Early KLRG1+ but Not CD57+CD8+ T Cells in Primary Cytomegalovirus Infection Predict Effector Function and Viral Control

This information is current as Aki Hoji, Iulia D. Popescu, Matthew R. Pipeling, Pali D. of September 24, 2021. Shah, Spencer A. Winters and John F. McDyer J Immunol published online 25 September 2019 http://www.jimmunol.org/content/early/2019/09/19/jimmun ol.1900399 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 25, 2019, doi:10.4049/jimmunol.1900399 The Journal of Immunology

Early KLRG1+ but Not CD57+CD8+ T Cells in Primary Cytomegalovirus Infection Predict Effector Function and Viral Control

Aki Hoji,*,1 Iulia D. Popescu,*,1 Matthew R. Pipeling,* Pali D. Shah,† Spencer A. Winters,* and John F. McDyer*

CMV remains an important opportunistic pathogen in high-risk lung transplant recipients. We characterized the phenotype and function of CD8+ T cells from acute/primary into chronic CMV infection in 23 (donor+/recipient2; D+R2) lung transplant recipients and found rapid induction of both KLRG1+ and/or CD57+ CMV-specific CD8+ T cells with unexpected coexpression of

CD27. These cells demonstrated maturation from an acute effector T cell (TAEFF) to an effector memory T cell (TEM) phenotype + + 2 + with progressive enrichment of KLRG1 CD57 CD27 cells into memory. CMV-specific KLRG1 TAEFF were capable of Downloaded from in vitro proliferation that diminished upon acquisition of CD57, whereas only KLRG1+ expression correlated with T-bet

expression and effector function. In contrast to blood TAEFF, lung mucosal TAEFF demonstrated reduced KLRG1/T-bet expres- + sion but similar CD57 levels. Additionally, increased KLRG1 TAEFF were associated with early immune viral control following primary infection. To our knowledge, our findings provide new insights into the roles of KLRG1 and CD57 expression in human T cells, forming the basis for a refined model of CD8+ T cell differentiation during CMV infection. The Journal of Immunology, 2019,

203: 000–000. http://www.jimmunol.org/

ytomegalovirus, a member of the b-herpesvirus family, antiviral prophylaxis strategies in the past decade in many transplant remains a significant opportunistic pathogen and cause of programs, D+R2 LTRs (6), which compose 25% of all LTRs, C morbidity and mortality in solid organ and hematopoietic continue to demonstrate increased risk for recurrent CMV viremia cell transplant recipients. Lung transplant recipients (LTRs), in and CMV end-organ disease and increased 5-year mortality (7). We particular seronegative recipients of allografts from seropositive have previously demonstrated heterogeneity of CMV-specific T cell donors (donor+/recipient2;D+R2), are at increased risk for CMV immunity among the D+R2 LTR population that is predictive of complications (1, 2). CMV infectious complications such as the capacity for early viral control following primary infection. pneumonitis and viremia have been implicated in LTRs as risk Specifically, we have shown important roles for induction of the by guest on September 24, 2021 factors for developing chronic lung allograft dysfunction and the major type-1 transcription factor T-bet, effector function, and pro- bronchiolitis obliterans syndrome, the major limiting factor for liferative capacity in CD8+ and CD4+ T cells as significant func- long-term survival in LTRs (3–5). Despite the adoption of extended tional immune correlates for establishing viral control during early chronic CMV infection (8–10). Recently, we showed that idiopathic pulmonary fibrosis lung recipients with short telomeres demonstrate *Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Med- impaired CMV-specific T cell immunity and T-bet induction that icine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and correlated with increased risk for CMV complications (11). How- †Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 ever, questions remain as to the optimal T cell marker(s) that could prospectively stratify high-risk lung recipients who are at risk for 1A.H. and I.D.P. contributed equally to this work. relapsing CMV following discontinuation of antiviral therapy ver- ORCIDs: 0000-0001-7093-3437 (A.H.); 0000-0003-4294-1223 (I.D.P.); 0000-0003- 0595-595X (J.F.M.). sus those with the capacity to establish immune control. Lung Received for publication April 5, 2019. Accepted for publication July 31, 2019. transplantation provides a unique opportunity to evaluate viral im- mune mechanisms, as the advent of primary CMV infection is often This work was supported by National Institutes of Health Grants R01 AI079175 and R01HL133184 (to J.F.M.) and funding from the Immune Transplant and Therapy known and both peripheral and allograft-derived resident T cells can Center of UPMC. be tracked into chronic infection (12, 13). A.H., I.D.P., and J.F.M. designed and analyzed experiments. A.H. and I.D.P. performed Similar to virus-specific CD8+ T cells in the mouse, a linear experiments and analyzed and interpreted results. A.H. wrote a majority of the manu- progression in differentiation is the current paradigm in human script, and I.D.P. contributed figure legends and methods. M.R.P. and P.D.S. provided clinical specimens and patients’ characteristics. I.D.P. and S.A.W. compiled patient data. T cells (14–16). However, although the phenotype and function of J.F.M. supervised the study and edited the manuscript. effector memory CMV-specific CD8+ T cells during chronic infec- Address correspondence and reprint requests to Dr. John F. McDyer, Division of tion has been widely investigated, the phenotypic correlates of CD8+ Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School T cells effector function during acute/primary CMV infection have of Medicine, 3459 Fifth Avenue, NW 628, Pittsburgh, PA 15213. E-mail address: [email protected] been less characterized. Early studies showed that CMV-specific + The online version of this article contains supplemental material. CD8 T cells during chronic infection are enriched predom- Abbreviations used in this article: BAL, bronchoalveolar lavage; ChIP, chromatin inantly in the mature effector memory T cell (TEM) phenotype 2 2 hi immunoprecipitation; D+R2, donor+/recipient2; KLRG1, killer cell lectin-like re- CD27 CD28 CD45RA , marked by the increased expression of ceptor subfamily G member 1; LMNC, lung mucosal mononuclear cell; LTR, lung granzymes A/B, perforin, and IFN-g but a diminished proliferative transplant recipient; T , acute effector T cell; T , effector memory T cell. AEFF EM capacity (17–19). In parallel, these cells acquire surface expression Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 of the terminal differentiation markers, coinhibitory receptor killer

