An expanded population of CD8dimT cells with features of mitochondrial dysfunction and senescence is associated with persistent HIV-associated Kaposi’s sarcoma under ART

14 HIV-associated Kaposi’s sarcoma (KS), which is caused by Kaposi’s sarcoma -associated 15 herpesvirus, usually arises in the context of uncontrolled HIV replication and 16 immunosuppression. However, disease occasionally persists in individuals with durable HIV 17 viral suppression and CD4 T cell recovery under anti-retroviral therapy ( ART). The 18 underlying mechanisms associated with this persistence are unclear. Suppression of viral 19 infections can be mediated by CD8 T cells that detect infected cells via their T cell receptor 20 and the CD8 co -receptor. However, CD8 T cells exhibit signs of functional exhaustion in 21 untreated HIV infection that may not be fully reversed under ART. To investigate whether 22 persistent KS under ART was associated with phenotypic and functional perturbations of 23 CD8 T cells, we performed a cross-sectional study comparing HIV-infected individuals with 24 persistent KS under effective ART (HIV+ KS+) to HIV -infected individuals receiving 25 effective ART with no documented history of KS (HIV+ KS neg). A subset of T cells with 26 low cell surface expression of CD8 (“CD8 dim T cells”) was expanded in HIV+ KS+ 27 compared with HIV+ KS neg participants. Relative to CD8 bright T cells, CD8 dim T cells 28 exhibited signs of senescence (CD57) and mitochondrial perturbations (PGC-1α, 29 MitoTracker) ex vivo. Mitochondrial activity (MitoTracker) was also reduced in 30 proliferating CD8 dim T cells. These findings indicate that a n expanded CD8 dim T cell 31 population displaying features of senescence and mitochondrial dysfunction is associated 32 with KS persistence under ART. CD8 co -receptor down-modulation may be symptomatic 33 of ongoing disease. 34 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 Running title: CD8dim T cells expanded in HIV-associated KS 35

T cells; CD8 co-receptor; KSHV; Kaposi’s sarcoma; Tumor immunity; HIV; 36 Metabolism; Senescence; Mitochondria; MitoTracker; PGC-1α; Proliferation. 37 38 Funding: This study was supported by a Young Investigator Pilot Award awarded to G.T.C. 39 by the AIDS and Cancer Specimen Resource, funded by the National Cancer Institute (UM1 40 CA181255). Initial sample collection was supported by NIH grants U01 AI117844, U01 41 AI095052, and R01 HL132791. The UNC Flow Cytometry Core Facility is supported in part 42 by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger 43 Comprehensive Cancer Center and by the Center for AIDS Research award number 44 5P30AI050410. Statistical expertise was provided by the University of North Carolina at 45 Chapel Hill Center for AIDS Research, an NIH funded program P30 AI050410. The content 46 is solely the responsibility of the authors and does not necessarily represent the official views 47 of the National Institutes of Health. 48 49 Abbreviations used in this article 50 ART (HIV) Anti-retroviral therapy 51 CFSE carboxyfluorescein succinimidyl ester 52 KS Kaposi’s sarcoma 53 KSHV Kaposi’s sarcoma-associated herpesvirus, also known as HHV-8 54 MTDR MitoTracker® Deep Red 55 PBMC Peripheral blood mononuclear cells 56 PHA Phytohaemagglutinin 57 TCR T cell receptor 58 59 60 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 3

61 Kaposi’s sarcoma ( KS), a cancer of epithelial and endothelial cells characterized by dark 62 plaques and nodules, is a common AIDS-related morbidity in individuals with untreated HIV 63 infection [1]. However, while the etiologic agent of KS, Kaposi’s sarcoma -associated 64 herpesvirus ( KSHV), generates a lifelong infection , it rarely causes disease in 65 immunocompetent individuals. The introduction of HIV antiretroviral therapy (ART), and 66 resulting immune recovery in treated individuals, has been accompanied by a steep decline 67 in HIV-associated KS cases [2, 3]. However, a minority of HIV-infected KSHV-seropositive 68 individuals experience persist ent KS despi te durable HIV suppression and CD4 T cell 69 recovery under ART [4-6]. The underlying causes of this failure to achieve KS remission are 70 not currently understood. The recent discovery that latent KSHV can be reactivated by 71 proteins from SARS-CoV-2 and some anti-COVID-19 drugs further underscores the need to 72 better understand the control and pathogenesis of KS [7]. 