Terminal Effector CD8 T Cells Defined by an IKZF2 +IL-7R− Transcriptional Signature Express Fc γRIIIA, Expand in HIV Infection, and Mediate Potent HIV-Specific This information is current as Antibody-Dependent Cellular Cytotoxicity of September 23, 2021. Prossy Naluyima, Kerri G. Lal, Margaret C. Costanzo, Gustavo H. Kijak, Veronica D. Gonzalez, Kim Blom, Leigh Anne Eller, Matthew Creegan, Ting Hong, Dohoon Kim,

Thomas C. Quinn, Niklas K. Björkström, Hans-Gustaf Downloaded from Ljunggren, David Serwadda, Elly T. Katabira, Nelson K. Sewankambo, Ronald H. Gray, Jared M. Baeten, Nelson L. Michael, Fred Wabwire-Mangen, Merlin L. Robb, Diane L. Bolton, Johan K. Sandberg and Michael A. Eller

J Immunol published online 13 September 2019 http://www.jimmunol.org/ http://www.jimmunol.org/content/early/2019/09/12/jimmun ol.1900422

Supplementary http://www.jimmunol.org/content/suppl/2019/09/12/jimmunol.190042 Material 2.DCSupplemental by guest on September 23, 2021

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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 The Authors. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 13, 2019, doi:10.4049/jimmunol.1900422 The Journal of Immunology

Terminal Effector CD8 T Cells Defined by an IKZF2+IL-7R2 Transcriptional Signature Express FcgRIIIA, Expand in HIV Infection, and Mediate Potent HIV-Specific Antibody-Dependent Cellular Cytotoxicity

Prossy Naluyima,*,†,1 Kerri G. Lal,†,‡,x,1 Margaret C. Costanzo,‡,x Gustavo H. Kijak,‡,x Veronica D. Gonzalez,† Kim Blom,† Leigh Anne Eller,‡,x Matthew Creegan,‡,x Ting Hong,{ Dohoon Kim,‡,x Thomas C. Quinn,‖,# Niklas K. Bjo¨rkstro¨m,† Hans-Gustaf Ljunggren,† David Serwadda,** Elly T. Katabira,†† Nelson K. Sewankambo,†† ‡‡ {,xx,{{ ‡

Ronald H. Gray, Jared M. Baeten, Nelson L. Michael, Fred Wabwire-Mangen,* Downloaded from Merlin L. Robb,‡,x Diane L. Bolton,‡,x Johan K. Sandberg,†,2 and Michael A. Eller‡,x,2

HIV-1 infection expands large populations of late-stage differentiated CD8 T cells that may persist long after viral escape from TCR recognition. In this study, we investigated whether such CD8 populations can perform unconventional innate-like antiviral effector functions. Chronic untreated HIV-1 infection was associated with elevated numbers of CD45RA+CD57+ terminal + effector CD8 T cells expressing FcgRIIIA (CD16). The FcgRIIIA CD8 T cells displayed a distinctive transcriptional profile http://www.jimmunol.org/ between conventional CD8 T cells and NK cells, characterized by high levels of IKZF2 and low expression of IL7R. This transcriptional profile translated into a distinct NKp80+ IL-7Ra2 surface phenotype with high expression of the Helios tran- scription factor. Interestingly, the FcgRIIIA+ CD8 T cells mediated HIV-specific Ab-dependent cellular cytotoxicity (ADCC) activity at levels comparable with NK cells on a per cell basis. The FcgRIIIA+ CD8 T cells were highly activated in a manner that correlated positively with expansion of the CD8 T cell compartment and with plasma levels of soluble mediators of antiviral immunity and inflammation such as IP-10, TNF, IL-6, and TNFRII. The frequency of FcgRIIIA+ CD8 T cells persisted as patients initiated suppressive antiretroviral therapy, although their activation levels declined. These data indicate that terminally differ-

entiated effector CD8 T cells acquire enhanced innate cell-like characteristics during chronic viral infection and suggest that HIV- by guest on September 23, 2021 specific ADCC is a function CD8 T cells use to target HIV-infected cells. Furthermore, as the FcgRIIIA+ CD8 T cells persist in treatment, they contribute significantly to the ADCC-capable effector cell pool in patients on antiretroviral therapy. The Journal of Immunology, 2019, 203: 000–000.

he exquisite sensitivity and specificity of TCR-mediated with initial viral decline; however, the response fails to completely sensing of infection is central to the function of T cells suppress or clear infection (1–4). Since the initial character- T but can also, in some situations, limit their ability to ization of HIV-specific cytotoxic CD8 T cells in the late 1980s provide effective immunity. This is evident in the context of HIV-1 (5–9), the limitations in their ability to control viral replication infection, in which the appearance of HIV-1–specific T cells coincides and clear infection are evident (10, 11). High mutation rates in

*Makerere University Walter Reed Project, Kampala, Uganda; †Center for Infectious Received for publication April 11, 2019. Accepted for publication August 20, 2019. Medicine, Department of Medicine, Karolinska Institutet, 17177 Stockholm, Swe- This work was supported by a cooperative agreement (W81XWH-07-2-0067) be- den; ‡U.S. Military HIV Research Program, Walter Reed Army Institute of Research, x tween the Henry M. Jackson Foundation for the Advancement of Military Medicine Silver Spring, MD 20910; Henry M. Jackson Foundation for the Advancement of { and the U.S. Department of Defense and with an Interagency Agreement (1Y-A1-26- Military Medicine, Bethesda, MD 20817; Department of Global Health, University ‖ 42-07) with the Division of AIDS, National Institute of Allergy and Infectious Dis- of Washington School of Public Health, Seattle, WA 98195; Laboratory of Immuno- eases, National Institutes of Health, the Swedish Research Council, the Swedish regulation, Division of Intramural Research, National Institute of Allergy and Infectious Cancer Society, and the Karolinska Institutet. Sample collection for the Couples Diseases, National Institutes of Health, Bethesda, MD 20852; #School of Medicine, Johns Observational Study was supported by the Bill and Melinda Gates Foundation (Grant Hopkins University, Baltimore, MD 21205; **Rakai Health Sciences Program, Uganda 41185). Virus Research Institute, Entebbe, Uganda; ††Faculty of Medicine, Makerere University College of Health Sciences, Kampala, Uganda; ‡‡Bloomberg School of Public Health, Address correspondence and reprint requests to Dr. Michael A. Eller, U.S. Military Johns Hopkins University, Baltimore, MD 21205; xxDepartment of Medicine, University of HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Washington School of Public Health, Seattle, WA 98195; and {{Department of Epidemi- Avenue, 1N11, Silver Spring, MD 20910. E-mail address: [email protected] ology, University of Washington School of Public Health, Seattle, WA 98195 The online version of this article contains supplemental material. 1The two first authors contributed equally and should both be considered first authors. Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; ART, 2The two last authors contributed equally and should both be considered senior antiretroviral therapy; COS, Couples Observation Study; Eomes, eomesodermin; authors. FDR, false discovery rate; HCV, hepatitis C virus; KIR, killer Ig-like ; MFI, mean fluorescence intensity; PCA, principal component analysis. ORCIDs: 0000-0002-8970-0198 (K.G.L.); 0000-0003-2043-1513 (L.A.E.); 0000- 0002-0404-1315 (T.C.Q.); 0000-0002-0967-076X (N.K.B.); 0000-0003-0908- This article is distributed under the terms of the CC BY 4.0 Unported license. 7387 (H.-G.L.); 0000-0001-9362-053X (N.K.S.); 0000-0001-5882-5548 (N.L.M.); 0000-0003-3949-9649 (M.L.R.); 0000-0002-6275-0750 (J.K.S.); 0000-0003-3905- Copyright Ó 2019 The Authors. 4877 (M.A.E.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900422 2 TERMINAL EFFECTOR FcgRIIIA+ CD8 T CELLS EXPAND IN HIV