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900399 2 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL cell lectin-like receptor subfamily G member 1 (KLRG1) (20) and (time point referred to as “primary CMV”). PBMCs were isolated from CD57 (21, 22). Acquisition of CD57 expression is thought to occur heparinized blood samples by density gradient centrifugation using Ficoll- increasingly over the course of chronic CMV infection (16, 23), Paque (GE Healthcare) to be used in subsequent assays. Study participants underwent BAL by use of a standard protocol with instillation of 180 ml of whereas persistence of CMV Ag is thought to drive progressive sterile normal saline in the right middle lobe of the lung. A BAL speci- downregulation of CD27 into the TEM phase (24). men was obtained on the same day for blood collection. Lung mucosal In the acute/primary LCMV mouse infection model, KLRG1hi mononuclear cells (LMNCs) were obtained simply via centrifugation of surface expression marks short-lived effector cells that are critical BAL fluid. All patients had therapeutic levels of calcineurin inhibitors at for rapid viral clearance, and its expression is T-bet dependent the time of sampling. Cells were then washed with PBS, aliquoted at 10 million cells in 1 ml of freezing medium (Invitrogen), and frozen in (25). Although both KLRG1 and CD57 (no mouse equivalent) are the liquid nitrogen tank. PBMCs were thawed rapidly in the presence of + expressed in human memory CD8 T cells (26), and most notably 2 U per ml of Benzonase (EMD Millipore). in CMV-specific CD8+ T cells (27, 28), expression and potential MHC class I dextramer and surface staining of PBMCs functional correlation of these markers of terminal differentia- and LMNCs tion have not been evaluated during acute/primary viral infection. Based on our previous findings showing early T-bet induction in Cryopreserved PBMCs and LMNCs were thawed in in the presence of ∼ 3 6 CD8+ T cells during acute/primary CMV infection and its im- 2 U/ml Benzonase (EMD Millipore), and 2 10 cells were labeled with FV-510 (BD Biosciences) in IMDM medium (Life Technologies) for portance in viral control (8, 9), we hypothesized that an early + 10 min at 37˚C. Immediately after incubation, cells were washed with the induction of KLRG1 and/or CD57 in CMV-specific CD8 T cells complete medium and stained with the MHC class I dextramer (Immudex, correlates with effector function and the early establishment of Copenhagen, Denmark) against known immunodominant CMV epitopes

CMV control. (Supplemental Table I) at 25˚C for 25 min. Cells were then washed and Downloaded from In this study, we characterized the phenotypic and functional incubated with a mixture of conjugated mAbs for 30 min at 4˚C in PBS + with a fish gelatin blocking reagent (Biotium, Fremont, CA) and 0.1% capacity of CMV-specific CD8 T cells during acute/primary sodium azide. After the final wash, cells were fixed with 1% paraformal- CMV infection into chronic CMV infection in a cohort of dehyde (Protein Sciences, Meriden, CT) for analysis. Available pretrans- D+R2 LTRs.WeobservedrapidinductionofbothCD57and plant PBMC samples (Pre-PBMC) were stained with matching MHC class KLRG1 surface expression in CMV-specific CD8+ acute effector I dextramers (Fig. 1B, Supplemental Fig. 1B), and frequencies of CD3+CD8+ dextramer positive cells were below 0.01%. The following T cells (T ) during primary CMV infection and de novo vi- http://www.jimmunol.org/ AEFF fluorescent conjugated Abs were used for staining surface phenotypic remia, surprisingly in conjunction with CD27 coexpression. Im- + + + markers: CD3 Alexa Fluor 700 (UCHT1), CD8–allophycocyanin-H7 portantly, both CD57 and KLRG1 CMV-specific CD8 TAEFF (SK1), CD14-BV605 (MSE2), CD16-BV605 (3G8), CD19-BV605 (SJ25C1), demonstrated the capacity for in vitro proliferation in response to CD27-PECy7 (MT271), CD28-PerCPCy5.5 (L293), CD38-BV711 (HIT2), CMV Ag; however, only KLRG1 expression significantly corre- CD45RA-PECy5 (H100), CD57-BV421 (NK-1), CD279 PE-CF594 (EH12.1), lated with T-bet and effector multifunction. Furthermore, only the CD69-BV650 (FN50), and CD103-BV605 (BerACT8) were purchased from BD Biosciences. T-bet-BV711 (4B10) was purchased from BioLegend. KLRG1 promoter was highly predicted and bound T-bet in a KLRG1-allophycocyanin (13F12F2) was kindly provided by Dr. H. Pircher chromatin immunoprecipitation (ChIP) assay and correlated with (Institute of Immunology, University of Freiburg). intracellular T-bet protein expression. Finally, acute KLRG1 in-

+ Flow cytometry by guest on September 24, 2021 duction, but not CD57, expression in CMV-specific CD8 TAEFF and total acute CD8+ T cells were predictive of the capacity for Stained PBMCs or BAL-derived LMNCs were analyzed by the BD Fortessa viral control in D+/R2 LTRs during early chronic infection. SORP high-throughput system. Prior to the acquisition, CountBright Beads (Invitrogen) were added to each well, and a fixed volume of the stained sample was acquired by high-throughput system. For visualization of ex Materials and Methods vivo–stained total and CMV-specific CD8+ T cells (Supplemental Fig. 1A), Study participants we used a compounded gating scheme previously described in (23) with a following sequential gate order: potential doublet exclusion based on an D+/R2 LTRs from the Johns Hopkins Lung Transplant Program were forward scatter area and forward scatter height plot, dead cell exclusion identified (Table I) and provided informed written consent for participation based on the live cell gate on FV-510 and forward scatter area plot, in- in a Johns Hopkins Medicine institutional review board–approved proto- clusion of CD3+ cells based on a CD3+ gate with a CD14+CD16+CD19+ col. All patients were treated with standard three-drug immunosuppres- exclusion gate on a CD3–Alexa-700 and CD14CD16CD19-QDot605 plot, sion. Antiviral prophylaxis with ganciclovir and/or valganciclovir was used and a lymphocyte gate on an forward scatter and side scatter log plot. for the initial 3 mo after transplant. Patients were prospectively monitored CMV-specific CD8+ T cells were gated further on MHC class I dextramer at least weekly for the development of primary CMV infection (defined as positive population on the MHC class I dextramer-PE and side scatter area de novo detection of viral replication by quantitative PCR). CMV viral plot (Supplemental Fig. 1A). Data analysis and graphic representations loads were determined using quantitative PCR of plasma by the Johns were done with FlowJo v.10 (TreeStar, Ashland, OR). Hopkins Hospital Clinical Virology Laboratory. Patients developing pri- mary CMV infection were treated with antiviral therapy (ganciclovir and/ In vitro short-term stimulation and intracellular or valganciclovir) until two consecutive weekly quantitative CMV PCR cytokine staining measurements revealed undetectable viremia. Following completion of antiviral therapy for primary infection, patients continued to be prospec- Single-pools of overlapping 15-mer peptides for pp65 (JPT Peptide tively monitored with quantitative CMV PCR measured at least biweekly, Technologies, Berlin, Germany) were used. PBMCs were cultured in round- as well as during any symptomatic or clinically indicated time points, for the bottom tissue culture tubes (Sarstedt) in the presence or absence (medium development of relapsing viremia (defined as the detection of .300 copies/ml alone) of pooled pp65 peptides (1 mg/ml). All stimulations for intracellular of CMV by quantitative PCR on two consecutive samples after the com- cytokine production were performed using 106 cells per condition for 6 h at pletion of antiviral therapy for primary infection). Patients with relapsing 37˚C with brefeldin-A (10 mg/ml) (Sigma-Aldrich) added for the final 4 h viremia received antiviral therapy (ganciclovir and/or valganciclovir) if of culture. Monensin (5 mg/ml) along with brefeldin-A and anti-CD107a– clinically indicated. Dates of chronic and acute viremic samples were at a Pacific Blue was added at the beginning of culture when CD107a was minimum of 6 mo apart (median 6 SEM, 379 6 69 d; range 18421096 d). measured. All cells were collected for flow cytometric analysis with a The study was approved by the institutional review board at the University range of 0.5–1 3 106 total events collected per condition. All gates for of Pittsburgh. cytokine frequencies were set using the medium alone control and sub- tracted from peptide restimulated samples frequencies. Preparation of PBMCs and lung mucosal mononuclear cells In vitro Ag-specific T cell proliferation Blood and bronchoalveolar lavage (BAL) samples from LTRs were obtained prior to the discontinuation of initial antiviral prophylaxis (time point re- Thawed frozen PBMCs were labeled with CytoTell Green (AAT Bioquest) ferred to as “Pre-CMV”) and within 5–14 d of detection of de novo viremia or 0.2 mM CFSE (Invitrogen, Carlsbad, CA) in IMDM medium (Life The Journal of Immunology 3