73 CD8 T cells are major mediators of anti -viral immunity, detecting virus-infected cells via 74 the T cell receptor (TCR) and CD8-co-receptor. KSHV-infected individuals harbor CD8 T 75 cells capable of secreting antiviral cytokines and killing cells expressing KSHV antigens in 76 vitro [8-14]. These KSHV-specific CD8 T cells are detected at higher frequencies in KSHV -77 seropositive individuals who do not have KS compared with individuals with active disease 78 [14]. Collectively, these observations suggest that in immunocompetent individuals, CD8 T 79 cells may play a lifelong role in preventing KSHV from causing disease. However, during 80 untreated progressive HIV infection, T cells exhibit signs of functional exhaustion that may 81 not be fully reversed by ART [15-19]. Notably, these defects include metabolic perturbations 82 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 4 such as impaired mitochondrial oxidative phosphorylation and increased reliance on 83 glycolysis [20, 21]. Mitochondrial metabolism is crucial for lasting CD8 T cell control of viral 84 infections: while effector T cells upregulate glycolysis to rapidly generate ATP, memory T 85 cells use mitochondrial oxidative phosphorylation to support their long-term persistence [22-86 26]. Indeed, loss of mitochondrial mass is associated with senescence, a state where T cells 87 lose pro liferative capacity [27]. Defects in CD8 T cell metaboli c fitness in HIV -infected 88 individuals on ART could be particularly detrimental in the tumor microenvironment, where 89 rapidly proliferating malignant cells create an environment of hypoxia and mitochondrial 90 stress [28-30]. 91 We investigated the possibility that altered CD8 T cell phenotype and metabolism could be 92 associated with persistent KS in HI V-infected individuals on ART by comparing HIV -93 infected individuals with and without KS. We observed an elevated frequency of CD8 dim T 94 cells in individuals with HIV -associated KS. These cells expressed elevated levels of the 95 senescence marker CD57, lower levels of the mitochondrial master-regulator PGC-1α, and 96 exhibited reduced mitochondrial activity. Persistent KS is therefore associated with the 97 expansion of a subset of CD8 T cells with metabolic hallmarks of senescence. 98 99 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 5

100 Study participants: 101 HIV-1-infected participants with biopsy-confirmed KS (“HIV+ KS+”) were recruited from 102 primary care practices in San Francisco and the adjacent counties, UCLA-related primary 103 care clinics, and the Study of the Consequences of the Protease Inhibitor Era (SCOPE) . 104 Participants had received ART (including protease inhibitors, NNRTIs, and early integrase 105 inhibitors) and maintained plasma viral loads <75 copies/ml for ≥2 years. CD4 T cell counts 106 were ≥340/µl. All participants had stage 1 tumors according to ACTG criteria (tumor 107 confined to skin and/or lymph nodes and/or minimal oral disease) [31]. The study was 108 approved by the Institutional Review Board of the University of California, San Francisco 109 (approval no. 10 -02850). HIV-1-infected participants with no documented history of KS 110 (“HIV+ KSneg”) were recruited from the UNC HIV Clinical Trials Unit. Participants had 111 received ART for ≥2 years and maintained plasma viral loads 300/µl for ≥6 months. Participant characteristics are detailed in Table 1. Initial 113 collection of UNC samples was approved by the UNC Institutional Review Board (ethics 114 numbers 11-0228; 14-0741; and 15-1626). Retrospective use of all samples was approved 115 by the UNC Institutional Review Board (ethics number 17-2415). 