HIV-1 contribute to the ability of the virus to escape adaptive Ethics statement T cell responses (3, 12–14). Also, HIV-specific T cells become The study was approved by the following institutional review boards in the functionally impaired during chronic infection, additionally United States and Uganda: the institutional Review Boards of Uganda’s limiting their ability to control viral replication (15–17). Indeed, National Council for Science and Technology and the National AIDS polyfunctional HIV-specific T cell responses are associated with Research Committee, as well as Division of Human Subjects Protection at better disease outcomes compared with those with a narrower the Walter Reed Army Institute of Research. All participants gave written- informed consent, or written-informed consent was obtained from the functional breadth (18–20). In chronic HIV-1 infection, the parent or legal guardian of those aged 17. For samples from the COS in replicating viral quasispecies have, to a large extent, mutated Kampala, Uganda, all participants gave written-informed consent, and away from the originally transmitted viral sequence under T cell ethical approvals for the study were obtained from Uganda’s National selection pressure, and this probably contributes to the accu- Council for Science and Technology and the National AIDS Research Committee and the University of Washington. mulation of late-stage effector CD8 T cells with a skewed maturational phenotype (21, 22). Flow cytometry and mAbs Persistent pathogen replication in chronic infections, such as Cryopreserved specimens were thawed and washed. Counts and viability untreated HIV-1 infection, engages T cell–mediated immune were assessed on the Guava PCA (Guava Technologies, Hayward, CA), responses continuously with sustained antigenic challenge. using Guava ViaCount reagent. Standard flow cytometry phenotyping was Interestingly, some chronic infections have been associated performed as previously described (46). Commercial mAbs (clone) used in with expansion of an unusual subset of CD8 T cells expressing flow cytometry were as follows: CCR5/CD195 BV421 (2D7), CCR7/ CD197 FITC (150503), CD14 allophycocyanin H7 (MFP9), CD14 CD16 (23–25). CD16 is the low-affinity IgG and Alexa Fluor 700 (M5E2), CD19 Alexa Fluor 700 (HIB19), CD16 allo- exists in two isoforms, FcgRIIIA (CD16a) and FcgRIIIB phycocyanin Cy7, PE-Cy5, Pacific Blue and BUV496 (3G8), CD161 PE- Downloaded from (CD16b). CD16b is expressed exclusively by neutrophils Cy5 (DX12), CD27 PerCP Cy5.5 (L128), PD-1/CD279 Alexa Fluor 647 and recognizes IgG-containing immune complexes, whereas and PE (EH12.1), CD3 AmCyan, allophycocyanin–H7, and PerCP-Cy5.5 (SK7), CD3 PE-CF594 (UCHT1), CD4 BV605 and allophycocyanin–H7 CD16a is best characterized for its role in mediating Ab- (SK3), CD38 allophycocyanin (HB7), CD45RA allophycocyanin (HI100), dependent cellular cytotoxicity (ADCC) as a function of the CD56 PE-Cy7 (NCAM16.2) and (B159), CD8 PE-Cy7 and PerCP-Cy5.5 innate (26, 27, and reviewed in 28). NK cells (SK1), CD8 PE and PerCP-Cy5.5 (RPA-T8), CD8b PE (2ST8.5H7), HLA-

are able to mediate a strong effector function in response to DR FITC (G46-6), IL-7R/CD127 FITC and Alexa Fluor 647 (HIL-7R- http://www.jimmunol.org/ signaling through CD16-mediated stimulation. Whereas Fc M21), KIR2DL2/DS2/DL3 PE (DX27), NKG2D/CD314 PerCP Cy.5 (ID11), TCRab FITC and allophycocyanin (T10B9.1A-31), TRAIL/ receptors are generally not expressed by T cells, CD16 can CD253 PE (RIK-2) (all from BD Biosciences, San Jose, CA); Aqua sometimes be expressed by subsets of TCRab T cells (29–32). LIVE/DEAD viability stain, CD3 PE Texas Red (7D6), CD14 PE-Cy5 Growing evidence suggests the potential importance of ADCC (Tuk4), and CD19 PE-Cy5 (SJ25-C1) were obtained from Invitrogen in protection from HIV-1 infection (33, 34). Additionally, (Carlsbad, CA); CD4 ECD (SFCL12T4D11), NKG2A allophycocyanin (Z199), and NKp46/CD335 PE (BAB281) were all from Beckman Coulter nonneutralizing Abs mediate an array of effector functions (Brea, CA); CD27 Alexa Fluor 700 (O323), NKp80 PE (5D12), CD45RA through their interactions with Fc receptors that may poten- BV785 (HI100), CD57 allophycocyanin, Pacific Blue and FITC (HCD57), tiate protection from HIV-1 infection or inhibit viral replica- CD8 allophycocyanin-H7 (SK1), CXCR3 FITC (G025H7), KIR3DL1 tion postinfection (35–40). Still, a better understanding of Alexa Fluor 700 (DX9), and T-bet FITC (4B10) were from BioLegend by guest on September 23, 2021 effector mechanisms, such as ADCC, involved in HIV-1 (San Diego, CA); eomesodermin (Eomes) PE (WD1928), Helios eFluor450 (22F6), KIR2DL1/DS1 PerCP Cy5.5 (HP-MA4), and perforin control is needed. FITC (DG9) were from eBioscience (San Diego, CA). For assessment of In this study, we hypothesized that late-stage differentiation of transcription factors, cells were washed, permeabilized and fixed using an CD8 T cells may be associated with transcriptional changes that optimized (FOXP3 transcription factor staining buffer set) before support innate-like effector functions in the T cell compartment. intranuclear stain. Flow cytometry data were acquired with a BD LSR II instrument or a BD FACSCanto II instrument (BD Biosciences). Sorting We demonstrate, in this study, that chronic, untreated HIV-1 in- was performed on a four-laser BD FACSAria II SORP (BD Biosciences) fection is associated with the expansion of a late-stage differen- contained in a biosafety cabinet. Clinical immunophenotyping tiated CD8 T cell population expressing FcgRIIIA and that this was performed using the FACS MultiSET System and run on a FACS- population mediates HIV-specific ADCC. Furthermore, we show Calibur using the single-platform Multitest four-color reagent in combi- that the FcgRIIIA+ CD8 T cells display a hybrid CD8 T cell and nation with Trucount tubes (BD Biosciences) (47). NK cell transcriptional profile characterized by high expression of Soluble factor analysis NKp80 and the transcription factor Helios. A custom multiplex cytokine array was used to quantify 16 analytes from cryopreserved plasma, including IFN-g, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-15, IL-17, IP-10, MCP-1, TNF, and Materials and Methods TNFRII, according to the manufacturer’s instructions (Quansys Biosci- Patients and samples ences, Logan, UT). Commercial single ELISAs were used to measure a Study participants aged 15–49 y were enrolled in a prospective neopterin (GenWay Biotech, San Diego, CA), IFN- , I-FABP, and sCD14 (R&D Systems). All samples were run in triplicate, and mean values were community-based cohort to assess the prevalence and incidence of HIV-1 used for data analysis. infection in Rakai District, Uganda, from 1998 to 2004 (Table I) (41–43). Infected subjects were identified between 1997 and 2002 with continued expression analysis annual follow up through 2008. Blood samples from 103 randomly selected HIV-1 seropositive individuals and 40 community-matched Targeted analysis was performed as previously de- seronegative controls were obtained. PBMCs were then isolated and scribed (48). Cells from seven donors were stained and four pheno- cryopreserved as described previously (44). None of the patients had typically distinct cell populations (CD8 T cells: CD45RA-CD572, received antiretroviral therapy (ART). HIV-1 testing was performed as CD45RA+CD57+FcgRIIIA2,CD45RA+CD57+FcgRIIIA+,aswellas described previously (43). Positive samples were subjected to the CD56dimFcgRIIIA+ NK cells) (500–1000 cells per well) were sorted Amplicor HIV-1 Monitor test, version 1.5 (Roche Diagnostics, Indianapolis, into wells containing 10 ml of reaction buffer (SuperScript III Reverse IN). The HIV-1–infected study participants initiating ART were from the Transcriptase/Platinum Taq Mix, CellsDirect 23Reaction Mix; Invi- Couples Observation Study (COS) in Kampala Uganda as previously de- trogen). Reverse transcription and specific transcript amplification were scribed (45). The index partner in each HIV-1–serodiscordant couple was performed using a thermocycler (GeneAmp PCR System 9700; Applied followed up after the initiation of ART. Samples were collected; CD4 T cell Biosystems) as follows: 50˚C for 15 min, 95˚C for 2 min, then 95˚C for counts determined and viral load assessments made at baseline, 6 and 12 mo 15 s, 60˚C for 30 s for 18 cycles. The amplified cDNA was loaded into after initiation of ART. Biomark 96.96 Dynamic Array chips using the NanoFlex IFC controller The Journal of Immunology 3

(Fluidigm). This microfluidic platform was then used to conduct Results quantitative PCR in nL reaction volumes. Threshold cycle, as a mea- FcgRIIIA+ CD8 T cells expand in chronic untreated surement of relative fluorescence intensity, was extracted from the Biomark real-time PCR analysis software. A panel of 96 preselected HIV-1 infection related to both NK cell and CD8 T cell biology was qualified as HIV-1 negative (n = 40) and HIV-1 positive (n = 103) individuals previously described, using a script provided courtesy of Mario Roe- from a cohort in Rakai, Uganda, were chosen for the investigation derer (49). Subsequent data analysis was performed using JMP software + (version 10). Initial analyses of the transcriptome data from the Fluid- of FcgRIIIA expression in CD8 T cells (Table I). The FcgRIIIA igm Biomark confirmed the quality of 74 of the 96 genes, although data CD8 T cell population was identified as positive for CD3, TCRab, on 22 genes were discarded because of lack of amplification. CD8, and FcgRIIIA and negative for CD14, CD19, and CD4 (Fig. 1A, Supplemental Fig. 1). FcgRIIIA expression was de- ADCC assays tectable in T cells from healthy donors at a median (range) Measurement of ADCC was performed using the PanToxiLux assay frequency of 3.8% (0.7–20.7%) of CD8 T cells (Fig. 1B). In- (OncoImmunin, Gaithersburg, MD) similar to the previously described terestingly, this population was nearly doubled in HIV-1– assay (50). rHIV-1 BaL gp120 (catalog no. 4961; obtained through the National Institutes of Health AIDS Reagent Program, Division of AIDS, infected donors, in which a median frequency of 5.9% (1.3–37. National Institute of Allergy and Infectious Diseases, National Institutes of 9%)ofCD8TcellsexpressedFcgRIIIA (p , 0.001) (Fig. 1B). Health) were used to coat target CEM.NKRCCR5 cells. Optimal concen- This expansion was positively associated with the overall tration used to coat target cells was determined for each gp120 through an CD8 T cell expansion in HIV-1–infected patients (p , 0.001, 11-point titration starting with 20 mg/ml and 2-fold serial dilution. After coating CEM.NKR target cells with gp120 in 0.5% FBS–RPMI me- rho = 0.546) (Fig. 1C). The HIV-1–associated expansion of CCR5 +