Technologies, Grand Island, NY) for 10 min at 37˚C. The cells were im- Statistical analysis and data visualization mediately washed and plated with IMDM supplemented with 10% FCS in a 96-well plate for 6 d. For Ag-specific stimulation, PBMCs were Statistical analysis was performed by using the GraphPad Prism 7 stimulated in the presence of 2 mM single CMV dextramer cognate pep- (GraphPad, La Jolla, CA), JMP12 (SAS, Cary, NC), and R (R Core Team, tides or the pool of peptide mix for 6 d and then were harvested and 2018), and cytokine coexpression was based on the Boolean gating analysis prepared for flow cytometric analysis. Proliferating cells were restimulated determined by using SPICE (29). Nonparametric statistical analyses, as , in vitro for additional 6 h with the CMV-specific pp65 peptide pool and indicated, were used, and a two-tailed p value 0.05 was considered assessed for proliferation via simultaneous CFSE dilution and cytokine statistically significant. production by intracellular cytokine staining. Based on the Boolean gating Online supplementary materials analysis, cytokine coexpression was determined by using SPICE (29). A list of single CMV peptide Ags used for in vitro proliferation experiments ChIP assay and MHC class I dextramer staining is found in Supplemental Table I. A list The ChIP assay was performed by using Zymo-Spin ChIP kit according to of primer sets used in ChIP assay was shown in Supplemental Table II. Supplemental Fig. 1 shows a compound gating strategy leading to the live the manufacturer’s recommendations, with modifications based on pub- + + + lished protocols (30, 31). Briefly, 5–6 3 106 cells were fixed in 1% CMV pp65-specific MHC class I dextramer CD3 CD8 cells and their paraformaldehyde for 10 min at 37˚C. A total of 2.5 M glycine was added phenotypic marker expressions from acute primary and chronic samples. to stop the fixation, and then nuclei were extracted. Chromatin in isolated Supplemental Fig. 2 shows predicted T-bet binding sites using KLRG1 nuclei were digested by Atlantis MNase (Zymo Research) and after di- promoter (Supplemental Fig. 2A) and using B3GAT1 (CD57) promoter gestion, isolated nuclei were mildly sonicated to release digested chro- (Supplemental Fig. 2B). matin. Digested chromatin was then incubated with the ChIP grade mouse mAb against human TBX21 (4B10 and D39) (Santa Cruz Biotechnology), Results or the control mouse IgG Ab (Santa Cruz Biotechnology) for overnight at Patient characteristics and clinical phenotypes during and Downloaded from 4˚C, and after the incubation, the bound chromatin was immunoprecipi- 2 tated by Magna ChIP protein A+G magnetic beads (EMD Millipore). After following primary CMV infection in D+R LTRs series of washes, bound DNA was isolated and stored at 280˚C until PCR + In this study we evaluated the acute CMV-specific CD8 TAEFF was performed with sets of primers (Supplemental Table II) to identity the 2 promoter region of the target gene. All the forward and reverse ChIP phenotype and functional responses in a cohort of 23 D+R LTRs primer pairs except IL-4 pairs were designed by the National Center for during acute primary CMV infection and a subset into chronic Biotechnology Information Primer-BLAST (32). Sequences of a IL-4 ChIP infection. The clinical characteristics of these patients during primer pair were derived from Zhou et.al (33). acute/primary de novo viremia are shown in Table I. Using close http://www.jimmunol.org/ Transcription factor binding site prediction prospective monitoring (see Materials and Methods), we detected primary CMV infection at a median of 165 d posttransplant fol- For prediction of TBX21 DNA binding sites on the KLRG1 promoters, we lowing discontinuation of CMV prophylaxis therapy. Prospective, used a bioconductor R package, TFBSTools (34), with the JASPAR 2018 Transcription Factor DNA binding motif database (35) and scanned through standard-of-care monitoring in this high-risk population continued the promoter region of KLRG1 genomic sequence (ENSG00000139187), and detected relapsing viremia in 10 LTRs (relapsers; 43.4%) 1272 bp upstream of the transcription initiation site (hg38;chr12+:8988175– within the first 6 mo of early chronic infection, in contrast to 13 8989637), and that of B3GAT1 (CD57) genomic sequence (ENSG00000109956), LTRs who demonstrated immune control (controllers; 56.5%) 1429 bp upstream of the transcription start (hg38;chr112:134413347– following discontinuation of antiviral therapy for acute/primary

134411717). We used the default parameter of a searchSeq tool for by guest on September 24, 2021 scanning potential TBX21 DNA binding sites with a minimum score CMV infection. All episodes of relapsing viremia occurred inde- parameter set at 80%. pendent of acute rejection episodes, augmented immunosuppression,

Table I. Patient characteristics and clinical phenotypes following primary CMV infection in D+R2 LTRs

