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

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 (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 10 cells per and cytokines in response to antigenic stimulation. Accordingly, 200 ml R-15 for 5.5 h in the presence of anti-CD28 (1 mg/ml) and anti- the major goal of this study was to more fully explore the ex- CD49d (1 mg/ml), anti-CD107a, 1 mM GolgiStop (BD Biosciences), pression and coexpression patterns of GzmA, B, and K in parallel brefeldin A (5 mg/ml; Sigma-Aldrich), and the appropriate antigenic stim- m with perforin in HIV-1–specific and non–HIV-1–specific CD8+ ulation: Gag-peptide pool (final concentration 3.5 g/ml of each peptide), or medium containing an equivalent amount of DMSO (peptide solvent) as the T cells in rectosigmoid mucosa during chronic HIV-1 infection. negative control. SEB (5 mg/ml) was used as positive control. Following incubation, cells were stained for surface markers and viability (Aqua Live/ Dead cell stain kit; Invitrogen) for 20 min at room temperature. Cells were Materials and Methods then fixed in 4% paraformaldehyde and permeabilized using FACS Perm 2 Participants and sample collection (BD Biosciences) prior to intracellular staining for CD3, cytotoxic effectors,

HIV-1 positive and seronegative (SN) participants were enrolled through the chemokines, and cytokines. Staining for cytotoxic effectors following anti- Downloaded from SCOPE study based at San Francisco General Hospital. Written informed genic stimulation used staining protocols and Ab clones previously dem- consent for phlebotomy and rectosigmoid biopsy was obtained through onstrated to reveal perforin and GzmB expression following TCR protocols approved by the Committee on Human Subjects Research, stimulation (21). Cells were resuspended in 1% paraformaldehyde and stored University of California, San Francisco (protocols 10-01218, 10-00263, at 4˚C in the dark no longer than 24 h until analysis. and 10-01330). Participants were classified in four groups defined by Data acquisition and analysis plasma viral load (VL) and ART as follows: controllers (C, n = 10) con- sistently maintained VL ,2000 copies per ml without ART; viremic un- An LSR II flow cytometer and FACSDiva software (Becton Dickinson http://www.jimmunol.org/ treated (V, n = 15) subjects maintained VL $2000 copies per ml without Immuno-Cytometry Systems) were used to collect flow cytometry data. ART; ART-treated (Tx, n = 13) participants had VL ,40 copies per ml; Flowjo software (Flowjo, Ashland, OR) was used to perform initial flow and SN (n = 10) controls were negative for HIV-1. SN participants were cytometric analysis and SPICE was used to visualize multifunctional T cell healthy volunteers from whom samples were collected for research pur- populations (22). CD8+ T cells were identified using the following gating poses only. Exclusion criteria included active sexually transmitted infec- strategy: lymphocytes [side scatter -A versus forward scatter (FSC)-A] → tions other than HIV-1; inflammatory bowel disease or other known singlets (FSC-H versus FSC-A) → live, CD3+ (CD3 versus viability) → inflammatory conditions affecting the gastrointestinal tract; colorectal CD8+, CD4Neg (CD8 versus CD4). Memory subsets within the CD8+ T cell cancer or other malignancies; and severe anemia or blood clotting disor- population were identified by first establishing positive gates for each ders. Additional demographic information (gender, race/ethnicity, age, VL, memory marker within the live, CD3+, CD4Neg, CD8+ T cell population CD4 count, and time since diagnosis) is supplied in Table I. In total, 20–40 followed by Boolean gating to define memory subsets as follows: naive 2 + + + + + ml of blood was collected by sterile venipuncture into tubes containing (CD45RO CD27 CCR7 ), central memory (CD45RO CD27 CCR7 ), by guest on October 3, 2021 + + 2 EDTA. Approximately 30 rectosigmoid biopsies were obtained by flexible transitional memory (TTM CD45RO CD27 CCR7 ), effector memory + 2 2 2 2 2 sigmoidoscopy 10–30 cm from the anal verge (18). Biopsies were col- (TEM CD45RO CD27 CCR7 ), and TEFF (CD45RO CD27 CCR7 ) lected in 50 ml conical tubes containing 15 ml of R-15 medium [RPMI (23). Polyfunctional cells and cells coexpressing cytotoxic effector mole- 1640 supplemented with FCS (15%), penicillin (100 U/ml), streptomycin cules were identified by first establishing positive gates for each response (100 mg/ml), and glutamine (2 mM)]. Blood and biopsy samples were analyte and/or cytotoxic effector molecules within live, CD3+, CD4Neg, transported at room temperature to the laboratory and processed imme- CD8+ T cell populations, followed by Boolean gating to define subsets diately upon arrival. coexpressing these effector molecules. We then used Boolean gating to define memory marker expression within each cytotoxic effector subset. Blood and tissue processing An in-house statistical algorithm was used to determine whether Ag- Ficoll-Paque (Pfizer-Pharmacia, New York, NY) was