dia, cells were labeled with TFL4 (OncoImmunin), a fluorescent target cell FcgRIIIA CD8 T cells was not associated with the expression Downloaded from marker, for 15 min at 37˚C and 5% CO2. Cells were then washed twice levels, measured as geometric mean fluorescence intensity 3 with 1 PBS and stained with the viability dye LIVE/DEAD Fixable Aqua (MFI), of FcgRIIIA on the surface of these cells (data not Dead Cell Stain (Life Technologies) for 30 min at room temperature. After washing in 0.5% FBS–RPMI media, cells were counted as above, then shown). There was no significant difference in FcgRIIIA ex- + resuspended to reach a final concentration of 8.0 3 105 cells/ml. At this pression levels (as measured by MFI) on FcgRIIIA CD8 T cells point, sorted effector cell populations (NK cells, CD45RA+CD57+ CD8 between HIV-1–infected and uninfected participants (data not 2 T cells, and CD45RA-CD57 CD8 T cells) were washed in 0.5% FBS– shown). Interestingly, the FcgRIIIA+ CD8 T cells were more 3 6 http://www.jimmunol.org/ RPMI media and resuspended to a final concentration of 24 10 cells/ml g 2 for an E:T ratio of 30:1. In a 96-well polypropylene plate, 25 ml of both activatedthantheirFcRIIIA counterparts, as assessed by target and effector cell suspensions were both added to each well along CD38 expression (p , 0.001) (Fig. 1D). They also expressed with 75 ml of granzyme B substrate (OncoImmunin). After incubation for less of the inhibitory receptor PD-1 (p , 0.001) (Fig. 1E). The 5 min at room temperature, 25 ml of HIV-Ig (North American Biologicals, CD38 expression levels were inversely associated with CD4 Miami, FL) at a 0.5 mg/ml dilution was added to each well, and the plate counts, albeit weakly (p = 0.02, rho = 20.367), suggesting that was incubated for another 15 min at room temperature. The plate was the FcgRIIIA+ CD8 T cells become more activated as disease then spun at 300 3 g for 1 min and placed at 37˚C and 5% CO2 for 1 h. Cells were washed twice with wash buffer and acquired on the LSR II progresses (Fig. 1F). (BD Biosciences) on the same day. Fluorophores were detected using To address the stability of the FcgRIIIA+ CD8 T cell pool over a 488-nm 50-mW laser with 515/20 filters to detect granzyme B time, we studied a second cohort of Ugandan HIV-1–infected by guest on September 23, 2021 substrate, a 406-nm 100-mW laser with 525/50 filters to detect Aqua LIVE/DEAD stain, and a 640-nm 40-mW laser with 670/30 filters to subjects (n = 32) located in Kampala, where longitudinal samples detect TFL4 stain. Because of the spectral properties of the fluorescent were available from before and after initiation of ART (Table I). molecules used in this panel, manual compensation of detected signals These patients displayed a stable population of FcgRIIIA+ CD8 was performed to analyze the data. Data were analyzed by using T cells over 12 mo of ART (Fig. 1G). However, of note, the ac- FlowJo 9.7.5 (Ashland, OR). tivation levels of these FcgRIIIA+ CD8 T cells declined over the Statistical analysis course of treatment, as measured by CD38 expression (p , 0.001) (Fig. 1H). These data show that HIV-1–infected Ugandans have an Statistical analysis was performed usingGraphPadPrism6.0(Version6) for Macintosh (GraphPad Software, La Jolla, CA) or JMP software expanded population of activated TCRab CD8 T cells expressing (version 10; SAS Institute, Cary, NC). Direct comparisons between two FcgRIIIA and that this population is stable over 12 mo of ART. groups were performed using the nonparametric Mann–Whitney U test. Next, multiplexed assays and ELISA were used to quantify a Associations between groups were determined by Spearman cor- suite of 20 soluble factors in plasma in relation to the size and relation. To correct for multiple comparisons, the Benjamini–Hochberg + false discovery rate (FDR) (51) was calculated for all observations. An activation level of the FcgRIIIA CD8 T cell population in HIV-1– FDR ,0.05 was considered statistically significant. For paired obser- infected individuals. Although none of the analytes measured vations, a paired t test was used. A p value ,0.05 was considered showed a relationship to the percentage of CD8 T cells expressing statistically significant. Flow cytometry analysis and presentation of FcgRIIIA, several markers were directly associated with the ac- distributions were performed using SPICE version 5–1.2, downloaded g + from http://exon.niaid.nih.gov/spice (52). Comparison of distributions tivation levels of Fc RIIIA CD8 T cells (i.e., cells coexpressing was performed using a Student t test and a partial permutation test as CD38) (Table II). Statistically significant correlations between the described previously (52). frequency of FcgRIIIA+ CD8 T cells expressing CD38 and plasma

Table I. Descriptive statistics for study population

HIV-1 Negative (n = 40) HIV-1 Positive (n = 103) HIV-1 Positive Initiating ART (n =32) Age (y), median (IQR) 30 (25–35) 31 (26–36) 32 (29–38) Gender, no. (%) Female 20 (50) 65 (63) 14 (44) Male 20 (50) 38 (37) 18 (56) a Viral load (log10/ml), median (IQR) NA 4.5 (4.1–5.12) 5.0 (4.1–5.3) CD4 count (cells/ml), median (IQR) NA 513 (375–670) 194 (139–240) Whole blood from these participants was used to measure the expression of CD16 on CD8 T cells and characterize their activation profile. aViral load was measured by Roche Amplicor Monitor version 1.5, limit of detection 400 copies/ml. IQR, interquartile range; NA, not applicable. 4 TERMINAL EFFECTOR FcgRIIIA+ CD8 T CELLS EXPAND IN HIV Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. FcgRIIIA+ CD8 T cells expand numerically and persist in Ugandans with untreated HIV-1 infection. (A) Bivariate pseudocolor flow cytometry plots of FcgRIIIA+ CD8 T cells after gating on small that are Aqua LIVE/DEAD2TCR a/b+, CD8+CD3+ T cells in healthy donors (HIV2)(n = 40) and HIV-1–infected (HIV+) individuals (n = 103). Overlay plots of FcgRIIIA+ CD8 T cells (in red) and bulk CD8 T cells in gray for representative HIV2 and HIV+ donors. (B) Scatter plot of the frequency of FcgRIIIA+ CD8 T cells in HIV+ versus HIV2 healthy donors with lines at the mean and SD shown. (C) Correlation of the FcgRIIIA+ CD8 T cell subset frequency with the overall CD8 compartment frequency. (D) CD38 MFI and (E) PD-1 MFI in FcgRIIIA+ CD8 T cells (orange) as compared with the overall CD8 compartment (green) with lines at the mean and SD. (F) Correlation between FcgRIIIA+ CD8 T cells and absolute CD4 T cell counts. Longitudinal graph of the FcgRIIIA+ CD8 T cell subset frequency (G) and the CD38 MFI of FcgRIIIA+ CD8 T cell subset (H) in patients starting ART (n = 32) at baseline, 6, and 12 mo after ART initiation. Gray circles and lines represent individuals and red line and outlined, filled circle represents the median level. (I) Correlation between activation levels in FcgRIIIA+ CD8 T cells and TNFRII levels in plasma. levels of the inflammatory cytokines IL-6 (p = 0.011, rho = 0.446, activation. In contrast, neither soluble markers of an innate antiviral FDR = 0.040), IP-10 (p , 0.001, rho = 0.582, FDR = 0.009), MCP-1 response, such as IFN-a, nor the common indices of microbial (p = 0.016, rho = 0.424, FDR = 0.048), TNF (p = 0.008, rho = 0.459, translocation sCD14 and IFABP were associated with the size of the FDR = 0.036), and TNFRII (p = 0.001, rho = 0.556, FDR = 0.009) FcgRIIIA+ CD8 T cell population or the extent of their activation. were observed (Fig. 1I, Table II). Similar correlations were observed + fortheMFIofCD38onFcgRIIIA+ CD8 T cells and IP-10 (p = 0.001, FcgRIIIA CD8 T cells are late-stage effector cells and rho = 0.547, FDR = 0.009), MCP-1 (p = 0.009, rho = 0.456, characterized by expression of Helios FDR = 0.032), TNF (p = 0.009, rho = 0.458, FDR = 0.032), Because of the significant expansion and activation of FcgRIIIA+ and TNFRII (p , 0.001, rho = 0.569, FDR = 0.009). Thus, CD8 T cells in HIV-1–infected individuals, we next investigated expansion and activation of the FcgRIIIA+ CD8 T cells is the detailed phenotype of these cells in HIV-infected subjects from associated with plasma markers of HIV-driven systemic immune the Rakai cohort. The combinatorial coexpression pattern of CCR7, The Journal of Immunology 5

Table II. Correlative analysis between plasma-derived soluble factors and CD16+ CD8+ T cells in HIV+ donors

CD16+CD8+ T cell (%) CD16+CD8+ CD38+ T Cell (%) CD16+CD8+ CD38+ T Cell (CD38 mfi)