Immunosuppression at Primary Primary CMV Relapsing LTR Age (y) Gender Primary Diagnosis CMV Onset Onseta Viremia 23 50 M Idiopathic pulmonary fibrosis CSA 175/100, RAPA 12, Pred 51 108 2 24 31 F Cystic fibrosis TAC 1.52, MMF 0.52, Pred 101 96 + 25 34 F Primary pulmonary hypertension TAC 42, MMF 0.52, Pred 101 129 2 28 33 F Cystic fibrosis TAC 62, MMF 0.52, Pred 101 210 2 29 62 F COPD TAC 2.52, MMF 0.52, Pred 101 168 + 31 55 M Cystic fibrosis TAC 32, AZA 501, Pred 51 248 2 33 51 F Idiopathic pulmonary fibrosis TAC 22, MMF 0.52, Pred 7.51 186 + 34 59 F COPD TAC 42, MMF 0.252, Pred 101 174 2 35 27 M Cystic fibrosis TAC 12, MMF 0.52, Pred 7.51 219 2 36 49 F Idiopathic pulmonary fibrosis TAC 42, MMF 0.252, Pred 101 167 + 37 56 F Obliterative bronchiolitis TAC 52, MMF 0.52, Pred 101 133 2 38 56 M COPD TAC 12, MMF 12, Pred 101 155 2 40 54 F COPD TAC 1.52, MMF 0.52, Pred 151 122 2 41 64 F Bronchiectasis TAC 42, MMF 0.52, Pred 51 184 2 43 51 M Sarcoidosis TAC 42, MMF 0.52, Pred 151 83 + 45 21 M Cystic fibrosis TAC 2.52, MMF 0.52, Pred 151 92 2 46 59 M COPD TAC 1.52, MMF 12, Pred 201 37 + 48 47 M Idiopathic pulmonary fibrosis TAC 22, MMF 0.252, Pred 101 162 + 51 41 F Pulmonary hypertension TAC 22, MMF 0.52, Pred 7.51 214 2 53 35 F Cystic fibrosis TAC 22, MMF 0.52, Pred 101 125 + 56 61 F COPD TAC 12, MMF 0.52, Pred 101 142 + 57 56 M Idiopathic pulmonary fibrosis TAC 22, MMF 0.252, Pred 101 118 + 60 62 M Idiopathic pulmonary fibrosis TAC 42, MMF 0.252, Pred 101 410 2 Summaryb 48.4(12.5) M 43.4% 160.1 (73.7) + 43.4% aDays posttransplant. bValues represent mean or percent of indicated group (SD). AZA, azathioprine (dose in milligrams); COPD, chronic obstructive pulmonary disease; CSA, cyclosporine (dose in milligrams); F, female; M, male; MMF, mycophenolate mofetil (dose in grams); Pred, prednisone (dose in milligrams); RAPA, rapamycin (dose in milligrams); TAC, tacrolimus (dose in milligrams with superscript times per day). 4 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL or active infections. Additionally, there was no clinical evidence CMV dextramer+ cells acquired the CD57+KLRG1+ phenotype of ganciclovir-resistant CMV in any of the LTR relapsers in this into chronic infection (Fig. 2D). We next wished to assess the cohort. in vitro proliferative capacities of CMV-dextramer+CD8+ T cells; however, staining was consistently poor for dextramer cells at day CD57 and KLRG1 are rapidly induced in CMV-specific + + 6 following restimulation with pooled peptides (data not shown). CD8 TAEFF (CD27 ) during primary infection and undergo 2 Therefore, we used the single HLA-restricted dextramer cognate phenotypic progression to T (CD27 ) into chronic infection EM peptides for restimulation of PBMC to assess the in vitro prolif- We first assessed the dynamics of CD57 and KLRG1 expression, erative capacities at day 6. We previously used this approach to along with other major phenotypic markers, in circulating total assess CMV-tetramer+ effector responses (9). Using this approach, + + CD8 T cell and CMV-specific CD8 TAEFF in our cohort of we assessed the phenotype of proliferating cells during acute D+R2 LTRs during acute/primary viremia. We observed in- (TAEFF) and chronic (TEM) CMV infection in 10 LTRs (Fig. 2E, + + + + creased frequencies of CD57 , KLRG1 , and CD57 CD27 total 2F). Under these stimulatory conditions, KLRG1+, CD57+, and + CD8 T cells during acute/primary CMV infection compared with T-bet+ CMV-dextramer+ cells all robustly proliferated, despite pre-CMV infection (i.e., before the detection of de novo viremia) the loss of CD27 expression in the majority of cells progressing levels determined in a subset of patients (Fig. 1A–D), along from the TAEFF into the TEM stage during chronic CMV infection. with de novo CMV-dextramer responses (Supplemental Fig. 1B, Collectively, these data show an enrichment of CMV-specific Supplemental Table I). We next determined CD57 expression in CD57+KLRG1+CD8+ T cells from primary into chronic CMV relation to KLRG1 and CD27 expression and found a substantial infection. Further, we find the in vitro proliferative capacities of + + enrichment of total CD8 CD57 (Fig. 1E, 1F) and CMV-specific CMV-dextramer+ KLRG1+CD8+ T cells and CD57+CD8+ T cells Downloaded from + + + + CD8 CD57 dextramer TAEFF in the CD27 subset (Fig. 1G, 1H) to be robust in response to dextramer cognate CMV peptides, + 2 during acute infection with evidence of progression to a CD57 CD27 whereas CD57+ cells demonstrated relatively reduced proliferative predominant subset into chronic infection. Similarly, propor- capacities in response to pooled peptides compared with CD572 + + + tions of CD57 KLRG1 cells increased in total CD8 T cells cells, perhaps due to factors such as multiclonal competition and + + + (Fig. 1E, 1F) and CMV-specific CD8 CD57 KLRG1 dex- limited Ag concentration. + tramer TAEFF (Fig. 1G, 1H) from primary into chronic infec- http://www.jimmunol.org/ KLRG1 expression, but not CD57, correlates with tion. We also assessed other phenotypic markers including + CD28, CD38, CD45RA and CD279 (programmed death-1; CMV-specific CD8 TAEFF multifunction PD-1) during primary infection and chronic infection. During A surface phenotypic correlate for effector function has not been primary infection, both total and CMV-specific TAEFF pre- well defined in humans. We previously have shown that induction dominantly expressed a CD27hiCD28+/2CD38+CD279+CD45RA2 of intracellular T-bet during primary CMV infection in D+R2 + TAEFF phenotype (Supplemental Fig. 1C–F) and progressed to a LTRs highly correlates with CMV-specific CD8 T cell effector lo/2 2 2 2 +/hi CD27 CD28 CD38 CD279 CD45RA TEM phenotype in function. Therefore, we next asked whether KLRG1 and/or CD57 chronic infection (Supplemental Fig. 1E, 1G). Together, our results surface expression were predictive of CMV-specific CD8+ effector demonstrate an unanticipated rapid induction of CMV-specific function, compared with T-bet, during acute primary infection by guest on September 24, 2021 + + + + CD8 CD57 TAEFF and CD8 KLRG1 TAEFF during acute/primary (i.e., TAEFF). Using pp65 pooled peptides, we measured ex vivo CMV infection, with a predominant subset of TAEFF that coexpress CMV-specific IFN-g, TNF-a, and CD107a expression by flow these markers in conjunction with CD27. We then find that these cytometric intracellular staining in a 6 h restimulation assay. We effector populations become progressively enriched into chronic in- found that KLRG1+ expression significantly correlated with CMV- fection, bearing a classical TEM phenotype. specific IFN-g, TNF-a, and CD107a TAEFF frequencies, similar to T-bet+ expression in CD8+ T cells (Fig. 3A, 3B). By comparison, CMV-specific KLRG1+ and CD57+ CD8+ T cells are enriched CD57+ expression was an inferior functional correlate for CMV- from primary infection into chronic infection CMV infection specific TAEFF responses and did not reach statistical significance and demonstrate in vitro proliferative capacities + (Fig. 3C). We next assessed CMV-specific CD8 TAEFF multi- + We have recently shown the importance of CMV-specific CD8 function (i.e., the capacity to produce more than one effector T cell proliferation during primary infection for the establishment molecule) using Boolean analysis. We found that KLRG1+CD8+ of immune viral control and upregulation of the major type-1 TAEFF demonstrated significantly increased CMV-specific effec- transcription factor, T-bet, in high-risk LTRs (9). Therefore, we tor multifunction (IFN-g, TNF-a, and CD107a) compared with next addressed changes in CD57 and KLRG1 expression in + KLRG1 CD8 TAEFF (Fig. 3D). Similar findings were observed + CMV-specific CD8 T cells over the course of CMV infection and when we compared T-bet+ versus T-bet2 CMV-specific effector determined their relative in vitro proliferative capacities during responses (Fig. 3E). However, in contrast, CMV-specific TAEFF acute and chronic CMV (12 or more months postprimary) in re- frequencies and multifunction were similar between CD57+ and sponse to CMV peptides. Using an overlapping 15-mer peptide CD572 CD8+ T cells (Fig. 3F). Taken together, our data show that pool of the major CMV Ag, phosphoprotein pp65, we measured KLRG1 expression is a significant functional correlate for both in vitro CMV-specific proliferation by CFSE dilution at 6 d in + individual and multifunctional CMV-specific CD8 TAEFF re- seven LTRs during acute primary CMV infection (Fig. 2A–C). We sponses, similar to T-bet, whereas CD57 expression is a relatively + + found that CMV-specific KLRG1 CD8 T cells from PBMC poor predictor of effector function. proliferated at levels similar to T-bet+CD8+ T cells (Fig. 2A), whereas CMV-specific CD8+CD57+ T cells comprised a signifi- KLRG1 expression correlates with T-bet expression and T-bet cantly reduced population(s) of proliferating cells compared with associates with the human KLRG1 promoter 2 2 KLRG1+ and KLRG1 CD8+ T cells that were CD57 (Fig. 2A–C). Because we found that KLRG1 expression correlated with CMV- + + + We also tracked CMV-dextramer CD8 T cells ex vivo in three specific CD8 TAEFF function, we then investigated whether there donors from acute CMV, during the contraction phase (1–2 mo was a relationship between T-bet and KLRG1 in human T cells. after resolution of viremia) and during chronic CMV infection (at An earlier murine study (25) showed that CD8+ T cells from least 12 mo after primary infection) and found that the majority of T-bet2/2 mice did not express significant surface KLRG1 during The Journal of Immunology 5