used to isolate PBMCs specific responses differed significantly from the negative control (12). from blood. Rectosigmoid mucosal mononuclear cells were liberated from For responses found to be statistically significant, net responses were then biopsies using enzymatic and mechanical disruption as previously described calculated by subtracting the negative control values from Ag-specific (19, 20). Briefly, biopsies underwent shaking incubation at 37˚C for 30 min responses. Statistical analyses were performed with GraphPad Prism V.6 in 25 mg/ml Liberase DL (Roche, Indianapolis, IN), followed by passage (GraphPad Software, San Diego, CA) or SPICE using nonparametric sta- through a 16-gauge blunt-end needle to disrupt tissue. Free cells were tistical test as follows: Wilcoxon matched-pairs signed rank test for paired U collected through a sterile 70 mm cell strainer. The disruption process was samples, Mann–Whitney test for unpaired samples, and Spearman repeated until all biopsy tissue was digested. Free rectosigmoid mucosal correlation and linear regression analysis for correlative analysis. mononuclear cells were washed three times in 20 ml of R-15. All isolated cells were rested overnight at 37˚C, 5% CO2 in R-15 supplemented with Results 2003 Zosyn (Pfizer-Pharmacia). Ex vivo expression of cytotoxic effector molecules differs Abs and peptide pools between blood and gut and across HIV-1 disease status The following fluorochrome-labeled monoclonal Abs were used in flow Expression of perforin, and to a lesser extent GzmB, is reduced in cytometry: CD107a (H4A3: PE-Cy5, PE-Cy7), CD8 (SK1: APC-H7, resting and stimulated CD8+ T cells in gastrointestinal mucosa FITC), CCR7 (3D12: PE-Cy7), GzmB (GB11: V450, PE-CF594), MIP- compared with blood, but whether this trend extends to other 1b (D21-1351: APC-H7, PerCP-Cy5.5), IFN-g (B27: PE-Cy7), TNF-a Gzms, or to their coexpression, has not been fully explored (13). It (Mab11: Ax700), CD69 (L78: PE) from BD Biosciences (San Jose, CA); GzmK (GM6C3: FITC) from Santa Cruz Biotechnology (Dallas, TX); is also unclear whether coexpression of these proteins varies with CD4 (T4D11: ECD) from Beckman Coulter (Brea, CA); CD4 (RPA-TA: HIV-1 disease status. To address these questions, we compared BV785), CD45RO (UCHL1: BV785), CD27 (O323: BV650), CD103 (Ber- intracellular expression of GzmA, B, K and perforin in resting ACT8: Ax488), GzmA (CB9: Ax647, PacBlue), and CD3 (UCHT-1: CD8+ T cells freshly isolated from blood and rectosigmoid mu- Ax700) from BioLegend (San Diego, CA). CD3 (S4.1: Qdot655) was cosal biopsies of participants in the following groups: HIV-1+ purchased from Invitrogen (Carlsbad, CA). Sphingosine-1-phosphate re- + + ceptor (S1PR1) (SW4GYPP: eFluor660) was purchased from eBioscience controllers (C); HIV-1 viremic (V) individuals; HIV-1 individ- (San Diego, CA); unlabeled CD28 (L293) and CD49d (L25) were from BD uals on ART (Tx); and SN controls (Table I). The Journal of Immunology 3

Table I. Participant characteristics

Plasma VL, RNA Copies per mla CD4 Count, Cells per mm3 Time Post–HIV+ Diagnosis, y Race/ (Median) (Median) (Median) Gender Ethnicity (Range) (Range) (Range) C(n = 10) M, 5 AA, 7 844 716 20.5 F, 3 C, 2 ,40–1727 353–2542 4–29.8 T, 2 Mx, 1 V untreated (n = 15) M, 15 AA, 7 20,544 506 2.75 C, 4 393–186,307 236–1075 1–22.67 HL, 3 Mx, 1 ART-Tx (n = 13) M, 11 AA, 3 ,40 603 18 F, 1 C, 7 NA 193–811 2.7–27.1 T, 1 HL, 3 SN (n = 10)b M, 10 AA, 1 NA 950 NA C, 6 NA 600–1211 NA HL, 2 Mx, 1 aMedian calculated using only detectable VL data points. bCD4 count unavailable for one subject. AA, African American; C, Caucasian; F, female; HL, Hispanic/Latino; M, male; Mx, mixed; NA, not applicable; T, transgender (M to F). Downloaded from

GzmB, GzmK, and perforin all displayed bimodal expression mucosa. In blood, HIV-1–specific CD8+ T cells have been pre- patterns with distinctive positive and negative populations (Fig. 1A). viously characterized as Perforin2/+ GzmB+ GzmA+ GzmK+ GzmA displayed a trimodal expression pattern, with a small frac- (24). As expression patterns of cytotoxic effectors were elevated tion of CD8+ T cells identified as GzmABright, expressing GzmA at in blood CD8+ TcellsofHIV-1+ individuals compared with SNs, high fluorescence intensity, and a large subset displaying interme- we predicted that their coexpression would likewise be elevated http://www.jimmunol.org/ diate intensity, designated as GzmAInt (Fig. 1A). The GzmABright in the mucosa of HIV-1–infected compared with healthy indi- population was particularly prominent in mucosa. Irrespective of viduals. To test this prediction, we used flow cytometry and HIV-1 disease status, GzmA was the most abundantly expressed SPICE software to analyze coexpression of cytotoxic effectors in cytotoxic effector protein in both blood and mucosa, whereas per- unstimulated CD8+ T cells from rectosigmoid mucosa and blood forin was the rarest (Fig. 1A, 1B). In agreement with our previous of chronically HIV-1–infected and SN adults (Fig. 2). This work, the proportion of CD8+ T cells expressing GzmB and perforin dataset captured information from all CD8+ T cells, regardless of was greater in blood compared with mucosa in both SN and HIV-1+ specificity. participants (11, 13). In contrast, the relative abundance of GzmAInt Irrespective of HIV-1 disease status, we observed significant by guest on October 3, 2021 cells in blood and gut differed between HIV-1+ and SN participants. differences in the percentages of CD8+ T cells coexpressing certain In HIV-1+ individuals, the proportion of GzmAInt CD8+ T cells was cytotoxic effectors between blood and mucosa, most notably in similar in blood and mucosa; in SNs, it was significantly greater in the proportions of Perforin+GzmAIntGzmB+ and GzmABright CD8+ mucosa. No significant differences were detected between blood T cells. Although in both tissues, perforin was typically coex- and gut in the proportion of GzmK+ CD8+ T cells in either HIV-1+ pressed with GzmAInt and GzmB, the percentage of Perforin+ or SN individuals (Fig. 1B). GzmAIntGzmB+ CD8+ T cells was significantly higher in blood Expression of cytotoxic effectors varied across HIV-1 disease compared with mucosa (Fig. 2A, 2B). This putative cytotoxic Bright status. With the exception of GzmA cells, which were most subset was one of the most abundant in blood (BloodHIV+ median + abundant in SNs, percentages of CD8 T cells expressing cyto- 17.1%, BloodSN median 6.17%) but one of the rarest in mucosa + toxic effectors were typically higher in HIV-1 groups compared (RectosigmoidHIV+ median 1.18%, RectosigmoidSN median with SN in both tissues, with the highest percentages observed in 0.508%). In contrast, the mucosa contained a significantly higher HIV-1+ individuals not on ART (Fig. 1C, Supplemental Fig. 1A). percentage of GzmAInt single-positive CD8+ T cells compared + Among HIV-1 groups, C and V individuals not on ART had with blood (RectosigmoidHIV+ median 17.6%, RectosigmoidSN Int higher percentages of GzmA and GzmK-positive rectosigmoid median 28.9%; BloodHIV+ median 2.69%, BloodSN median CD8+ T cells compared with ART-Tx participants. V individuals 1.43%). A similar trend was observed with GzmABright CD8+ not on ART also had greater GzmB expression in the gut com- T cells. Interestingly, in addition to differences in abundance, the pared with ART-Tx subjects (Fig. 1C). Similar trends were ob- coexpression pattern of GzmABright also differed between blood served in blood but not as pronounced (Supplemental Fig. 1A). and mucosa. In mucosa, GzmABright CD8+ T cells typically lacked Taken together, these data demonstrate elevated expression of expression of any other cytotoxic effectors; in contrast, blood GzmAInt, B, K, and perforin in rectosigmoid CD8+ T cells from GzmABright CD8+ T cells typically coexpressed perforin and HIV-1+ individuals, particularly those not on ART, compared with GzmB (Fig. 2A, 2B). SNs, and to our knowledge highlight for the first time a novel Perhaps not surprisingly, HIV-1+ participants also had higher subset of CD8+ T cells expressing GzmA at high fluorescence median frequencies of CD8+ T cells coexpressing cytotoxic ef- intensity in the mucosa. fectors compared with SNs in both blood and gut, including a large proportion of CD8+ T cells coexpressing GzmAInt, B, and Coexpression of cytotoxic effectors is reduced in rectosigmoid K (RectosigmoidHIV+ median 21.7%, RectosigmoidSN median CD8+ T cells compared with PBMC but elevated in HIV-1 6.89%; BloodHIV+ median 15.9%, BloodSN median 5.89%) infection (Fig. 2B). To elaborate on this observation, we compared the Differences in the abundance of individual cytotoxic effectors led abundance of coexpressing subsets across HIV-1 disease status us to investigate the coexpression patterns of these molecules in (Fig. 2C, Supplemental Fig. 1B). In mucosa, the proportions of 4 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Expression of cytotoxic effector molecules in unstimulated blood and rectosigmoid CD8+ T cells across HIV-1 disease status. (A) Repre- sentative flow cytometry plot displaying intracellular protein staining for GzmAInt/Bright, GzmB, GzmK, and perforin in unstimulated blood and rec- tosigmoid CD8+ T cells. (B) Differences in the intracellular expression of GzmAInt/Bright, GzmB, GzmK, and perforin between unstimulated blood and rectosigmoid CD8+ T cells in HIV-1–infected (HIV-1+) and SN participant groups (n = 25 and 10, respectively). (C) Differences in the intracellular ex- pression of GzmAInt/Bright, GzmB, GzmK, and perforin in unstimulated rectosigmoid CD8+ T cells across HIV-1 disease (Figure legend continues) The Journal of Immunology 5