Cytokine rho p Value FDR rho p Value FDR rho p Value FDR IFN-g 20.217 0.234 0.904 20.082 0.655 0.737 0.003 0.986 0.986 IL-1a 20.137 0.455 0.904 20.228 0.210 0.344 20.154 0.400 0.600 IL-1b 20.079 0.669 0.904 0.015 0.935 0.935 0.062 0.735 0.882 IL-2 NA NA NA NA NA NA NA NA NA IL-4 20.213 0.242 0.904 0.139 0.448 0.620 0.201 0.271 0.443 IL-5 20.077 0.673 0.904 0.118 0.522 0.671 0.045 0.807 0.908 IL-6 0.149 0.417 0.904 0.446 0.011 0.040 0.378 0.033 0.099 IL-8 0.067 0.714 0.904 0.032 0.864 0.915 0.018 0.921 0.975 IL-10 20.095 0.604 0.904 0.505 0.003 0.018 0.465 0.007 0.032 IL-12p70 20.034 0.854 0.904 0.338 0.059 0.133 0.290 0.108 0.216 IL-15 0.044 0.812 0.904 0.373 0.036 0.093 0.242 0.182 0.328 IL-17 NA NA NA NA NA NA NA NA NA IP-10 20.006 0.975 0.975 0.582 0.001 0.009 0.547 0.001 0.009 MCP-1 20.055 0.765 0.904 0.424 0.016 0.048 0.456 0.009 0.032 TNF-a 20.176 0.336 0.904 0.459 0.008 0.036 0.458 0.009 0.032 TNFR-II 20.199 0.276 0.904 0.556 0.001 0.009 0.569 0.001 0.009

IFABP 0.041 0.823 0.904 20.157 0.390 0.585 20.136 0.457 0.633 Downloaded from sCD14 20.077 0.674 0.904 0.307 0.087 0.157 0.312 0.082 0.200 IFN-a 20.071 0.706 0.904 0.094 0.616 0.737 0.069 0.714 0.882 Neopterin 0.106 0.563 0.904 0.317 0.077 0.154 0.305 0.089 0.200 mfi, geometric MFI; NA, not applicable. Bold indicates statistical significance, p , 0.05. http://www.jimmunol.org/ CD27, and CD45RA was significantly different between CD8 T cells KIR coexpression profile very similar to that of NK cells (p , 0.001 positive or negative for FcgRIIIA (Fig. 2A, Supplemental Table I) for FcgRIIIA+ CD8 T cells in HIV-1–uninfected donors compared (p , 0.001). Expression of CD45RA in the absence of CCR7 and with HIV-1–infected donors; p =0.250forFcgRIIIA+ CD8 T cells CD27 was the dominant pattern among the FcgRIIIA+ CD8 T cells, compared with NK cells in HIV-1–infected donors). consistent with a terminally differentiated status, whereas this phe- T cell differentiation and maturation are controlled by a set of notype was less common among CD8 T cells lacking FcgRIIIA (74% transcription factors, including T-bet, Eomes, and Helios. PBMC from versus 18%, respectively) (p , 0.001). Next, the expression patterns HIV-infected donors were stained intracellularly for these transcription of CD57, NKG2A, and NKG2D were evaluated, and the frequency of factors, and their expression patterns were analyzed in CD8 T cells the subsets defined by these receptors were different in CD8 T cells lacking or expressing FcgRIIIA, as well as in NK cells (Fig. 3A). by guest on September 23, 2021 expressing FcgRIIIA compared with those that did not (Fig. 2B) FcgRIIIA+ CD8 T cells displayed a T-bet, Eomes, and Helios ex- (p , 0.001). The majority of FcgRIIIA+ CD8 T cells expressed CD57 pression pattern distinct from both the general CD8 T cell population while maintaining NKG2D expression. In fact, all Boolean subsets and from CD56dim NK cells, with higher levels of coexpression as containing CD57 expressing cells were higher in FcgRIIIA+ CD8 compared with FcgRIIIA2 CD8 T cells. Coexpression of all three T cells compared with FcgRIIIA2 CD8 T cells (all p # 0.001) (data transcription factors was common in FcgRIIIA+ CD8 T cells and also not shown). The next panel examined CD161 and perforin, and relatively frequent in NK cells but uncommon in the general CD8 comparison of the distribution of cell subsets expressing combina- T cell pool. Notably, 61% of the FcgRIIIA+ CD8 T cells tions of these two markers again revealed differences between expressed Helios, and this was significantly higher compared the FcgRIIIA+ and FcgRIIIA2 CD8 T cells (Fig. 2C) (p , 0.001). with the FcgRIIIA2 CD8 T cells and NK cells (p , 0.001), in The vast majority of FcgRIIIA+ CD8 T cells expressed perforin which a median of 10 and 28% expressed Helios, respectively. as compared with∼20% of FcgRIIIA2 CD8 T cells. In summary, Characterization of T-bet and Eomes can be discriminated, based FcgRIIIA+ CD8 T cells are distinct from their FcgRIIIA2 CD8 on a continuum of expression and varies on lymphocyte subsets (53). T cell counterparts by lack of CD27 expression, higher proportion of FcgRIIIA+ CD8 T cells were dominated by a high T-bet expression cells expressing CD57, and their predominantly perforin positivity. profile with variable Eomes expression that was very similar to The patterns of expression of maturation markers observed in CD16+ NK cells (Fig. 3B, 3C). HIV-1 infection status had minimal FcgRIIIA+ CD8 T cells in HIV-1–infected donors were not signifi- effect on T-bet and Eomes in these populations. FcgRIIIA2 CD8 cantly different from HIV-1–uninfected control subjects (all p values T cells showed a much more variable expression pattern of both .0.05) (data not shown), suggesting that the elevated levels of transcription factors, which may reflect the different states of mat- FcgRIIIA+ CD8 T cells in infected individuals represent an expansion uration and differentiation within this compartment. of a phenotypic cell subset retaining relatively normal characteristics. Altogether, these data indicate that the FcgRIIIA+ CD8 T cell To address this question further, we investigated the expression of population expanded in HIV-1–infected people is characterized by killer Ig-like receptors (KIRs) in CD8 T cells and NK cells expressing Helios expression and has a late-stage differentiated effector FcgRIIIA, as well as in late-stage differentiated CD8 T cells defined phenotype. This population mostly retains the characteristics seen by coexpression of CD45RA and CD57 and memory CD8 T cells in healthy donors as it expands during HIV-1 infection, although negative for these markers (Fig. 2D). In uninfected donors, T cell KIR expression is significantly elevated. populations lacking FcgRIIIA had low levels of KIR expression, + whereas NK cells had high KIR levels in diverse combinations. The FcgRIIIA CD8 T cell transcriptome reveals a mixed The FcgRIIIA+ CD8 T cells displayed a pattern intermediate be- effector CD8 T cell and NK cell character tween T cells and NK cells. Strikingly, this pattern was altered in To better understand the identity of the FcgRIIIA+ CD8 T cells, we HIV-1–infected subjects whose FcgRIIIA+ CD8Tcellshadadopteda next analyzed their transcriptional profile by Fluidigm Biomark. A 6 TERMINAL EFFECTOR FcgRIIIA+ CD8 T CELLS EXPAND IN HIV Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. FcgRIIIA+ CD8 T cells display a late-stage effector phenotype in chronic untreated infection. A detailed phenotype of FcgRIIIA+ CD8 T cells after gating on small lymphocytes, singlets, Aqua LIVE/DEAD2, CD8+CD3+ T cells in HIV+ (n = 15) and HIV2 (n = 15) individuals was examined. (A) Expression of CD27, CCR7, and CD45RA in CD8 T cell subsets having or lacking FcgRIIIA surface expression. (B) Expression of CD57, NKG2A, and NKG2D in CD8 T cell subsets having or lacking FcgRIIIA surface expression. (C) Expression of CD161 and perforin in CD8 T cell subsets having or lacking FcgRIIIA surface expression. (D) Analysis of KIR surface expression patterns in CD45RA+CD57+FcgRIIIA+ CD8 T cells, CD45RA+CD57+FcgRIIIA2 CD8 T cells, CD56dim NK cells, and CD45RA2CD572 CD8 T cells. panel of 96 genes involved in T cell function or NK cell function per population: 1) CD45RA+CD57+ CD8 T cells expressing was selected (Supplemental Table II), and the expression of these FcgRIIIA, 2) CD45RA+CD57+ CD8 T cells lacking expression genes was analyzed in cell populations purified by flow cytometry of FcgRIIIA, 3) CD45RA-CD572 CD8 T cells not expressing sorting. For these analyses, cells from seven HIV-1–infected FcgRIIIA, and 4) CD56dimCD16+ NK cells. The data for 74 out donors were sorted into four populations, 500–1000 cells of the 96 genes passed quality control, and principal component The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Transcription factors T-bet, Eomes, and Helios expression in CD8 T cells with or without FcgRIIIA and NK cells. In HIV+ (n = 10) and HIV2 (n = 10) individuals (A) expression of T-bet, Eomes, and Helios, assessed by intracellular staining, is presented for CD8 T cell subsets having or lacking FcgRIIIA (CD16) surface expression in comparison with NK cells. FcgRIIIA2 CD8 T cells (blue), FcgRIIIA+ CD8 T cells (green), and NK cells (purple) are displayed on a bar graph with individual points shown. Asterisks denote statistically significant differences of the FcgRIIIA+ CD8 T cells, with a p value ,0.05). Major populations expressing all three transcription factors (orange box) and positive for T-bet and Eomes in the absence of Helios (lavender) are presented and correspond to slices of the pie chart. Individual expression of each transcription factor is shown by an arc (Eomes in red, Helios in gold, and T-bet in green). A p value is presented for comparison of distribution of each part of the pie between groups. (B) Example flow cytometry plots showing the coordinated expression of T-bet and Eomes for FcgRIIIA2 CD8 T cells, FcgRIIIA+ CD8 T cells, and NK cells. (C) Scatter plot for FcgRIIIA2 CD8 T cells (blue), FcgRIIIA+ CD8 T cells (green), and NK cells (purple) for subsets of cells expressing high, medium, or low levels of T-bet and positive or negative for Eomes, with lines at the mean and SD shown. Line with * denotes statistical significance between cell populations. *p , 0.05. analysis (PCA) was performed on the total data set of expres- overlapped with both the CD45RA+CD57+ CD8 T cells lacking sion of these 74 genes in all four of the cell subsets (Fig. 4A). expression of FcgRIIIA and the CD56dimCD16+ NK cells, Notably, the transcriptional profile of FcgRIIIA+ CD8 T cells whereas the CD45RA-CD572 memory CD8 T cell subset was 8 TERMINAL EFFECTOR FcgRIIIA+ CD8 T CELLS EXPAND IN HIV Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Transcriptome analysis reveals a mixed CD8 T cell and NK cell character in the FcgRIIIA+ CD8 T cells. Supervised expression analysis of 74 genes involved in the regulation and function of innate and adaptive immune responses in seven HIV-1–infected donors using the Fluidigm Biomark system. (A) PCA of the transcriptional data from four sorted cell populations reflecting CD45RA2CD572 (blue), CD45RA+CD57+FcgRIIIA2 (red), CD45RA+CD57+FcgRIIIA+ (green), as well as CD56dimFcgRIIIA+ NK cells (purple). Polygons represent 95% confidence intervals in the data. (B) Ex- pression of 10 selected genes in the same sorted subsets. (C) Successive flow cytometry gating strategy used for confirmation of IL-7Ra and KLRF1 genes at the level. Offset histograms showing the relative expression of IL-7Ra (CD127) and KLRF1 (NKp80) on CD8 T cells: CD45RA2CD572 (blue), CD45RA+CD57+FcgRIIIA2 (red), CD45RA+CD57+FcgRIIIA+ (green/filled), as well as CD56dimFcgRIIIA+ NK cells (purple). most distant. Principal component 1 contributed 26% of the pattern intermediate between FcgRIIIA2 CD45RA+CD57+ CD8 variability in the data set, and component 2 contributed 14.7%. TcellsandtheCD56dimCD16+ NK cells. In fact, KLRF1 encoding Expression of genes GZMB, LAIR1, GZMK, PRF1,andCD244 the NK cell–associated receptor NKp80, expressed at the highest contributed most to principal component 1, and genes GZMK, levels by CD56dimCD16+ NK cells, was expressed at significantly IL-6ST, TGFB1, CD38,andCD160 contributedmosttoprin- higher levels when compared with the FcgRIIIA2 terminal effector cipal component 2. CD8 T cells and effector memory CD8 T cells. Compared with their A subset of genes showed expression patterns that segregated the FcgRIIIA2 counterparts, the FcgRIIIA+ CD8 T cells also expressed FcgRIIIA+ CD8 T cell population from the NK cells and the higher levels of genes involved in regulating T cell function, in- FcgRIIIA2 CD8 T cell populations (Fig. 4B, Supplemental Fig. 2). cluding TNFSF13B. Additionally, the FcgRIIIA+ CD8 T cells had Notably, the FcgRIIIA+ CD8 T cell displayed significantly higher significantly lower expression of TGFBR1 than the CD56dim NK IKZF2 expression than any of the three other reference populations cells, but levels were above that of the other CD8 T cells pop- and lower IL-7R expression than the other T cell populations and at ulations. Altogether, the gene expression analysis indicates that levels similar to CD56dimCD16+ NK cells. Regarding a range of FcgRIIIA+ CD8 T cells have a transcriptional profile intermediate genes encoding NK cell–associated receptors, including KIR2DS2; between effector CD8 T cells and CD56dim NK cells. KIR2DS1, KIR3DL1;KIR3DS1, KLRC2L;KLRC3, KLRD1, KLRF1, Because of the distinct transcriptional signature of FcgRIIIA+ CD8 KLRK1;KLRC4-1,andNCR1,theFcgRIIIA+ CD8Tcellsshoweda T cells, we were interested in confirming expression of the IL-7R and The Journal of Immunology 9