FIGURE 1. Rapid Induction of KLRG1+ and CD57+ surface expres- sion in CMV-specific CD8 TAEFF during acute primary CMV infection evolve into TEM during chronic infec- tion. (A)Representativeflowcyto- metric plots show the expression of KLRG1+ (upper panel) and CD57+ (lower panel) in total CD8+ T cells from D+R2 patient during Pre-CMV and acute CMV infection. Total CD8+ T cells were gated on live CD3+ T cells after excluding CD14+, CD16+, and CD19+ cells. (B) Cu- mulative data showing distributions of total KLRG1+CD8+ (left graph) and CD57+CD8+ (right graph) T cells from patients during Pre-CMV and acute CMV infection (n =8).Statis- Downloaded from tical analysis was performed using Wilcoxon matched pair signed rank test. Also, a two-sided Mann–Whitney U test was performed, with p , 0.05 considered statistically significant. (C) Cumulative data shows distributions of total CD3+CD8+ T cells KLRG1+ http://www.jimmunol.org/ and CD57+ for (n = 23) patients dur- ing acute CMV infection. (D)Repre- sentative flow cytometric plots show expression of CD57+ and CD27+ on CD8+ T cells during Pre-CMV (left plot) and acute CMV (right plot). (E) Representative flow cytometric plots showing coexpression of CD57+ and + + CD27 (upper panels) or KLRG1 by guest on September 24, 2021 and CD57+ (lower panels) on the gated population of total CD3+CD8+ T cells ex vivo from D+R2 patient during acute CMV (left plots) and chronic CMV (right plots) infection. (F) Cumulative data graph showing frequency of total CD8+ T cells expressing phenotypic markers as labeled. PBMCs from acute CMV (orange circles) and chronic CMV (green squares) (n = 14 dextramer responses) D+R2 patients were stain- ed ex vivo and analyzed by flow cytometry. (G)Representativeplots show CD57+ and KLRG1+ expression on MHC class I dextramer cells and coexpression of CD57+, and CD27+ or KLRG1+ on the gated population dextramer+ cells CD3+CD8+ Tcells. (H) Cumulative data graph shows proportions of total CD8+ T cells proportions of MHC class I dextramer + cells from acute CMV (n =14dex- tramer responses) (orange circles) and chronic CMV (n = 14 dextramer responses) (green squares) infection, expressing distinct phenotypic markers are shown. 6 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Phenotype and proliferation of CD57+ and/or KLRG1+ CMV-specific CD8+ T cells from primary into chronic CMV infection. (A) Rep- resentative flow cytometry plots from the acute CMV showing phenotype and CMV pp65-specific proliferation in response to pp65 pooled peptides (lower panels) or medium alone (negative control) (upper panels), KLRG1+ (middle panels), and T-bet+ (right panels). (B) Representative flow plot from (A) showing KLRG1+/2 and CD57+/2 phenotypes in CD8+CFSElo subset (right panel). (C) Cumulative data showing CMV pp65-specific proliferation at 6 d for CD8+ T cells with respect to CD57+ (orange bars), CD572 (green bars) of CFSElo events (n = 7). Bars represent median values of CFSElo events for CD57+KLRG12, CD57+KLRG1+, CD572KLRG1+, and CD572KLRG12, respectively. Statistical analysis was performed using a two-sided Mann– Whitney U test, with p , 0.05 considered statistically significant. (D) Graphs showing frequencies of CMV-dextramer (Figure legend continues) The Journal of Immunology 7