GzmAIntGzmB+GzmK+ and GzmAIntGzmB+ CD8+ T cells were and/or CD107, and again found differences between Gag- and significantly greater in all HIV-1+ subgroups compared with SNs. SEB-responding CD8+ T cells. Similar to previous observations The proportion of GzmAIntGzmB+GzmK+ CD8+ T cells was also in blood (7), the proportion of Gag-responding CD8+ T cells greater in V individuals compared with those on ART. Both C and expressing perforin and/or GzmB tended to increase as cytokine V individuals not on ART had significantly higher percentages of polyfunctionality decreased in both blood and gut (Fig. 3D). This phenotypically cytotoxic CD8+ T cells, defined as those coex- trend was especially pronounced in mucosa, where the majority of pressing perforin and Gzms, compared with SNs. Specifically, CD8+ T cells responding to Gag with three or four soluble markers C and V individuals not on ART had higher percentages of Per- and/or CD107, did not express perforin or GzmB (medians 60.18 forin+GzmAIntGzmB+GzmK+ CD8+ T cells compared with SNs; and 67.33% for three- and four-function cells, respectively) C had higher percentages of Perforin+GzmAIntGzmB+ CD8+ (Fig. 3D). In contrast, single-function mucosal Gag-responding T cells compared with SNs. In contrast, SNs displayed greater CD8+ T cells displayed the greatest GzmB expression (Fig. 3D). frequencies of GzmAInt and GzmABright single-positive CD8+ This was not observed for blood SEB-responding CD8+ T cells, in T cells compared with all HIV-1+ subgroups (Fig. 2C). Similar which the proportion of perforin and/or GzmB-expressing cells trends were observed in the blood (Supplemental Fig. 1B). appeared greater in polyfunctional compared with single-function Taken together, these data reveal that, in general, the rec- CD8+ T cells (Supplemental Fig. 2C, 2D). No obvious trend tosigmoid mucosa contains a lower percentage of phenotypically emerged for SEB in the mucosa, as the majority of rectosigmoid cytotoxic CD8+ T cells compared with blood, but an abundance of SEB-responding CD8+ T cells did not express perforin or GzmB, GzmA single-positive CD8+ T cells. Furthermore, individuals with irrespective of cytokine expression (Supplemental Fig. 2). chronic HIV-1 infection, particularly those not on ART, have Taken together, these data demonstrate an apparent inverse re- greater coexpression of cytotoxic effector proteins in mucosal lationship between polyfunctionality (defined as coexpression of Downloaded from CD8+ T cells compared with uninfected individuals. multiple cytokines/chemokines and CD107a) and cytotoxic po- tential (defined as coexpression of perforin and GzmB) in rec- Expression of perforin and GzmB in Ag-specific CD8+ T cells tosigmoid HIV-1 Gag-specific CD8+ T cells, and highlight differs with anatomical compartment, antigenic stimulation, discordance in the expression of perforin and GzmB between and cytokine polyfunctionality Gag- and SEB-responding CD8+ T cells in the mucosa. + http://www.jimmunol.org/ We next turned our attention to Ag-specific CD8 T cells. To + explore the range of functionality exhibited by Ag-specific rec- Expression of cytotoxic effectors in Ag-responding CD8 tosigmoid CD8+ T cells, lymphocytes isolated from blood and T cells differs by anatomic location and by antigenic mucosa of HIV-1+ individuals during early and chronic infection stimulation or SN controls (Table I) were stimulated with an HIV-1 Gag The data presented in Fig. 3 suggest that most cytokine/ peptide pool or SEB in a 5.5 h ex vivo stimulation assay followed chemokine-producing HIV-1–specific rectosigmoid CD8+ T cells by intracellular staining for MIP-1b, IFN-g, and TNF-a along do not express perforin and GzmB. The abundance of mucosal with CD107a, GzmB, and perforin as surrogates of cytotoxicity. CD8+ T cells expressing Gzms independently of perforin led us to For these experiments we used reagents and protocols previously re-examine this result using a broader range of cytotoxic effectors. demonstrated to reveal perforin newly produced in response to As before, lymphocytes isolated from blood and rectosigmoid by guest on October 3, 2021 TCR stimulation (21). Expression of perforin and GzmB was mucosa of chronically HIV-1–infected and SN adults were stim- measured within Gag- and SEB-responding CD8+ T cells as de- ulated with an HIV-1 Gag-peptide pool or SEB in a 5.5 h ex vivo fined by one or more of the following responses: degranulation stimulation assay. However, in this series of experiments, stimu- (CD107a), MIP-1b, IFN-g, or TNF-a production (Fig. 3A). lation was followed by intracellular staining for perforin, GzmA, In both HIV-1–infected and SN participant groups, a large GzmB, and GzmK, along with MIP-1b and CD107 (Fig. 4). As fraction of rectosigmoid CD8+ T cells responding to antigenic CD107a and MIP-1b expression were the two strongest responses stimulation did not express detectable perforin or GzmB previously detected in rectosigmoid mucosa in response to HIV-1 (medianGag 49.6%, medianSEB 74.5%) (Fig. 3B). For both Ags, the Gag and SEB stimulation, they were used in this study as indi- proportion of total responding CD8+ T cells coexpressing perforin cators of Ag responsiveness (10, 12). and GzmB was significantly greater in blood compared with For both HIV-1 Gag and SEB stimulation, the mucosa displayed mucosa (Fig. 3B); in contrast, the proportion of total responding a greater proportion of responding CD8+ T cells not expressing