KLRF1 genes at the protein level. We further examined 10 chronically indicating that nonneutralizing Ab-mediated effects may contrib- HIV-1–infected and 10 uninfected individuals for surface expression ute to HIV vaccine efficacy have spurned a renewed interest in of these receptors by flow cytometry. The majority of FcgRIIIA+ ADCC as a protective mechanism (54, 55). The present observa- CD45RA+CD57+ CD8 T cells expressed NKp80 (median 68%) and tion that HIV-1 infection drives the expansion of late-stage ef- lacked expression of the IL-7 receptor, CD127 (median 2%) (Fig. 4C). fector CD8 T cells with a hybrid NK cell–CD8 T cell character, No differences were observed in FcgRIIIA+ CD45RA+CD57+ CD8 including FcgRIIIA and lytic protein expression, suggests that T cells expressing NKp80 or IL-7Ra between HIV-1 positive and CD8 T cells might actually mediate ADCC. To test this possibility, negative individuals, and no relationship was observed between ex- effector cell populations from HIV-1–infected donors were sorted pression and markers of HIV-1 disease progression. IL-7Ra protein by flow cytometry, and the ability of these cells to mediate ADCC + + expression was similar between NK cells and CD45RA CD57 CD8 against HIV BaL gp120-coated CEM.NKRCCR5 target cells was T cells, irrespective of FcgRIIIA+ expression. Interestingly, NKp80 evaluated by the PanToxiLux granzyme B substrate cytotoxicity was only found at appreciable levels in the T cells with the FcgRIIIA+ assay (Fig. 5A). To avoid FcgRIIIA downregulation or blocking CD45RA+CD57+ phenotype. Together, the FcgRIIIA+ CD8 T cells because of staining, CD45RA+CD57+ CD8 T cells were sorted to have a distinct NKp80+ IL-7Ra2 character different from other enrich for FcgRIIIA+ cells (9–21% FcgRIIIA+) and then com- effector CD8 T cells and more akin to CD56dim NK cells. pared with FcgRIIIA2 CD45RA2CD572 memory CD8 T cells

+ and with NK cells sorted from the same donors. In the pres- Potent HIV-specific ADCC activity mediated by FcgRIIIA ence of HIV-Ig, the CD45RA+CD57+ cells from three HIV+ CD8 T cells donors clearly mediated ADCC, as did the NK cells, whereas 2 2 ADCC is part of the repertoire of effector functions employed by the CD45RA CD57 CD8 T cell population did not (Fig. 5A, 5B). Downloaded from NK cells to detect and target HIV-1–infected cells. Recent data As such, bulk CD45RA+CD57+ CD8 T cells performed ADCC http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. HIV-specific ADCC mediated by FcgRIIIA+ CD8 T cells. (A) Representative FACS plots of the cytolysis PanToxiLux assay from HIV+ (n = 3) individuals. (B) HIV-1 gp120-specific ADCC mediated by HIV-Ig. (C)ComparisonofADCC mediated by FcgRIIIA+ CD8 T cells and NK cells on a per FcgRIIIA+ cell basis. 10 TERMINAL EFFECTOR FcgRIIIA+ CD8 T CELLS EXPAND IN HIV lower than the NK cells (Fig. 5B). However, after adjusting for the protein levels for KLRF1, encoding the activating NKp80 receptor, frequency of FcgRIIIA expression in these populations, 9–21% in were expressed at high levels similar to NK cells compared with CD45RA+CD57+ CD8 T cells and 69–96% in CD56dim NK cells, effector memory or FcgRIIIA2 CD8 T cells. NKp80 has recently ADCC capacity of FcgRIIIA+ CD8 T cells was similar to that of been shown to associate with the development and maturation FcgRIIIA+ NK cells (Fig. 5C). Interestingly, the FcgRIIIA+ MFI of fully functional NK cells (60). Whereas FcgRIIIA+ CD8 T cells on FcgRIIIA+ CD8 T cells was significantly lower compared show some features similar to CD56dim NK cells, PCA revealed with FcgRIIIA+ MFI on CD56dim NK cells (p , 0.001). that FcgRIIIA+ CD8 T cells, FcgRIIIA2 CD8 T cells, and FcgRIIIA+ CD8 T cell ability to mediate ADCC, based on normal- CD56dim NK cells were distinct from the CD45RA2CD572 ized FcgRIIIA+ MFI or the integrated MFI (frequency multiplied by memory CD8 T cell population. Consistent with this notion, when the MFI), was as good as NK cells (data not shown). These data the genes differentially expressed between the FcgRIIIA+ CD8 demonstrate that the FcgRIIIA+ CD8 T cell population expanding T cell and the effector memory T cell population were entered into during chronic HIV-1 infection can mediate HIV-specific ADCC the Reactome pathway analysis database, the DAP12 pathway, at levels comparable to NK cells. implicated in activation of NK cells, was indicated as enriched in the FcgRIIIA+ CD8 T cells (Supplemental Fig. 2, Supplemental Discussion Table II) (61, 62). Furthermore, we observed the upregulation of CD8 T cells use a range of effector functions to combat viral 10 genes in FcgRIIIA+ CD8 T cells compared with the effector infections, including cytolysis and effects mediated by cytokines memory CD8 T cell population that are associated with NK-like and chemokines. A hallmark of these antiviral functions is that they rapid effector function and the “innateness gradient” defined by depend on the exquisite Ag specificity of TCRs and their recog- Gutierrez-Arcelus et al. (63) including GZMB, PRF1, KIR3DL1, Downloaded from nition of viral Ag in an MHC-restricted manner. In this study, we KLRK1, KLRD1, KLRF1, NCR1, KLRC2L;KLRC3, KIR2DS2, and demonstrate that late-stage effector CD8 T cells acquire FcgRIIIA ITGAM (Supplemental Fig. 2, Supplemental Table II). Although expression in HIV-1–infected individuals and use this Fc receptor the overall pattern is that FcgRIIIA+ CD8 T cells overlap with to mediate HIV-specific ADCC in the absence of TCR recognition both FcgRIIIA2 T cells and CD56dim NK cells, these cells also of Ag. Using a commercial in vitro assay, commonly used in manifest distinctive features somewhere between innate and