LCMV infection, suggesting an important role in regulation. In effector function and phenotypic correlate that differentiates the CD8+ T cells from our cohort, we found a high level of T-bet and capacity of D+R2 LTRs to establish early immune viral control. KLRG1 coexpression in contrast to CD57 expression (Fig. 4A). Total and CMV-specific lung mucosal CD8+ T from BAL Using Spearman rho analysis, we then assessed intracellular T-bet AEFF express reduced levels of KLRG1 and T-bet compared with the and surface KLRG1 expression and found they were significantly blood during acute/primary CMV infection correlated in CD8+ T cells in our cohort during primary CMV + + infection (Fig. 4B), in contrast to CD57. To further demonstrate a In contrast to circulating CD8 T cells, mucosal CD8 resident direct association between T-bet and the KLRG1 promoter, we memory T cells were recently reported to express reduced levels next searched for a putative T-bet binding site within the promoter of T-bet (37, 38). Whereas we have previously demonstrated high region of the KLRG1 gene (Supplemental Fig. 2A, 2C), using a effector function in CMV-specific lung mucosal cells derived from consensus T-bet DNA binding matrix and a transcription factor BAL during acute primary CMV infection (13), little is known binding site analysis tool (TFBS tools) and identified the T-bet about KLRG1 and T-bet expression in lung mucosal CMV-specific + binding site with the highest TFBS score (13.4) comparable to the CD8 TAEFF during acute/primary CMV infection. Therefore, we well-defined T-bet binding site in the IFN-g promoter (TFBS score assessed KLRG and T-bet expression lung mucosal versus blood + = 11.6; data not shown). In contrast, we did not identify putative CMV-specific CD8 TAEFF during acute primary CMV infection in binding sites within the CD57 promoter (Supplemental Fig. 2B). the context of other key phenotypic markers. As shown in Fig. 6, + Based on this analysis, we performed a ChIP assay using DNA both total lung CD8 T cells and CMV-specific CD8 lung TAEFF extracted from PMA/anti-CD3–activated Jurkat T cells, previously express substantially reduced levels of both T-bet and KLRG1 as shown to have rapid upregulation of T-bet with activation (36). As compared with these cells in the blood. In contrast, CD57 and Downloaded from + shown in Fig. 4C, the pulldown fragment of chromatin containing CD27 were similar in total CD8 T cells and CMV-specific + the KLRG1 promoter proximal to the highest likely putative T-bet CD8 TAEFF between these compartments, whereas CD69 and binding site, bound T-bet ChIP chromatin, indicating a direct as- CD103 expression were strikingly increased in the lung compartment. + sociation between T-bet and KLRG1 promoter. Similarly, the IFN- Thus, lung mucosal CMV-specific CD8 TAEFF demonstrated a pre- lo lo +/2 +/2 + + g promoter (positive control) bound T-bet ChIP, whereas the IL-4 dominant KLRG1 T-bet CD103 CD57 CD69 CD27 phenotype + promoter (negative control), GAPDH promoter (housekeeping), or in contrast to systemic CMV-specific CD8 TAEFF which exhib- http://www.jimmunol.org/ + + 2 + 2 + isotype IgG did not bind T-bet ChIP. Taken together, our data iting a predominant KLRG1 T-bet CD103 CD57 CD69 CD27 supports a direct association between T-bet and the KLRG1 pro- phenotype. Together, these data demonstrate significant differ- + moter, but not the CD57 promoter, in human T cells and supports ences in total and CMV-specific CD8 TAEFF in the lung versus the a role for T-bet in the regulation of KLRG1 gene expression. blood compartments during acute/primary CMV infection (Fig. 7). D+R2 LTR controllers demonstrate increased total and + + Discussion CMV-specific acute/primary KLRG1 CD8 TAEFF than those In this study to our knowledge, we demonstrate for the first time of LTR relapsers that two surface phenotypic markers, CD57 and KLRG1, long

Having found a direct association between T-bet and KLRG1, we associated with terminal T cell differentiation and immunose- by guest on September 24, 2021 + next analyzed KLRG1 surface expression in regard to the capacity nescence, are rapidly induced in CMV-specific CD8 TAEFF during for D+R2 LTRs to establish durable early viral control in the first primary infection. Our findings provide what are to our knowledge 6 mo following primary CMV infection. We initially screened 14 novel insights into the phenotypic differentiation and function of + patients for all of the cell surface markers we evaluated in Fig. 1 CMV-specific human CD8 T cells progressing from the TAEFF to + + + and found that only KLRG1 expression differentiated CMV the TEM stage. We show that both CD57 and KLRG1 total and + + + +/hi controllers from relapsers in total CD8 T cells (Fig. 5A). Next, we CMV dextramer CD8 TAEFF are unexpectedly CD27 and compared the frequencies of total KLRG1+ and CD57+CD8+ progress to the well-described CD272 phenotype into the memory T cell frequencies versus T-bet+CD8+ T cell frequencies in our phase. However, despite coexpression with the immature marker, entire D+R2 cohort and found similarly increased levels in con- CD27, our data show that cells which acquire CD57+ demon- trollers for both KLRG1+ and T-bet+ but not CD57+CD8+T cell strate relatively inferior proliferative capacity following in vitro frequencies (Fig. 5B). We then evaluated the frequencies of CMV restimulation using pp65 15-mer pooled peptides, compared with + + pp65-specific KLRG1 CD8 TAEFF producing IFN-g, TNF-a,or cells that express KLRG1 alone or neither marker. These findings CD107a in our cohort and found significantly increased frequen- suggest an unexpected rapid acquisition of senescence in CD57+CD8+ + + + 2 cies of pp65-specific KLRG1 CD8 TAEFF producing these ef- TAEFF, relative to the expansion capacity of KLRG1 CD57 Tcells, fector molecules in LTR controllers compared with relapsers, and under conditions of acute viremia and pooled viral Ag exposure. + + similar to T-bet CD8 TAEFF responses (Fig. 5C, 5D). Last, we Furthermore, our observed proliferative differences among subsets + + 2 + assessed CMV dextramer CD8 TAEFF and found significantly suggest that the CD57 KLRG1 TAEFF population, and likely to a + + + increased surface KLRG1 expression differentiated LTR con- lesser extent, the CD57 KLRG1 TAEFF population, give rise to the + + trollers from relapsers compared with other TAEFF surface phe- predominant CD57 KLRG1 TEM population over time. Importantly, notypic markers (Fig. 5E, 5F). Collectively, our data demonstrate however, we also observed robust intrinsic proliferative capacities in + + + + that acute/primary surface KLRG1 CD8 TAEFF expression is an CMV-specific CD57 dextramer TAEFF and TEM when restimulated

CD8+ T cells and KLRG1+/2 CD57+/2 subsets in three patients during acute CMV, contraction phase, and chronic CMV infection. Samples for the contraction phase were obtained 1–2 mo following clearance of acute viremia and chronic CMV at least 1 y after acute CMV. (E) Representative flow cytometry plots of CMV dextramer proliferation in response to cognate peptide restimulation or medium measured at 6 d using CFSE dilution. Shown are responses during acute CMV and chronic CMV and phenotypic markers CD57+ (upper left), CD27+ (upper right), KLRG1+ (lower left), and T-bet+ (lower right) plots. (F) Cumulative data from (E) showing respective phenotypes of CFSElo events in dextramer+ cells during acute CMV (orange bars) and chronic CMV (green bars). Bars represent median values of CFSElo frequencies. Statistical analysis performed using a two-sided Mann–Whitney U test, with p , 0.05 considered statistically significant. 8 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. CMV pp65-specific CD8+ effector responses reciprocally correlate with T-bet and KLRG1 expression but not with CD57 and CD8+KLRG1+ effectors have higher quantity and quality of multifunction during acute primary CMV infection similar to CD8+T-bet+ (Figure legend continues) The Journal of Immunology 9

FIGURE 4. T-bet expression significantly correlates with KLRG1 in CD8+ T cells during primary CMV infection and T-bet interacts with the KLRG1 promoter in human T cells. (A) Representative flow cytometric plots showing coexpression of T-bet+ and KLRG1+ or CD57+ gated on live CD3+CD8+ Downloaded from T cells. This is a representative of n = 23 individuals. (B) Cumulative correlation data (n = 23) by scatter plot analysis of T-bet+ and CD8+CD57+ (left panel), and CD8+KLRG1+ (right panel), frequencies during acute CMV infection from the LTR cohort. A black line indicates the regression line as well as R and p values that were generated using the Spearman rank correlation test. (C) Agarose gel images showing bands of semiquantitative PCR amplicons that reside in the promoter sequence of T-bet targets, KLRG1, CD57 (B3GAT1), and IFN-g (positive control) as performed by ChIP assay. Also shown are non– T-bet targets, IL-4 (negative control), and GAPDH (negative control) genes. ChIP assay was performed using human Jurkat T cell line using mouse anti- human TBX21 Ab (clone 39D; IgG1). Immunoprecipitated DNA was subject to PCR amplification using primer sets for KLRG1, CD57(B3GAT1), IFN-g,