any CD8+ T cells expressing neither perforin nor GzmB was greater in cytotoxic effectors compared with blood. These Perforin2GzmA2 mucosa compared with blood, reaching statistical significance for GzmB2GzmK2 responses dominated in the mucosa and were SEB (Fig. 3, Supplemental Fig. 2). As no statistically significant particularly striking for SEB-responding CD8+ T cells (Fig. 4C). differences were observed between HIV-1+ subgroups, HIV-1+ Rectosigmoid mucosa also exhibited greater proportions of GzmA participant data were consolidated into a single group. Interest- single-positive Ag-responding CD8+ T cells compared with blood. ingly, in mucosa but not blood, the proportion of responding CD8+ In contrast, blood had greater proportions of Perforin+/2GzmA+ T cells expressing GzmB in the absence of perforin (PerforinNeg GzmB+–responding CD8+ T cells compared with mucosa GzmB+) was significantly greater for Gag compared with SEB (Fig. 4C). Differences between antigenic stimulations were also stimulation (medians 38.7% and 19.9% respectively) (Fig. 3B). observed. In mucosa but not blood, Gag-responding CD8+ T cells The Gag-specific PerforinNegGzmB+ CD8+ T cell response was exhibited a larger proportion of GzmA+GzmB+GzmK+ CD8+ also greater in mucosa compared with blood (Fig. 3B). T cells compared with SEB-responding CD8+ T cells (Fig. 4C, We next assessed the expression of perforin and GzmB within data not shown). In contrast, rectosigmoid SEB-responding CD8+ CD8+ T cell subsets producing multiple cytokines/chemokines T cells included a greater proportion of cells not expressing any

status as follows: C n = 8, V (untreated) n = 7, Tx (ART-suppressed) n = 10, SN n = 10. In (B and C), wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 6 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 2. Coexpression patterns of cytotoxic effectors vary with anatomical compartment and HIV-1 disease status. (A) Representative flow cytometry plot displaying the intracellular coexpression patterns of GzmAInt/Bright, GzmB, GzmK, and perforin in unstimulated blood and rectosigmoid CD8+ Tcells. (B) Differences in the proportions of CD8+ T cells coexpressing GzmAInt/Bright, GzmB, GzmK, and perforin between blood and rectosigmoid mucosa in HIV-1– infected (HIV-1+) and SN participants (n = 25 and 10, respectively). Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: *p , 0.05, **p , 0.01, ***p , 0.001. (C) Differences in the percentages of rectosigmoid CD8+ Tcells coexpressing cytotoxic effectors across HIV-1 disease status as follows: V n =7,Cn =8,Txn = 10, and SN n = 10. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; horizontal bars between participant subgroups indicate significance as follows: *p , 0.05, **p , 0.01. cytotoxic effectors compared with Gag-responding CD8+ T cells displayed proportionately greater expression of GzmA, B, and K (Fig. 4C, data not shown). compared with SEB-responding cells. These findings expand upon the data presented in Figs. 1 and 3, and further support the interpretation that rectosigmoid CD8+ Memory phenotype varies with cytotoxic effector expression T cells are typically primed for cytokine expression rather than and anatomic location cytotoxic effector production. However, this observation may be Based on observations in blood, it is thought that CD8+ T cells partly Ag dependent, as rectosigmoid Gag-specific CD8+ T cells acquire expression of cytotoxic effectors as they mature into The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. Expression of cytokines, chemokines, perforin, and GzmB in blood and rectosigmoid CD8+ T cells responding to ex vivo stimulation. (A) Representative flow cytometry plot displaying intracellular expression of perforin and GzmB in polyfunctional HIV-1 Gag-responding CD8+ T cells (four functions: CD107a+MIP-1b+IFN-g+TNF-a+ and three functions: CD107a+MIP-1b+IFN-g+) in blood and rectosigmoid mucosa. (B) Expression of perforin (Perf) and/or GzmB in HIV-1 Gag- or SEB-responding CD8+ T cells showing differences related to stimulus and anatomical compartment, n = 27. (C) Differences in intracellular expression of perforin and GzmB within mono- and polyfunctional HIV-1 Gag-responding CD8+ T cells in blood and rectosigmoid mucosa visualized with a SPICE pie chart. Responsiveness was defined as the expression of one or more of the following effectors: CD107a, MIP-1b, IFN-g, and TNF-a. Percentages indicate the median total HIV-1 Gag CD8+ T cell response. Wedge colors correspond to the number of si- multaneous functions, as indicated in the legend (perforin, GzmB, CD107a, MIP-1b, IFN-g, and TNF-a), n = 27. (D) Differences in expression of perforin and/or GzmB between mono- and polyfunctional HIV-1 Gag-specific CD8+ T cells in blood and mucosa. Numbers on the x-axis correspond to the number of simultaneous functions (CD107a, MIP-1b, IFN-g, and TNF-a), n = 27. In (B and D), wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 8 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 4. Expression/coexpression patterns of