assessing HIV-1 ADCC activity (50), we measured the effector adaptive immune cells (64). A pattern that stands out is the high http://www.jimmunol.org/ capacity, on a per cell basis, of FcgRIIIA+ CD8 T cells to mediate expression by FcgRIIIA+ CD8 T cells of the transcription factor Ag-specific ADCC against gp120-coated targets as efficiently as Helios, encoded by the IKZF2 gene, both at the protein and gene NK cells from the same donors. These findings indicate that in the levels. These cells also have very low expression of IL-7Ra. The context of chronic uncontrolled HIV-1 infection, a significant low IL-7Ra expression level is consistent with a model in which subset of CD8 T cells acquires innate characteristics and performs these cells are either maintained by non–IL-7–dependent factors a function in the immune system normally associated with NK or, rather, short-lived in vivo. Our finding that patients initiating cells. Functional diversification of adaptive CD8 T cells may be ART largely maintain the expanded FcgRIIIA+ CD8 T cell important as therapeutic strategies evolve to include Ab-mediated population over 12 months suggests that these cells are not mechanisms to eliminate HIV-1 reservoirs (56–58). intrinsically short-lived, and thus may even be maintained by by guest on September 23, 2021 In the Ugandan population studied in this work, expression of IL-7–independent mechanisms. This interpretation is sup- FcgRIIIA occurs on∼5% of CD8 T cells from healthy donors, and ported by the recent finding of expansion of long-lived effector this frequency is doubled in patients with chronic untreated HIV-1 CD45RA+ CD8 T cells that are IL-7Rlo KLRG1high in latent CMV infection. In fact, some patients have more than 30% of their CD8 and EBV infection, a population which phenotypically overlaps T cells expressing FcgRIIIA. The finding that the size of this with the FcgRIIIA+ CD8 T cell identified in this study (65). population is positively associated with the global CD8 T cell The expansion of FcgRIIIA+ CD8 T cells we observe in this expansion in these patients suggests that the FcgRIIIA+ CD8 study is reminiscent of the expansion of CD8 T cells with a similar T cells expand in response to the chronic uncontrolled viral rep- phenotype in hepatitis C virus (HCV)–infected patients (23). lication. These expanded cell populations have a terminally dif- Whereas HIV-1 and HCV differ in target cell tropism and mech- ferentiated phenotype with frequent expression of CD45RA, anisms of pathogenesis, for example, they have in common es- CD57, and perforin but little expression of CD27 and CCR7, tablishment of chronic infections that are very difficult for the further supporting this notion. The phenotypic profile of these immune system to control. This is partly because of the shared cells is similar between HIV-1–infected patients and healthy do- features of rapid viral replication and high mutation rates. These nors [data not shown and (23)]. However, we found one exception features lead to selection of immune escape variants that to this observation; the FcgRIIIA+ CD8 T cells adopt a KIR ex- allow these viruses to avoid efficient recognition by clonally ex- pression profile similar to NK cells in HIV-1–infected subjects, an panded populations of T cells. Viral quasispecies mutate away observation not seen in healthy donors (Fig. 2D). The conditions from the originally transmitted viral sequence under T cell se- in vivo during HIV-1 infection thus seem to drive not only an lection pressure and some of the early responding epitope-specific expansion of these cells but also expression of surface receptors T cell populations may thus lose their efficiency in targeting in- beyond FcgRIIIA normally associated with NK cells and reflec- fected cells. Future studies are warranted to test the hypothesis tive of the rise in terminally differentiated CD8 T cells in chronic that accumulation of FcgRIIIA+ CD8 T cells may be a clonally viral infections (59). driven process and this could be addressed by TCR repertoire These functional and, to some extent phenotypic, similarities analysis. The FcgRIIIA+ CD8 T cells have a phenotype that would with NK cells led us to ask how the FcgRIIIA+ CD8 T cells relate be expected from a T cell population expanded by Ag recognition, to FcgRIIIA2 T cell subsets as well as FcgRIIIA+ NK cells on the because they are largely negative for CD27 and CCR7, but posi- transcriptional level. Based on a supervised transcriptional anal- tive for CD57, perforin and CD45RA. In the yellow fever virus ysis of 74 genes in seven donors, the FcgRIIIA+ CD8 T cells vaccine model, the yellow fever vaccine–specific CD8 T cells are appear to have a transcriptional program intermediate between CD45RO+ during the peak of the effector response and then revert late-stage effector CD8 T cells lacking FcgRIIIA and CD56dim back to CD45RA expression as the Ag is cleared and memory is NK cell expressing FcgRIIIA. Most interestingly, transcript and established (66, 67). This is consistent with a model in which The Journal of Immunology 11

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Supplemental Material

Supplemental Table 1. Boolean subset comparison across groups (Wilcoxon Rank Test Result)

1. HIV+ CCR7, CD27, and CD45RA expression Category P-value CD8 CD16+ CCR7 CD27 CD45RA vs. CD16- + + + 0.000 + + - 0.000 + - + 0.000 + - - 0.013 - + + 0.001 - + - 0.000 - - + 0.000 - - - 0.038

2. HIV+ CD57, NKG2A, and NKG2D expression Category P-value CD8 CD16+ CD57 NKG2A NKG2D vs. CD16- + + + 0.000 + + - 0.001 + - + 0.000 + - - 0.000 - + + 0.281 - + - 0.130 - - + 0.000 - - - 0.000

3.HIV+ Perforin and CD161 expression Category P-value CD8 CD16+ vs. CD16- NKG2A NKG2D + + 0.000 + - 0.000 - + 0.000 - - 0.000

4. HIV+ Transcription Factors (Eomesodermin, Helios, and T-bet) expression Category P-value CD8 CD16+ FDR of CD8 CD16+ P-value CD8 CD16+ FDR of CD8 CD16+ Eomes Helios T-bet vs. CD16- vs. CD16- vs. NK cell vs. NK cell + + + 0.000 0.001 0.019 0.0338 + + - 0.028 0.045 0.000 0.0015 + - + 0.007 0.014 0.001 0.0023 + - - 0.112 0.138 0.000 0.0015 - + + 0.001 0.002 0.000 0.0015 - + - 0.880 0.880 0.496 0.5669 - - + 0.059 0.079 0.049 0.0713 - - - 0.001 0.002 0.597 0.6368

5. HIV+ Killer Immunoglobulin receptor (KIR) Expression Category P-value CD8+CD16+ FDR of CD8+CD16+ P-value CD8+CD16+ FDR of CD8+CD16+ P-value CD8+CD16+ FDR of CD8+CD16+ 2DL1/DS1 2DL2/3/DS2 KIR3DL1 vs. CD8+CD16- vs. CD8+CD16- vs. CD8+CD45- vs. CD8+CD45- vs. NK cell vs. NK cell + + + 0.208 0.357 0.462 0.504 0.001 0.005 + + - 0.793 0.827 0.248 0.372 0.016 0.038 + - + 0.916 0.916 0.401 0.481 0.001 0.005 + - - 0.462 0.504 0.401 0.481 0.027 0.059 - + + 0.248 0.372 0.141 0.260 0.012 0.032 - + - 0.093 0.186 0.001 0.005 0.294 0.392 - - + 0.003 0.012 0.012 0.032 0.294 0.392 - - - 0.005 0.017 0.001 0.005 0.001 0.005

6. CD8+CD16+ Killer Immunoglobulin receptor (KIR) Expression Category P-value HIV- vs. 2DL1/DS1 2DL2/3/DS2 KIR3DL1 HIV+ + + + 0.058 + + - 0.142 + - + 0.270 + - - 0.426 - + + 0.298 - + - 0.178 - - + 0.951 - - - 0.006