116 CD8 T cell phenotyping: 117 Cryopreserved peripheral blood mononuclear cells ( PBMC) were rested in R10 medium 118 (RPMI 1640 supplemented with 10% fetal bovine serum; penicillin/streptomycin; 2 mM L-119 glutamine; 10 mM sodium pyruvate; and 10 mM HEPES) at 37°C overnight. PBMC were 120 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 6 stained with Zomb ie NIR viability dye , then cell surface antibodies CD3 -PerCP-Cy5.5 121 (clone UCHT1); CD4 -PE-Cy5 (OKT4); CD8-Brilliant Violet 510 (SK1); CD14 (M5E2), 122 CD16 (3G8), CD19 (HIB19), and CD56 (HCD56)-Brilliant Violet 650 ; CD45RO -PE 123 (UCHL1); and CD57-PE-Dazzle 594 ( HNK-1) (all Biolegend). Cells were stained 124 intracellularly with T -bet-Brilliant Violet 421 ( 4B10; Biolegend); Eomes -eFluor 660 125 (WD1928; eBioscience); polyclonal anti-PGC-1α antibody (Santa Cruz Biotechnology); and 126 PE-Cy7 secondary antibo dy. Samples were acquired on an LSRII flow cytometer and 127 analyzed using FlowJo 10 (BD Biosciences). Live lymphocytes were defined by dim 128 staining with Zombie viability dye, forward scatter height vs area (to identify single events), 129 and forward scatter versus side scatter. CD8 T lymphocytes were defined as CD3+ CD4- 130 CD14/16/19/56- and CD8 bright or dim. For phenotypic markers, positive events were gated 131 using fluorescence minus one controls (Supplementary Figure 1). 132 Table 1: Participant Characteristics 133 HIV+ KS+ (n = 8) HIV+ KSneg (n = 12) Age in years; median (range) 57 (35 – 65) 48 (33 – 66) Male sex 8/8 9/12 Viral load (copies/ml) <70 (<70 - 97) <50 CD4 count (cells/µl) 677 (340 – 1331) 769 (491 – 1289) KS stage ACTG stage 1 N/A 134 CD8 T cell proliferation: 135 Cryopreserved PBM C were rested overnight , then pulsed under rotation with 5 µM 136 carboxyfluorescein succinimidyl ester (CFSE, Biolegend). Staining was quenched with ice-137 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 7 cold R10. Cells were stimulated with vehicle (0.5% DMSO) or 3 µg/ml phytohaemagglutinin 138 (PHA, Sigma) for 5 days at 37° C. PBMC were stained with Zombie NIR; CD3-PE-Cy7; 139 CD4-BV421; CD8-BV510; CD14, CD16, CD19, and CD56 -BV650 (clones as previously; 140 Biolegend). To assess mitochondrial polarization, cells were stained with 25 nM 141 MitoTracker® Deep Red (MTDR, Molecular Probes) at 37°C. Cells were acquired on an 142 LSR Fortessa and a nalyzed using FlowJo 10 and Modfit LT 4 (Verity Software House). 143 Proliferation was assessed using prol iferation index , defined as the mean number of 144 proliferative cycles underg one by each proliferating cell [32]. MTDR high cells were gated 145 using a previously described method [33]. Briefly, after excluding outliers (the brightest and 146 dimmest 0.1% of events), the fluorescence intensities of the brightest and dimmest cells were 147 used to calculate the fluorescence range (brightest – dimmest). Cells that fell within the top 148 90% of this range were considered MTDRhigh (Supplementary Figure 2). 149 Statistical analysis: 150 Data were analyzed using GraphPad Prism version 8. Between-group differences were 151 analyzed using a n exact, two -sided Mann-Whitney U test. Within-individual differences 152 between CD8bright and CD8dim T cells were analyzed using an exact, two -sided Wilcoxon 153 signed-rank test. The monotonic (strictly increasing or decreasing) relationship between 154 variables such as CD8dim percentage and proliferation index was assessed using Spearman’s 155 rank correlation coefficient. 156 157 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 8

158 CD8dim T cells are expanded in HIV+ individuals with persistent KS, and exhibit features of 159 senescence 160 We compared the phenotype of CD8 T cells betwe en HIV -infected indivi duals with 161 persistent KS under ART (HIV+ KS+) and HIV-infected individuals receiving ART with no 162 documented history of KS (HIV+ KS neg). CD8 T cells were defined as CD3+ CD8+ CD4 - 163 CD14/CD16/CD56- to exclude NKT cel ls (Supplementary Figure 1). W e observed two 164 populations of CD8 T cells: a CD8bright subset with high cell surface expression of CD8 and 165 a CD8 dim subset with lower surface expression (Fig. 1A). CD3ε, which forms part of the 166 TCR complex, was also expressed at a lower level on the surface of CD8dim cells compared 167 with CD8 bright cells ( median of differences = -4358 MFI; Wilcoxon signed -rank test p = 168 0.007; Supplementary Figure 3 ). The CD8 dim subset was significantly expanded, as a 169 percentage of total CD8 T cells, in HIV+ KS+ compared wi th HIV+ KS neg participants 170 (difference of medians = 12. 