cytotoxic granule proteins in ex vivo stimulated CD8+ T cells from blood and rectosigmoid mucosa. (A) Representative flow cytometry plot displaying expression of GzmA, B, K, and perforin in CD8+ T cells responding to ex vivo Gag-peptide stimulation as indicated by degranulation (CD107a) and MIP-1b expression. (B) Differences in expression of cytotoxic effectors between blood and rectosigmoid mucosal CD8+ T cells responding to an HIV-1 Gag-peptide pool or SEB, summarized with SPICE pie charts. Responsiveness was defined as the expression of one or more of the following: CD107a and MIP-1b, n = 18. (C) Differences in the expression of cytotoxic effectors between blood and rectosigmoid mucosal CD8+ T cells responding to an HIV-1 Gag-peptide pool or SEB, detailing individual response combinations, n = 18. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: **p , 0.01, ***p , 0.001, ****p , 0.0001. effector subsets. For example, Perforin2GzmA2GzmB2, GzmA+ CD27 on unstimulated blood and rectosigmoid CD8+ T cells from GzmB+GzmK+, and Perforin+GzmA+GzmB+ subsets are associ- chronically HIV-1–infected and SN adults. ated with central memory, transitional, and effector maturation Not surprisingly, blood CD8+ T cells not expressing any cytotoxic stages, respectively (24–26). However, despite housing a high effectors primarily had a naive phenotype (Supplemental Fig. 3), proportion of TEM cells (27), the mucosa displays an abundance of whereas in rectosigmoid mucosa most quadruple-negative Per- CD8+ T cells expressing either only GzmA or not expressing any forin2GzmA2GzmB2GzmK2 CD8+ T cells were memory cells, cytotoxic effectors (Fig. 1). To address this apparent discrepancy, with a large fraction displaying a TTM phenotype (Fig. 5). Because we assessed the intracellular expression of cytotoxic effectors in the proportion of GzmAIntGzmB+GzmK+ was elevated in the conjunction with T cell memory markers CD45RO, CCR7, and mucosa of HIV-1+ individuals not on ART (Fig. 2C), we were The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 5. Expression of memory/effector markers by prominent CD8+ T cell subsets in rectosigmoid mucosa. (A) Representative flow cytometry plot displaying expression of memory markers CD45RO and CD27 on the following rectosigmoid CD8+ T cells subsets: GzmAIntGzmB+GzmK+ (purple), GzmAIntGzmK+ (orange), GzmABright (green), GzmAInt (brown), Perforin+GzmAIntGzmB+ (red), and cells lacking expression of cytotoxic effectors 2 2 2 2 2 + 2 (Perforin GzmB GzmA GzmK ) (blue). The proportions of TTM,TEM,TEFF, and CD45RO CD27 CCR7 cells within each subset are displayed within 2 + 2 the quadrants. (B) Differences in the percentages of naive (Tnaive), central memory (TCM), TTM,TEM,TEFF, and CD45RO CD27 CCR7 cells within GzmAIntGzmB+GzmK+, GzmAIntGzmK+, GzmAInt/Bright single-positive, Perforin+GzmAIntGzmB+, and (Figure legend continues) 10 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 interested in the memory phenotype of these cells. Rectosigmoid rectosigmoid CD8+ T cells, which may be of particular interest in subsets expressing GzmK, such as GzmAIntGzmB+GzmK+ and the light of recent literature demonstrating novel extracellular, Int + + GzmA GzmK CD8 T cells, exhibited a more pronounced TTM proinflammatory functions of Gzms (14, 15, 17, 30, 31). phenotype than their counterparts in blood (Fig. 5, Supplemental As previously reported, the percentages of CD8+ T cells Fig. 3). Mucosal single-positive GzmAInt/Bright CD8+ T cells were expressing perforin and GzmB were significantly reduced in rec- Bright predominantly TEM cells (Fig. 5). In blood, GzmA cells were tosigmoid mucosa compared with blood (11–13, 29). In contrast, Int + absent, and GzmA cells were distributed among TTM,TEM, and the percentages of GzmK- and GzmA-positive CD8 T cells were + TEFF categories. The relatively small subset of rectosigmoid CD8 similar between the two tissues. These observed differences in T cells expressing perforin, primarily Perforin+GzmAIntGzmB+ expression levels of cytotoxic effectors suggest differential regu- + CD8 T cells with or without GzmK, was comprised of TEFF cells lation at the transcriptional and/or posttranscriptional level. In- with some TTM and TEM (Fig. 5B), a distribution similar to that of terestingly, the for human GzmB is located on Perforin+GzmAIntGzmB+ CD8+ T cells in blood (Supplemental 14 whereas for GzmA and GzmK, whose expression was Fig. 3). Taken together, these data reveal differences between not deficient in the gut, are located in close proximity to one blood and gut regarding the memory phenotype of CD8+ T cells another on (30, 32). Little is known about the lacking expression of cytotoxic effectors, and identify the transcriptional regulation of GzmA and GzmK but their spatial Bright + GzmA CD8 T cell subset, limited to mucosa, as TEM cells. separation from GzmB might suggest differing transcriptional regulation (33). Differences in posttranscriptional regulatory GzmABright CD8+ T cells resemble tissue-resident T cells mechanisms may also account for the