1 Version 29 (16 August 2019) Journal of Immunology

Supplemental Table 2. Gene expression RT-PCR reagents Gene symbol Gene Name Gene ID Assay ID CD226 CD226 molecule 10666 Hs00170832_m1 EOMES eomesodermin 8320 Hs00172872_m1 NCR1 natural cytotoxicity triggering receptor 1 9437 Hs00183118_m1 IKZF2 IKAROS family zinc finger 2 (Helios) 22807 Hs00212361_m1 KLRF1 killer cell -like receptor subfamily F, member 1 51348 Hs00212979_m1 LAIR1 leukocyte-associated immunoglobulin-like receptor 1 3903 Hs00253790_m1 ITGAM integrin, alpha M (complement component 3 receptor 3 subunit) 3684 Hs00355885_m1 KIR2DL4;LOC100287534 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 4 3805 Hs00427106_m1 killer cell immunoglobulin-like receptor 2DL4-like 100287534 KIR3DL2 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 3812 Hs00601497_gH KIR3DX1 killer cell immunoglobulin-like receptor, three domains, X1 90011 Hs00736082_g1 KIR3DL1;KIR3DS1 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 3811 Hs00744448_s1 killer cell immunoglobulin-like receptor, three domains, short cytoplasmic tail, 1 3813 IL7R interleukin 7 receptor 3575 Hs00902334_m1 KLRG1 killer cell lectin-like receptor subfamily G, member 1 10219 Hs00929964_m1 IL2RG interleukin 2 receptor, gamma 3561 Hs00953624_m1 IKZF1 IKAROS family zinc finger 1 (Ikaros) 10320 Hs00958474_m1 IL18R1 interleukin 18 receptor 1 8809 Hs00977691_m1 IL18RAP interleukin 18 receptor accessory protein 8807 Hs00977695_m1 GZMA granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) 3001 Hs00989184_m1 IFNG interferon, gamma 3458 Hs00989291_m1 TLR6 toll-like receptor 6 10333 Hs01039989_s1 LAIR2 leukocyte-associated immunoglobulin-like receptor 2 3904 Hs01102514_m1 LILRB1 leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 10859 Hs01848117_s1 KIR2DL3;KIR2DL2;KIR2DS2 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 3 3804 Hs03407415_gH killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 3803 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 100132285 KIR2DS2;KIR2DL5A;KIR2DL2;KIR2DL3;KIR2DS4 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 100132285 Hs04190776_gH killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 3803 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 3 3804 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 5A 57292 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 4 3809 KIR2DS2;KIR2DS1;KIR2DS3;KIR2DL1;KIR2DS4 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 100132285 Hs04190778_mH killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 1 3802 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 1 3806 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 3 3808 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 4 3809 KIR2DS3;KIR2DS5 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 3 3808 Hs04190781_mH killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 5 3810 KIR2DS3;KIR2DL2;KIR2DS2;KIR2DS5;KIR2DL3 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 3 3808 Hs04190782_gH killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 3803 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 3 3804 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 100132285 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 5 3810 KLRC2;KLRC3 killer cell lectin-like receptor subfamily C, member 2 3822 Hs04192492_gH killer cell lectin-like receptor subfamily C, member 3 3823 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 8743 Hs00921974_m1 GZMK granzyme K (granzyme 3; tryptase II) 3003 Hs00157878_m1 IL12RB1 interleukin 12 receptor, beta 1 3594 Hs00538167_m1 IL12RB2 interleukin 12 receptor, beta 2 3595 Hs01548202_m1 TLR8 toll-like receptor 8 51311 Hs00152972_m1 LAMP1 lysosomal-associated 1 3916 Hs00174766_m1 NCR2 natural cytotoxicity triggering receptor 2 9436 Hs00183113_m1 ICOS inducible T-cell co-stimulator 29851 Hs00359999_m1 PDCD1 programmed cell death 1 5133 Hs01550088_m1 TLR1 toll-like receptor 1 7096 Hs00413978_m1 CD33 CD33 molecule 945 Hs01076281_m1 CXCR1 chemokine (C-X-C motif) receptor 1 3577 Hs00174146_m1 TLR3 toll-like receptor 3 7098 Hs01551078_m1 IL10 interleukin 10 3586 Hs00961622_m1 TBX21 T-box 21 30009 Hs00203436_m1 CXCR3 chemokine (C-X-C motif) receptor 3 2833 Hs00171041_m1 KLRK1;KLRC4-KLRK1 killer cell lectin-like receptor subfamily K, member 1 22914 Hs00183683_m1 KLRC4-KLRK1 readthrough 100528032 IRF7 interferon regulatory factor 7 3665 Hs01014809_g1 KLRC1 killer cell lectin-like receptor subfamily C, member 1 3821 Hs00970273_g1 CCL3 chemokine (C-C motif) ligand 3 6348 Hs00234142_m1 LILRB2;LILRB1 leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 2 10288 Hs00601427_g1 leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 10859 CCR7 chemokine (C-C motif) receptor 7 1236 Hs01013469_m1 MKI67 antigen identified by monoclonal antibody Ki-67 4288 Hs01032443_m1 B3GAT1 beta-1,3-glucuronyltransferase 1 ( P) 27087 Hs00218629_m1 BIRC3 baculoviral IAP repeat containing 3 330 Hs00154109_m1 CCL2 chemokine (C-C motif) ligand 2 6347 Hs00234140_m1 CCL4 chemokine (C-C motif) ligand 4 6351 Hs99999148_m1 CCL5 chemokine (C-C motif) ligand 5 6352 Hs00174575_m1 BCL2 B-cell CLL/lymphoma 2 596 Hs99999018_m1 CD244 CD244 molecule, natural killer cell receptor 2B4 51744 Hs00900271_m1 CD38 CD38 molecule 952 Hs01120071_m1 CD69 CD69 molecule 969 Hs00934033_m1 CLEC2B C-type lectin domain family 2, member B 9976 Hs00192860_m1 CTSB cathepsin B 1508 Hs00947433_m1 CXCR4 chemokine (C-X-C motif) receptor 4 7852 Hs00237052_m1 CXCR6 chemokine (C-X-C motif) receptor 6 10663 Hs00174843_m1 DPP4 dipeptidyl-peptidase 4 1803 Hs00175210_m1 ENTPD1 ectonucleoside triphosphate diphosphohydrolase 1 953 Hs00969559_m1 FASLG (TNF superfamily, member 6) 356 Hs00181225_m1 CD160 CD160 molecule 11126 Hs00199894_m1 GAPDH glyceraldehyde-3-phosphate dehydrogenase 2597 Hs99999905_m1 GNLY granulysin 10578 Hs00246266_m1 GZMB granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) 3002 Hs01554355_m1 HLADRA major histocompatibility complex, class II, DR alpha 3122 Hs00219575_m1 IL16 interleukin 16 3603 Hs00189606_m1 IL21R interleukin 21 receptor 50615 Hs00222310_m1 IL2Ra interleukin 2 receptor, alpha 3559 Hs00907777_m1 IL6ST interleukin 6 signal transducer (gp130, oncostatin M receptor) 3572 Hs00174360_m1 KLRD1 killer cell lectin-like receptor subfamily D, member 1 3824 Hs00233844_m1 LGALS9 lectin, galactoside-binding, soluble, 9 3965 Hs00371321_m1 KLRF1 killer cell lectin-like receptor subfamily F, member 1 51348 Hs00212979_m1 NCR3 natural cytotoxicity triggering receptor 3 259197 Hs00394809_m1 POLR2A polymerase (RNA) II (DNA directed) polypeptide A, 220kDa 5430 Hs00172187_m1 PRDM1 PR domain containing 1, with ZNF domain 639 Hs00153357_m1 PRF1 perforin 1 (pore forming protein) 5551 Hs00169473_m1 PSMG2 proteasome (prosome, macropain) assembly chaperone 2 56984 Hs00220315_m1 SLAMF5 CD84 molecule 8832 Hs01547121_m1 SLAMF7 SLAM family member 7 57823 Hs00221793_m1 SMAD4 SMAD family member 4 4089 Hs00232068_m1 SPATA2 spermatogenesis associated 2 9825 Hs00195835_m1 TGFB1 transforming growth factor, beta 1 7040 Hs00998133_m1 NKG7 natural killer cell group 7 sequence 4818 Hs01120688_g1 TGFBR1 transforming growth factor, beta receptor 1 7046 Hs00610318_m1 TIA1 TIA1 cytotoxic granule-associated RNA binding protein 7072 Hs00254561_m1 TNF tumor necrosis factor 7124 Hs00174128_m1 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 7128 Hs00234713_m1 TNFRSF9 tumor necrosis factor receptor superfamily, member 9 3604 Hs00155512_m1 TNFSF13B tumor necrosis factor (ligand) superfamily, member 13b 10673 Hs00198106_m1

2 Version 29 (16 August 2019) Journal of Immunology

A. Initial CD8 CD16+ survey Donor 1 Donor 2

60.5 24.2 250K 52.5 14.4 250K 5 250K 105 105 10 105 42.4

200K 200K 200K 75.9 4 4 4 A 4

A 10 A 10 10 10

150K 150K 150K 37.2

5.18 C- C-

C- 3 1b FITC 03 103 10 103 100K 100K 100K

2 SS 59.4 2 2 SS 2 SS 10 10 10 10 50K 50K 92.8 50K 44.5 0 0 0 0 CRa

CD38 APC 10.5 4.78 CD38 APC 32.1 0.974 0 0 0 T CD8 PE-Cy7 2 3 4 5 2 3 4 5 0 50K 100K 150K 200K 250K 0 102 103 104 105 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 FSC-A Aqua CD3 PPCy5.5 CD16 PB PD-1 PE CD16 PB PD-1 PE B. Homing Panel

