Differential Expression of CD8+ T Cell Cytotoxic Effector Molecules in Blood and Gastrointestinal Mucosa in HIV-1 Infection This information is current as Brenna E. Kiniry, Peter W. Hunt, Frederick M. Hecht, Ma of October 3, 2021. Somsouk, Steven G. Deeks and Barbara L. Shacklett J Immunol published online 19 January 2018 http://www.jimmunol.org/content/early/2018/01/19/jimmun ol.1701532 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/01/19/jimmunol.170153 Material 2.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 January 19, 2018, doi:10.4049/jimmunol.1701532 The Journal of Immunology Differential Expression of CD8+ T Cell Cytotoxic Effector Molecules in Blood and Gastrointestinal Mucosa in HIV-1 Infection Brenna E. Kiniry,* Peter W. Hunt,† Frederick M. Hecht,‡ Ma Somsouk,x Steven G. Deeks,‡ and Barbara L. Shacklett*,{ We previously reported that CD8+ T cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak or impaired cytotoxic capacity compared with their counterparts in blood. Nevertheless, these cells degranulate and express cyto- kines and chemokines in response to cognate Ag. In addition to weak expression of perforin, earlier studies suggested differential regulation of perforin and granzymes (Gzms), with GzmA and B expressed by significantly higher percentages of mucosal CD8+ T cells than perforin. However, this topic has not been fully explored. The goal of this study was to elucidate the expression and coexpression patterns of GzmA, B, and K in conjunction with perforin in rectosigmoid CD8+ T cells during HIV-1 infection. We Downloaded from found that expression of both perforin and GzmB, but not GzmA or GzmK, was reduced in mucosa compared with blood. A large fraction of rectosigmoid CD8+ T cells either did not express Gzms or were single-positive for GzmA. Rectosigmoid CD8+ T cells appeared skewed toward cytokine production rather than cytotoxic responses, with cells expressing multiple cytokines and chemokines generally lacking in perforin and Gzm expression. These data support the interpretation that perforin and Gzms are differentially regulated, and display distinct expression patterns in blood and rectosigmoid T cells. These studies may help inform the development of strategies to combat HIV-1 and other mucosal pathogens. The Journal of Immunology, 2018, 200: http://www.jimmunol.org/ 000–000. ucosal tissues are primary targets for HIV (HIV-1) CD8+ T cells exhibit strikingly low perforin expression and re- infection, and the gastrointestinal mucosa is a major duced cytotoxicity compared with blood T cells, suggesting CD8+ M site of virus transmission and pathogenesis. Accord- T cells housed in the gut are primed for cytokine release rather ingly, understanding mucosal immune responses to HIV-1 is im- than cytotoxicity (11). portant for the development of strategies to fight infection. CD8+ Despite significantly lower perforin levels in the gut compared with + T cells are thought to play a crucial role in containing HIV-1 in- blood, HIV-1 individuals not on antiretroviral therapy (ART) display by guest on October 3, 2021 fection, as depletion of these cells results in viral rebound (1, 2). greater proportions of perforin and granzyme (Gzm) B–positive However, the specific effector functions by which CD8+ T cells CD8+ T cells in gastrointestinal mucosa compared with those on mediate viral containment remain unclear. In blood, both perforin- ART and HIV-1–negative adults (10–12). Whether this indicates mediated cytotoxicity and polyfunctionality, defined as the si- expression of cytotoxic effector proteins by polyfunctional HIV-1– multaneous production of multiple cytokines and chemokines, specific CD8+ T cells, the emergence of a cytotoxic HIV-1–specific have been identified as possible correlates of immune-mediated effector T (TEFF) cell subset, and/or bystander activation-induced protection from disease progression (3–9). CD8+ T cell poly- perforin and GzmB expression in non–HIV-1–specific CD8+ functionality, including robust production of macrophage inflam- T cells in the mucosa during chronic HIV-1 infection is unknown. matory protein (MIP)-1b, has also been identified as a potential Interestingly, despite low perforin expression, gastrointestinal correlate of protection in mucosa (10). However, gastrointestinal CD8+ T cells express GzmB, albeit at lower levels compared with *Department of Medical Microbiology and Immunology, School of Medicine, Uni- the University of California San Francisco–Gladstone Center for AIDS Research versity of California Davis, Davis, CA 95616; †Division of Experimental Medicine, (P30 AI027763). Additional support was provided by the Delaney AIDS Research Department of Medicine, Zuckerberg San Francisco General Hospital and Trauma Enterprise (AI096109 and AI126611) and the American Foundation for AIDS Center, University of California San Francisco, San Francisco, CA 94110; Research Institute for HIV Cure Research (109301). ‡Positive Health Program, Department of Medicine, Zuckerberg San Francisco Gen- B.L.S. conceived and directed the project; M.S., P.W.H., F.M.H., and S.G.D. char- eral Hospital and Trauma Center, University of California San Francisco, San Fran- x acterized the participant cohort and procured clinical samples; B.E.K. conducted cisco, CA 94110; Division of Gastroenterology, Department of Medicine, laboratory assays and data analysis; B.E.K. and B.L.S. prepared, wrote, and edited Zuckerberg San Francisco General Hospital and Trauma Center, University { the manuscript; all authors read and approved the manuscript. of California San Francisco, San Francisco, CA 94110; and Division of In- fectious Diseases, Department of Medicine, School of Medicine, University of Address correspondence and reprint requests to Dr. Barbara L. Shacklett, Depart- California Davis, Davis, CA 95616 ment of Medical Microbiology and Immunology, University of California Davis, School of Medicine, 3146 Tupper Hall, Davis, CA 95616. E-mail address: ORCIDs: 0000-0001-6371-747X (S.G.D.); 0000-0002-7067-732X (B.L.S.). [email protected] Received for publication November 6, 2017. Accepted for publication December 15, The online version of this article contains supplemental material. 2017. Abbreviations used in this article: ART, antiretroviral therapy; C, controller; FSC, This work was supported by National Institutes of Health/National Institute of Al- forward scatter; Gzm, granzyme; MIP, macrophage inflammatory protein; SEB, lergy and Infectious Diseases Grant R01 AI057020. The University of California Staphylococcal enterotoxin B; SN, seronegative; S1PR1, sphingosine-1-phosphate Davis Flow Cytometry Shared Resource Laboratory was supported by grants from receptor; T , effector T; T , effector memory; T , transitional memory; Tx, the National Institutes of Health National Cancer Institute (P30 CA0933730), EFF EM TM treated; V, viremic untreated; VL, viral load. the National Institutes of Health National Center for Research Resources (C06- RR12088, S10-RR12964, S10-RR026825, and S10-OD018223-01A1), and Ó the James B. Pendleton Charitable Trust. The SCOPE cohort was supported by Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701532 2 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 blood, and display ample degranulation, measured by expression Pharmingen (San Diego, CA). Perforin (B-D48: PE) was from Cell Sci- of CD107, suggesting that these mucosal CD8+ T cells may pro- ences (Canton, MA). This perforin Ab detects de novo as well as pre- duce and release cytotoxic granules containing little or no perforin formed perforin and is suitable for monitoring perforin expression following TCR stimulation (21). The HIV-1 Gag peptide pool (Consensus (11, 13). Numerous in vitro and mouse in vivo studies have Clade B) consisted of 123 15-mers overlapping by 11 residues (JPT In- demonstrated noncytolytic, extracellular functions of GzmA, B, novative Peptide Solutions, Berlin, Germany). Staphylococcal enterotoxin and K, including extracellular matrix degradation and the direct B (SEB) was from Sigma-Aldrich. and indirect processing and release of proinflammatory cytokines Ag stimulation and intracellular cytokine staining (14–17). The concept of Gzms functioning in a noncytolytic manner to elicit a proinflammatory state appears consistent with Staining was performed as previously described with slight modifications (12) + on ex vivo blood and mucosal mononuclear cells rested overnight at 37˚C, the strong propensity of gut CD8 T cells to produce chemokines 6 5% CO2. For stimulation assays, cells were incubated at 2 3