IL-4, and GAPDH targets (lane 1) for each corresponding region of promoter sequences in proximity to the T-bet binding site for each gene. Results of PCR http://www.jimmunol.org/ amplifications of input DNA (lane 3) or mouse IgG1 isotype control Ab (lane 5) are also shown. Lanes 2, 4, and 6 show nontemplate PCR negative controls.

in vitro with single 8-mer cognate peptides. Although this was phenotype that progresses to a mature TEM phenotype in chronic somewhat unexpected, it further supports the capacity for self- CMV infection. renewal in these cells under more optimal stimulatory conditions We and others have demonstrated the correlation between T-bet (i.e., higher Ag concentration, reduced multiclonal competition that expression and CD8+ effector function playing an important role is present following restimulation with peptide pool). Notably, we in CMV, HIV, hepatitis B virus, and hepatitis C virus immune previously reported robust effector responses in response to single control (9, 40–42). We sought to determine whether either CD57 cognate peptide in CMV-tetramer+ CD8+ T cells (9). Overall, our and/or KLRG1 represented a functional immune correlate and by guest on September 24, 2021 + findings demonstrate a relative, but not absolute, proliferative found KLRG1 expression in CMV-specific CD8 TAEFF to be a defect in CD8+CD57+ T cells as reported in an earlier study by superior immune correlate for IFN-g, TNF-a, and CD107a ex- Brenchley et al. (21). However, there are important experimental pression and effector multifunction, similar to T-bet expression, differences between these studies, including our assessment of when compared with CD57. Moreover, we compared KLRG1+ + + + + CD57 TAEFF during primary CMV infection, our use of total and CD57 surface expression to T-bet and found that KLRG1 PBMC for restimulation versus sorted CD8+CD57+ T cells, and frequencies, similar to T-bet+ frequencies, differentiated CMV our use of single-peptide restimulation to study CMV-specific controller versus relapser clinical phenotypes in our D+R2 LTR dextramer+ cells. Conversely, our findings demonstrate substan- cohort, as well as effector molecule expression. Importantly, we + + tial proliferative capacities in CMV-specific CD8 KLRG1 TAEFF, previously have shown in a similar D+R2 LTR cohort that the unlike previous studies (20, 39) of CD8+KLRG1+ T cells, albeit frequencies of CMV-specific tetramer+ cells did not differentiate with similar experimental differences as highlighted above. controller versus relapse phenotypes, but rather the function of Our findings also indicate that the proliferative capacities of these tetramer+ cells, using single cognate peptide restimulation + + + + CMV-specific CD8 KLRG1 TAEFF more closely approximates (9). In this study, when we compared KLRG1 and CD57 fre- + + that of CMV-specific CD8 T-bet TAEFF, with a subset of quencies to other surface phenotypic markers expressed in total + + + + expanding KLRG1 TAEFF coexpressing CD57. Taken together, CD8 and CMV dextramer TAEFF, only KLRG1 expression our findings demonstrate rapid induction of CD57 and/or significantly differentiated the CMV controller versus relapser + + KLRG1intotalandCMV-specificCD8 TAEFF with differen- phenotypes. Collectively, our data point to KLRG1 surface ex- + tial proliferative capacities in conjunction with an immature pression in the blood CD8 TAEFF pool as a surrogate for T-bet,

effectors. (A) Cumulative correlation data (n = 23) by scatter plot analysis of KLRG1+, T-bet+ (B) and CD57+ (C), frequencies and CMV-specific pp65 frequencies for CD8+IFN-g+ (left panels), TNF-a CD8+TNF-a (middle panels), and CD8+CD107a+ (right panels) during acute CMV infection from the LTR cohort. Correlation coefficient (R) and p values were calculated using Spearman rank correlation test. (D) Individual pie charts of PBMC reflecting CD8+KLRG1+ (left pie) and CD8+KLRG12 (right pie) of T cell effector multifunction (E) CD8+T-bet+ (left pie) and CD8+T-bet2 (right pie) of CMV pp65– specific T cell effector multifunction and (F) CD8+CD57+ (left pie) and CD8+CD572 (right pie) of CMV pp65–specific T cell during acute primary CMV infection responses (IFN-g, TNF-a, CD107a) from the LTR cohort effector multifunction. Using Boolean analysis, the percentage of total and individual effector multifunctional subset responses for CD8+ T cells from KLRG1+ (gray bars) and KLRG12 (black bars) or T-bet+ (gray bars) and T-bet2 (black bars) or CD57+ (gray bars) and CD572 (black bars) are shown in the bar graph for each of the multifunctional subsets. *p , 0.05. All p values were determined by the Kruskal–Wallis one-way ANOVA or Wilcoxon signed rank test. Data analyzed using the program SPICE. *Significant differences when comparing mean frequencies of single and multifunctional responses, p # 0.05. 10 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL Downloaded from

FIGURE 5. Significantly higher frequencies of KLRG1+ total CD8+ and CMV-specific CD8+ T cells are found in viremic controllers compared http://www.jimmunol.org/ with relapsers. (A) Cumulative distributions of KLRG1 frequency and additional six phenotypic markers expressed on the total CD8+ T cell among controllers (blue circles) (n = 7) and the relapsers (red squares) (n = 8) are shown (mean 6 SEM). Only the KLRG1 expression exhibits the significant difference (p , 0.002) between controllers and relapser as indicated. (B) Cumulative data shows frequency of KLRG1+, T-bet+, or CD57+ total CD8+ T cells among controllers (blue columns) (n = 13) and relapsers (red columns) (n = 10). (C) Cumulative data shows Tbet+CD8+ frequencies and (D) KLRG1+CD8+ effector functions (IFN-g+, TNF-a+, CD107a+) in controllers (blue columns) and relapsers (red columns). (E) Representative flow cytometric plot shows KLRG1+ expression on CMV-specific dextramer+CD8+ T cells. Numbers show proportion of KLRG1+ subset within the gated dextramer+CD8+ T cell in a controller (left panel) versus a relapser (right panel). (F) Cumulative distributions of KLRG1+ frequency and additional six phenotypic markers expressed on the Dextramer+CD8+ T cell among controllers (blue circles) (n = 8 dextramer responses assed) and the relapsers

(red squares) (n = 7 dextramer responses assed) are shown (mean 6 SEM). Statistical analysis performed using a two-sided Mann–Whitney U test, with by guest on September 24, 2021 p , 0.05 considered statistically significant between the controller and the relapser throughout the course of statistical analyses. effector function, and the capacity for early CMV control in high- sought to determine its molecular regulation compared with CD57 risk D+R2 LTRs. (B3GAT1). Only the KLRG1 promoter demonstrated high putative Because we observed a high level of correlation between binding site scores for T-bet, in contrast to CD57 (B3GAT1), and KLRG1+ and T-bet+ expression in both proliferation and effector we expectedly found coprecipitation of T-bet and KLRG1 by ChIP function of CD8+ T cells during acute/primary CMV infection, we assay, similar to IFN-g, whereas we did not detect any binding to