250K 250K 250K 43.9 105 5 250K 10 250K 250K 250K

200K 200K 200K 200K 97.6 4 200K A 10 200K 10 4 5.5 200K A A

150K 150H K 150K 150K A 150K 150K 150K 99.7 C- 103 10 3 C- 100K 100K 100K C- 61.1 100K

C- 100K 100K 2 100K 2

SC- 10

SS 10 50K 50K 50K 8 PE-Cy7 50K SS SS 50K F 0 50K 50K 0 0 0 0 SS 42.8 0 36.6 6.12 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 102 103 104 105 0 102 103 104 105 43.9 0 CD 0 102 103 104 105 0 0 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 FSC-A FSC-A Aqua CD3 AH7 CD16 PE-Cy5 CD8 PE-Cy7 250K 250K 250K 250K

A 200K 200K 200K 200K

150K

C- 150K 150K 150K

100K 100K 100K 100K SS

50K 50K 50K 50K 74.8 54.1 1.07 57.8 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 0 10 10 10 10 CCR5 BV421 CXCR3 FITC PD-1 AF647 TRAIL C. NK cell receptor, perforin, and IL-7 receptor Panel

250K 250K 250K 34.3 250K 250K 250K 105 105 250K 3.39

200K 200K 200K 200K 200K 200K 200K 104 104 A A A A A A H 150K 150K 150K 150K 150K 150K 150K 97.4

3 6 PB 3 C- C- C- C- C- 69.4 C- 10 10 100K 100K 100K 100K 100K 100K 100K SC- 2 2 50K 50K 50K 10 10 SS SS SS SS SS SS F 50K 50K 50K 50K 0 0 84 CD1 20 0 0 0 0 13.8 0.0863 3.06 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 10 10 10 10 CD8 PE-Cy7 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 FSC-A FSC-A Aqua CD3 AH7 CD8 PE-Cy7 IL-7R AF647 Perforin FITC NKp46 PE CD161 PPCy5.5

D. NK cell receptor

250K 250K 250K 5 5 10 44.4 10 250K 47.3 3.11 105 200K 200K 200K 104 10 4 5.5 200K 104 A A A H 96.4 150K 150K 150K 88.8 59.7 3 3 150K 10 10 6 PB 3 C- C- C- 10 100K 100K 100K 7 FITC

SC- 2 100K 102 10

8 PE-Cy7 2 SS SS SS F 10 50K 50K 50K 0

CD1 0 0 50K CD5 CD 0 0 0 84.1 46.9 2.68 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 10 3 10 4 105 0 102 103 104 105 0 10 2 10 3 10 4 10 5 2 3 4 5 0 102 103 104 105 0 10 10 10 10 FSC-A FSC-A Aqua CD3-AH7 CD8 PE-Cy7 NKG2A APC NKG2D PerCP

E. Differentiation/Maturation Panel

250K 250K 250K 250K 5 5 10 45.9 10 5 24.7 7.87 2.14 10 200K 200K 200K 200K 4 4 10 10 104 A A A 150K H 150K 150K 150K 3 103 103 10

C- 62.7 94.1 C- C- C- 93.4 100K 100K 100K 100K 16 PB

102

8 PE-Cy7 2 2 SS SS SS 50K FS 50K 50K 10 10 45RA APC 0 50K

0 CD 0 54.5 0 0 0 CD 62.2 5.25 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD 2 3 4 5 0 102 103 104 105 0 10 10 10 10 FSC-A FSC-A Aqua CD3 AH7 CD8 PE-Cy7 CCR7 FITC CD27 PerCP

F. Transcription Factor Panel

250K 250K 5 48.4 5 4.6 250K 10 10 250K 250K

200K 200K 4 200K 4 10 200K 200K 10 A A A A 150K 150K 3 150K 150K 150K 98.3 10

C- 3 C- C- C- 68.4 10 100K 100K APC-C7 100K 100K 100K 10 2 FSC-H SS 2 SS SS SS 50K 50K 10 0 50K 50K 50K 0

69.7 80.3 70.5 0 0 CD8 PP-Cy5.5 CD16 0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 102 10 3 104 10 5 0 102 10 3 104 10 5 2 3 4 5 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 FSC-A FSC-A CD3 AmCyan CD8 PP-Cy5.5 Eomes PE T-bet FITC Helios eF450

G. Killer Immunoglobulin like Receptor Panel

250K 250K 250K 250K 105 5 10.9 10 250K 250K 22.7 250K 250K

200K 200K 200K 200K 87.5 4 97.2 10 4 200K 10 200K 200K 200K

3 A A

150K 150K 150K 150K A

92.4 10 A

A 150K H A H 150K 150K 150K 46.8 21.3 103 64.1 C- 100K 100K 100K 100K 2 C- C-

10 C- 100K C- C- 100K 100K 100K 0 2 35.9 10 57.7

SC- 41.9 50K SC- 50K 50K 50K 50K 50K 50K SS

SS 50K SS

0 SS SS SS F F 0 0 0 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 2 3 4 5 2 3 4 5 2 3 4 5 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CD57 APC 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 0 10 10 10 10 FSC-A FSC-A Aqua CD14/19 CD8 APC-H7 CD3 CD45 BV785 CD16 PB KIR3DL1 DL2/DS2/DL3 2DL1/S1 PE-Cy5 PE-CF594 A700 PE PP-Cy5.5 7 250K 250K 250K 250K 5 250K 250K 10 5 250K 16.2 10 87.5 200K 200K 200K 200K 4 200K 200K 97.2 10 200K 104 A A A A H A H 27.3 150K 150K 150K 150K 150K 3 150K 26.8 92.4 10 150K 21.3 3

C- 10 C- C- C- C- 100K 100K 100K 100K 100K 2 100K 10 100K SC- SC- 0 64.3 2 3.41 10 50K 50K SS SS SS SS SS F

50K F 50K 50K 50K 50K 0 0 0 0 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 CD8 APC-H7 FSC-A FSC-A Aqua CD14/19 CD3 CD56 PE-CY CD16 PB KIR3DL1 DL2/DS2/DL3 2DL1/S1 PE-Cy5 PE-CF594 A700 PE PP-Cy5.5

Supplemental Figure 1. Flow cytometry panels. Successive gating strategy used to characterize subpopulations of FcγRIIIa-expressing cell subsets using 7 flow cytometry panels. Gating strategies began by first identifying small lymphocytes through forward and side scatter profiles, single cells, and dead cell exclusion using an amine reactive dye. An initial survey identified an expanded FcγRIIIa+CD3+CD8+ cell subset within HIV+ donors, with differential PD-1 expression, as represented here (A). Five additional flow cytometry panels were designed to further characterize this subset. Homing markers were characterized in CD16 positive versus negative CD8 T cells, with CCR5, CXCR3, PD-1 and TRAIL gated individually within each CD16 expression subset (B). Additionally, CD16+ CD8 T cells were screened for surface and intracellular NK cell, innate-like markers through individual gating of IL-7R, Perforin, NKp46, and CD161 (C), single gating on NKG2D, and all combinations of NKG2A and CD57, individually or together (D). CD16+ CD8 T cells were also characterized for their differentiation and maturation markers through single gating on CD27, or all combinations of CD45RA and CCR7 (E). Transcription factors T-bet, Eomes, and Helios were characterized alone or in combination within CD16+ CD8 T cells, CD16- CD8 T cells, as well as CD56dim FcγRIIIa+ NK cells (F). Finally, surface KIR3DL1, KIRDL2/DS2/DL3, and KIR2DL1/S1 expression was measured in FcRγIIIa+ CD8 T cells and NK cells. CD8 T cells were gated on as CD3+CD8+ T cells, enriched for late-stage effector phenotype by gating on CD57+CD45+ cells, and further gated as CD16+. NK cells were identified by their CD3 negativity, followed by gating on CD56dimCD16+ cells. All KIR gates were drawn as individual gates (G).

3 Version 29 (16 August 2019) Journal of Immunology

Color Key

0123 4

IRF7 SMAD4 CTSB CD226 SLAMF7 FASLG TNFSF10 CD38 EOMES LGALS9 BIRC3 IL16 SLAMF5 IL21R IL6ST TNFRSF9 KIR3DL1.KIR3DS1 KLRC1 KIR2DS2.KIR2DL5A B3GAT1..CD57. KLRF1 IFNG TGFB1 CCL4 PSMG2 CLEC2B HLADRA GZMB PRF1 KLRD1 POLR2A PRDM1 KLRG1 CXCR4 IKZF1 IL2RG TGFBR1 CD244 CCL3 ITGAM IKZF2 KLRC2L.KLRC3 KIR2DS2.KIR2DS1. CD69 GZMA KLRK1.KLRC4.KLRK1 NKG7 CCL5 IL12RB1 TLR1 TNFSF13B SPATA2 CD160 TLR3 TIA1 TNF IL18R1 TLR6 BCL2 LAIR1 LAIR2 NCR1 LILRB1 IL18RAP DPP4 CXCR3 PDCD1 ICOS GZMK CCR7 IL7R

NK cells

CD45RA-CD57- CD8 T cells CD45RA+CD57+ CD45RA+CD57+ CD16+ CD8 T cellsCD16- CD8 T cells Supplemental Figure 2. Hierarchecal clustering of gene expression in sorted cell subsets. Two-way hierarchical clustering of gene expression in sorted subsets of CD8 T cells; CD45RA-CD57-,

CD45RA+CD57+FcγRIIIA-, CD45RA+CD57+FcγRIIIA+, as well as CD56dim FcγRIIIA+ NK cells. Data is normalized 2^DCt values.

4