential expression analysis was performed using Deseq2. Gene

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Set Enrichment Analysis (Subramanian et al., 2005) was performed using CD45RBhi versus CD45RBlo memory and central and effector memory gene sets (Milner et al., 2017), or progressively stimulated memory populations (Wirth et al., 2010). GSEA normalized enrichment scores (NES) of each population was displayed using corrplot package and R Studio. CD45 isoform analysis was performed using a custom python script that identifies intron spanning reads that overlap exon 2 of the Ptprc gene. CD45RO expression was determined by comparing the ratio of exon 2 to exon 6 junctions versus total exon 2 originating junctions. T Cell Receptor Repertoire Analysis TCR beta chain CDR3 repertoire analysis was performed on DNA from the same sorted np396-specific populations as above (Adaptive Biotechnology, Inc). Multiplex polymerase chain reaction (PCR) was used to amplify the TCR beta chain CDR3 region using a standard quantity of DNA as the template (Robins et al., 2009). PCR products were sequenced using the Illumina HiSeq platform. The sequences were aligned to a reference genome, and variable, diversity, and joining (VDJ) gene definitions were based on the ImMunoGeneTics (IMGT) system. PCR amplification bias was corrected using a synthetic repertoire of TCRs to establish an amplification baseline and adjusting the assay to minimize primer bias, as well as barcoded, spiked-in synthetic templates to assess sequence coverage. Resulting data were filtered and clustered using the relative frequency ratio between similar clones and a modified nearest neighbor algorithm to merge related sequences and remove PCR and sequencing errors, as previously described (Robins et al., 2009; Carlson et al., 2013). Clone sequences were analyzed using the ImmunoSeq platform. Analysis of clonal space homeostasis and inverse Simpson diversity index was performed using the tcR package and displayed using ggplot2 in R Studio. The inverse Simpson index provides a quantitative measure of the diversity (number of clones and abundance of each) of populations, whereby higher values indicate greater diversity. Flow Cytometry of Human T Cell Populations Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors with existing immunity to EBV and CMV following protocols approved by the Emory University Institutional Review Board. The HLA haplotype of donors was assessed using Class I haplotype matched tetramers for the EBV BMLF1259-267 epitope (GLCTLVAML), CMV pp65417-426 eptiope (TPRVTGGGAM), or CMV pp65495-503 epitope (NLVPMVATV) were provided by the NIH Tetramer Core and stained at 100 mg/mL (1:200 from provided concentration). Cells were stained with LIVE/DEAD AQUA and combinations of the following antibodies obtained from BD Biosciences or Biolegend against the following antigens: CD3ε, CD4, CD8, CD14, CD20, CD27, CD57, CD45RA, CD45RO, CD45RB, CD62L, and CCR7 for 30-60 min at room temperature. Data were acquired on a BD Fortessa and analyzed using FlowJo software (Tree Star). QUANTIFICATION AND STATISTICAL ANALYSIS All data points represent individual donors, and where individual data points are not depicted the value of n is provided in the corresponding figure legend. For analysis of absolute numbers and expression levels, paired or unpaired Student’s t tests (two-tailed) were performed where appropriate. Error bars represent standard error measurements. For functional avidity assessment, EC50 was calculated using a non-linear line of best fit (agonist versus normalized response). Ratios of Granzyme B expression were calculated for CD62L or CD45RB populations, respectively, by dividing %Granzyme B+ Marker Lo population / %Granzyme B+ Marker Hi population. The ratio of CD62Lhi cells Month 6 / Week 6 was assessed by first calculating the average frequency of CD62Lhi cells among CD45RBhi and CD45RBlo populations of 20 mice (4 independent experiments) at week 6 post infection. The ratio was calculated by dividing %CD62Lhi Month 6 / Average %CD62Lhi Week 6 for CD45RBhi and CD45RBlo populations, respectively. The ratio of CD45RBhi and CD45RBlo memory cells after parking was calculated as the absolute number of CD45RBlo cells / Average number of CD45RBhi cells (per experiment). Proliferation index was assessed using FlowJo software (Tree Star). Statistics were performed using GraphPad Prism 7.0c. All summary data depict the mean ± standard error measurement (SEM). Significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DATA AND CODE AVAILABILITY The RNASeq data from CD45RBhi and CD45RBlo memory populations are available at NCBI Gene Expression Omnibus website (GEO: GSE141772). The custom python script that enables quantification of Ptprc isoform abundance is available from the corresponding author upon reasonable request.

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