variations in expression + The human rectosigmoid mucosa houses a large fraction of CD8 levels. For example, although resting murine NK cells express T cells with a tissue-resident phenotype, including in chronic HIV- mRNA for perforin, GzmB, and GzmA, they must be activated to Downloaded from Bright + 1 infection (28). As GzmA CD8 T cells were abundant in express perforin and GzmB protein; in contrast, both resting and mucosa but not blood, and primarily displayed a TEM phenotype, activated NK cells express high levels of GzmA protein (34). we hypothesized that these cells might also express markers of Although GzmA was the most abundantly expressed cytotoxic tissue residency. To test this, we analyzed intracellular expression effector in both blood and gut, we observed discordance in its of GzmA and GzmB in conjunction with the canonical tissue- expression pattern between tissues. In mucosa, a large percentage resident markers CD69, integrin aE (CD103), and S1PR1 on of CD8+ T cells were single positive for GzmA; a subset of these http://www.jimmunol.org/ + Bright unstimulated blood and rectosigmoid CD8 T cells from chroni- expressed GzmA at high levels (GzmA ) and displayed a TEM, cally HIV-1–infected and SN adults. tissue-resident phenotype (CD103+CD69+S1PR12) (35, 36). In Irrespective of HIV-1 disease status, the overwhelming majority blood, however, GzmABright CD8+ T cells were scarce and GzmA of rectosigmoid GzmABright CD8+ T cells were CD69+CD103+ 2 was nearly always coexpressed with other cytotoxic effectors. S1PR1 , indicative of tissue residency (Fig. 6). In contrast, the These data suggest that similar to perforin and GzmB, GzmA + Int expression of tissue-resident markers on GzmB and GzmA expression may be regulated differently in rectosigmoid CD8+ + CD8 T cells varied across HIV-1 disease status. In ART-Tx and T cells compared with blood. + Int + healthy adults, GzmB and GzmA CD8 T cells were primarily In contrast to the abundance of GzmA, the percentage of CD8+ + + 2 + by guest on October 3, 2021 CD69 CD103 S1PR1 . In contrast, in HIV-1 individuals not on T cells coexpressing perforin and Gzms was sharply reduced in + Int + ART, GzmB and GzmA CD8 T cells were primarily single- the gut compared with blood, further evidence of the diminished positive for CD69 or lacked expression of all three markers cytotoxic potential of rectosigmoid CD8+ T cells (11). We pre- (Fig. 6). Together, these data support the interpretation that viously speculated that perforin and GzmB are tightly regulated in Bright + GzmA CD8 T cells are likely a subset of tissue-resident mucosa to avoid bystander damage to the fragile mucosal epi- + CD8 T cells unique to the mucosa, whose functions and anti- thelium (11). As Gzms are believed to be unable to induce target- genic specificity remain to be determined. cell without perforin (37), one interpretation of these data is that limited perforin expression enables rectosigmoid CD8+ Discussion T cells to use Gzms for noncytolytic functions. Consistent with The gastrointestinal mucosa is an important site of HIV-1 trans- this interpretation, although the cytotoxicity of GzmB and perforin mission and viral replication, and is generally considered as one of are well established, controversy remains as to whether other the front lines of defense against mucosal pathogens. Under- Gzms contribute significantly to target-cell apoptosis in vivo standing how mucosal CD8+ T cells respond to microbial chal- (15, 38). Rather, GzmA and GzmK may function in extracellu- lenge is important for developing therapeutic and preventative lar, noncytotoxic capacities in the mucosa. For example, GzmA strategies targeted to mucosal tissues. Previous reports have and GzmK have been shown to facilitate cytokine responses to the demonstrated that rectosigmoid CD8+ T cells are robust producers microbial cell-wall component LPS (16, 39). Additionally, GzmA, of chemokines and cytokines but have limited perforin expression, B, and K have demonstrated extracellular activities that promote supporting the interpretation that gut CD8+ T cells are primed for cellular migration and inflammation (14, 15, 17, 30, 31). cytokine and chemokine secretion rather than cytotoxicity (11–13, Proinflammatory functions of Gzms appear consistent with the 29). However, missing from earlier studies of mucosal T cells polyfunctional nature of rectosigmoid CD8+ T cells, whose ex- were detailed analyses of cytolytic effector proteins GzmA, B, pression of cytokines and b-chemokines likely have similar and K and their patterns of expression or coexpression with cy- proinflammatory effects (40, 41). tokines and chemokines. In the current study we provide a more Disparities in the expression levels of cytotoxic effectors varied comprehensive view of the effector molecules produced by with HIV-1 disease status. The percentages of CD8+ T cells

