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 25, 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 Supplementary http://www.jimmunol.org/content/suppl/2019/09/20/jimmunol.190039 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 25, 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.