FIGURE 6. CMV-specific dextramer+CD8+ T cells in BAL express lower levels of KLRG1 and T-bet transcription factor than PBMC. (A) Representative flow cytometry plots of CMV dextramer+CD8+ T cells BAL cells (upper panels) and blood (lower panels) during acute/primary CMV infection (n = 6) for a panel of six markers: KLRG1+, T-bet+, CD27+, CD103+, CD69+, and CD57+ plots. (B) A cumulative data of summary of phenotypic frequency analysis of (A) showing BAL cells (orange bars) and PBMC (green bars). Statistical analysis performed using a two-sided Mann–Whitney U test, with p , 0.05 considered sta- tistically significant between the BAL and blood compartments. The Journal of Immunology 11

CD57 (B3GAT1). Although these data support direct regulation of consistent with it being a surrogate for T-bet expression. Indeed, KLRG1 by T-bet, they also highlight a distinct role for CD57 in our results also show that KLRG1 expression is reduced in lung + T cell differentiation. In fact, the precise functional role of CD57, mucosal CD8 TAEFF expressing lower levels of T-bet, as re- which does not have a counterpart in the mouse, remains in- cently reported in the mouse (49), providing further ex vivo completely understood. Although our studies support CD57 as an evidence of regulation by this transcription factor. Collectively, + early marker of senescence in CD8 TAEFF, alternative functions our data provide, to our knowledge, new insights into the ki- have been reported. As CD57 is a glycoepitope, several studies netics of KLRG1 expression, its regulation, and the functionality have suggested a role for tissue adhesion, including it being a in human T cells in the context of acute primary into chronic ligand for laminin (43) and L- and P-selectins (44). Thus, CD57 CMV infection. surface expression could mark, via cellular adhesion, terminally There are several caveats to our study. Although our studies differentiated cells with broad tissue migratory licensure that are focused on T cell responses to pp65, we acknowledge that the total important for CD8+ immune surveillance. effector response is significantly broader, including CD4+ T cells To our knowledge, our study provides new insights into the role (50). However, because our earlier study found that CMV pp65- + of KLRG1 expression in CMV-specific TAEFF and TEM. Similar to specific CD8 effector responses were greater than IE1-specific CD57, KLRG1 has been associated with terminal differentiation CD8+ effector responses during acute primary infection, we fo- and immune senescence in human T cells, with the observation of cused on pp65 responses (12). We also recognize that our study accumulation of KLRG1+CD8+ T cells with age (27, 45) and prior size is somewhat small; however, capturing D+R2 LTRs during studies having demonstrated that sorted KLRG1+CD8+ T cells primary CMV infection and prospectively assessing their clinical exhibited replicative senescence in response to mitogen stimula- phenotype in addition to collecting serial blood samples is chal- Downloaded from tion (20, 46, 47). KLRG1 has also been shown to be a ligand lenging. Nonetheless, our study demonstrated statistically signif- for the cadherins expressed on epithelial cells, suggesting an im- icant findings in our study of CD57 and KLRG1 in TAEFF and TEM portant role for effector trafficking, cross-talk, and a potential over the course of CMV infection that advance our understand- mechanism to limit cytotoxicity (45, 48). Importantly, our studies ing of T cell differentiation. Future studies will test whether + + show that both CMV-specific CD8 KLRG1 TAEFF and TEM ro- measurement of KLRG1 expression, in particular, is useful to

bustly proliferate during acute primary and chronic infection differentiate the capacity for viral control and determining clinical http://www.jimmunol.org/ similar to T-bet+ cells, the transcription factor that binds the phenotypes in LTRs at high-risk for recurrent CMV infection, KLRG1 promoter, indicating that KLRG1+ expression alone, does which may be useful in guiding clinical antiviral decision-making. not necessarily indicate senescence. However, our studies do In summary, our study provides, to our knowledge, new insights support the concept that the acquisition of CD57 coexpression is into roles of KLRG1 and CD57 in the function and differentiation associated with cells that demonstrate reduced proliferative ca- of human T cells from the TAEFF to the TEM stage during CMV pacity in response to CMV pooled peptides. Interestingly, our infection. Based on these findings, we propose a revised model for findings in the human differ from the mouse studies with persis- T cell differentiation that incorporates these markers and relates tence of KLRG1 expression into the memory phase, although phenotype with function (Fig. 7). We show that rapid induction of the LCMV infection mouse model used by Joshi et al. was not KLRG1, but not CD57, in CMV-specific CD8+ T cells differen- by guest on September 24, 2021 the chronic LCMV model. Thus, we propose a revised model tiates the capacity for effector function and viral control. Fur- of human T cell differentiation in the human, in which early thermore, we provide evidence that KLRG1, but not CD57, is + +/2 + KLRG1 CD57 CD27 TAEFF more likely reflect the previously regulated by, and a surrogate for, T-bet in human T-broad mi- described mouse memory precursor effector cells rather than gratory capacities via tissue adhesion that are important for CD8+ short-lived effector cells and establish KLRG1+CD57+CD272 immune surveillance. Additionally, another study by Cebo et al. TEM population in the context of chronic CMV infection (Fig. 7). (51). suggested a potential role for CD57 in IL-6R signaling. We should also point out that, similar to earlier mouse studies, Collectively, these alternate functions are consistent with our KLRG1 expression marks viral-specific TAEFF with potent multi- findings of rapid induction of CD57 during acute primary infec- function and strongly correlates with the capacity to establish viral tion, its similar expression on lung mucosal CMV-specific TAEFF control. The correlation of KLRG1 expression with effector compared with the blood, and its progressive enrichment into function that we observed in peripheral blood TAEFF is also chronic infection.

FIGURE 7. CMV-specific CD8+ T effector cell differentiation model. A figure depicts our simplified linear model of the CMV-specific CD8+ effector T cell differentiation. According to the model, Ag primed CMV-specific CD8+ naive T cells (blue circle) undergo extensive clonal expansion and concurrent effector differentiation upon viral Ag encounter during CMV primary/acute viremia. An expanded pool of TAEFF subsets, including a predominant + + + + + KLRG1 CD27 CD57 T-bet subset (green circle) are generated. TAEFF, particularly KLRG1 TAEFF is central to the antiviral cell immunity during primary/ + 2 + acute infection and the subsequent formation of KLRG1 CD27 CD57 TEM (red circle) in the chronic phase. These TAEFF, despite expressing two effector markers, KLRG1 and CD57, exhibit differential proliferative capacities (middle panel) with increasing expression of KLRG1 and CD57, and declining expression of CD27 over the course of TEM differentiation. Although each phases of infection is represented by a few dominant subsets, in reality such stages likely made up of multiple subsets albeit low frequencies. 12 CMV KLRG1+CD8+ T CELLS PREDICT FUNCTION AND VIRAL CONTROL

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