Perforin2GzmB2GzmA2GzmK2 rectosigmoid CD8+ T cells. The large asterisk identifies memory phenotypes with a significantly greater median fre- quency compared with all other memory subsets, n = 11. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; small asterisks show level of significance as follows: *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 6. Expression of tissue-resident markers on GrzABright, GrzAInt, and GrzB-expressing CD8+ T cells in rectal mucosa. (A) Representative flow cytometry plot showing expression of the tissue-resident markers early leukocyte activation marker CD69 and integrin aE (CD103) on GrzABright, GrzAInt, and GrzB-expressing CD8+ T cells in rectal mucosa. (B) Proportion of rectal GrzABright, GrzAInt, and GrzB-positive CD8+ T cells expressing the tissue residency markers CD69, CD103, and S1PR1 in HIV-1 C n =4,Vn =5,Txn = 7, and SN n = 4. Gray box outlines the canonical tissue residency phenotype. Coexpression analysis was generated using SPICE software. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; symbols indicate level of significance as follows: *p , 0.05, **p , 0.01. 12 GASTROINTESTINAL CD8+ T CELLS IN HIV-1 expressing GzmAInt, GzmB, GzmK, and perforin were higher in the coexpression of cytotoxic effectors with cytokines/chemokines HIV-1–infected compared with SN adults, with the highest per- confers a protective advantage against HIV-1 will require further centages observed in HIV-1+ individuals not on ART. Interest- analysis. No differences were observed between HIV-1 C and non- ingly, the opposite trend was observed for single-positive C in the proportion of Gag-specific CD8+ T cells expressing GzmABright cells. One interpretation is that HIV-1 infection re- perforin and/or Gzms, although this analysis was limited by small sults in upregulation of other cytotoxic effectors in GzmABright sample size. CD8+ T cells. The overwhelming majority of GzmABright CD8+ Taken together, these data demonstrate that rectosigmoid T cells expressed aE integrin (CD103), which is associated with CD8+ T cells produce Gzms independently of perforin, likely proximity to epithelial cells expressing the CD103 ligand, E- complementing dominant cytokine and chemokine responses to cadherin (42–44). A characteristic of HIV-1 infection is loss of promote an inflammatory response. The work described in this epithelial barrier integrity (45, 46). Accordingly, an alternative study supports a model in which rectosigmoid CD8+ T cells are explanation is that lower proportions of GzmABright CD8+ T cells skewed toward proinflammatory responses rather than direct in HIV-1 infection reflects a loss of CD103+ CD8+ T cells, due in cytotoxicity, suggesting new insights into the mucosal adaptive part to reduced availability of epithelial-derived E-cadherin. immune response that may inform the development of thera- Notably, the proportion of CD8+ T cells coexpressing GzmA, B, peutics to treat mucosal infections such as HIV-1. and K was significantly greater in HIV-1–infected compared with SN participants in both blood and gut. In blood, coexpression of GzmA, B, and K is associated both with an intermediate memory Acknowledgments maturation status and HIV-1 specificity (24–26). Similarly, rec- We thank Rebecca Hoh, Montha Pao, Monika Deswal, and the clinical staff at San Francisco General Hospital for assistance with participant recruit- Downloaded from tosigmoid GzmA+GzmB+GzmK+ CD8+ T cells primarily dis- ment and sample collection. We thank the participants for willingness to played a TTM phenotype and a substantial fraction of rectosigmoid + contribute to this study. We thank the University of California Davis Flow Gag-responding CD8 T cells coexpressed GzmA, B, and K. Cytometry Shared Resource Laboratory staff members Bridget McLaughlin + Whether coexpression of GzmA, B, and K in Gag-specific CD8 and Jonathan Van Dyke for assistance with flow cytometry. T cells is a reflection of maturation status or a consequence of HIV-1–specific priming of the immune response will require fur- Disclosures http://www.jimmunol.org/ ther exploration. However, previous work has demonstrated that The authors have no financial conflicts of interest. blood HIV-1–specific CD8+ T cells are skewed toward an inter- mediate maturation stage, suggesting the former (47). Consistent with the hypothesis that rectosigmoid CD8+ T cells References are skewed toward cytokine production rather than cytotoxicity, 1. Cartwright, E. K., L. Spicer, S. A. Smith, D. Lee, R. Fast, S. Paganini, + B. O. Lawson, M. Nega, K. Easley, J. E. Schmitz, et al. 2016. CD8(+) lympho- the proportions of Gag- and SEB-responding CD8 T cells cytes are required for maintaining viral suppression in SIV-infected macaques expressing cytotoxic effectors were significantly lower in rec- treated with short-term antiretroviral therapy. Immunity 45: 656–668. tosigmoid mucosa compared with blood. Furthermore, we ob- 2. Di Stefano, M., A. Favia, L. Monno, P. Lopalco, O. Caputi, A. C. Scardigno, G. Pastore, J. R. Fiore, and G. Angarano. 2001. Intracellular and cell-free (in- served that perforin and GzmB expression increased in

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