28%; Mann-Whitney test p = 0.0006; Fig. 1B). Since highly 171 differentiated T cells accumulate during chro nic untreated infections [34, 35] , we next 172 compared the differentiation state of CD8 bright and CD8dim cells within participants. CD57 173 expression was higher on CD8 dim than CD8 bright T cells (median of differences = 8%; 174 Wilcoxon signed-rank test p = 0.008) , indicating that late -differentiated or senescent cells 175 were overrepresented in the CD8 dim population (Fig. 1C and Supplementary Figure 3 ). 176 Supporting this observation, Eomes odermin (Eomes) , a transcription factor expressed in 177 terminal memory cells, was expressed in a higher percentage of CD8 dim than CD8bright cells 178 (median of differences = 13.5%; Wilcoxon signed -rank test p = 0.008, Fig . 1D and 179 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 9 Supplementary Figure 3) [36, 37]. T-bet, a transcription factor expressed by effector cells, was 180 expressed at similar levels in CD8bright and CD8dim cells (Supplementary Figure 3). 181 182 Figure 1. CD8dim cells with low mitochondrial activity are expanded in individuals 183 with persistent KS. A) Representative plots showing CD8bright and CD8dim T cells in a 184 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 10 HIV+ KSneg and a HIV+ KS+ participant. B) The frequency of CD8dim T cells (as a 185 percentage of total CD8 T cells) is significantly elevated in HIV+ individuals with 186 persistent KS under ART (KS+; n = 7) compared with the KSneg group (n = 8) (difference 187 of medians= 12.28%; Mann-Whitney test). C) A significantly higher percentage of CD8dim 188 T cells express CD57 compared with CD8bright T cells (median of differences = 8%; 189 Wilcoxon signed-rank test). D) A significantly higher percentage of CD8dim T cells 190 express Eomes compared with CD8bright T cells (median of differences = 13.5%; Wilcoxon 191 signed-rank test). E) Expression of the mitochondrial master regulator PGC-1α is 192 significantly reduced in CD8dim T cells (median of differences = -789 MFI; Wilcoxon 193 signed-rank test). F) The frequency of MitoTracker Deep Red high cells is significantly 194 lower for CD8dim T cells compared with CD8bright T cells (median of differences = -8.99%; 195 Wilcoxon signed-rank test). Gray open squares, HIV+ KS+ participants; black circles, 196 HIV+ KSneg participants. 197 198 CD8dim T cells have an altered mitochondrial phenotype 199 The development, persistence, and recall function of memory CD8 T cells is highly 200 dependent on mitochondrial metabolism [24, 38]. Compared with CD8bright T cells, expression 201 of the mitochondrial master regulator PGC-1α was significantly reduced in CD8 dim T cells 202 (median of differences = -789 MFI; Wilcoxon signed -rank test p = 0.001 ; Fig. 1E). To 203 further investigate mitochondrial phenotype in CD8 bright vs CD8 dim T cells , we used 204 MitoTracker® Deep Red (MTDR), which selectively binds actively respiring mitochondria 205 [39]. MTDRhigh cells were defined using a previously described objective gating strategy ([33]; 206 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 11 Supplementary Figure 2). The frequency of MTDRhigh cells was lower in the CD8dim T cell 207 population than CD8 bright T cells (median of differences = -8.99%; Wilcoxon signed -rank 208 test p = 0.002; Fig. 1F). These observations indicate that individuals with HIV-associated 209 KS have an expanded population of CD8 dim T cells with a highly differentiated/senescent 210 phenotype and reduced mitochondrial activity. 211 212 Mitochondrial activity is reduced in CD8dim proliferating cells 213 We next examined whether cell surface expression of the CD8 co-receptor was related to 214 replicative capacity and mitochondrial activity in proliferating cells. PBMC were stimulated 215 with the polyclonal stimulus PHA for five days and proliferation was measured by CFSE 216 dilution (Fig. 2A) . Proliferative capacity was reported as proliferation index ( the mean 217 number of proliferative cycles undergone by each responding cell). 218 CD8 T cells that had proliferated (CFSElow) exhibited greater mitochondrial activity ( % of 219 cells MTDRhigh) than non-proliferating (CFSEhigh) cells (median of differences = 23.25%; 220 Wilcoxon signed -rank test p = 0.002; Fig. 2B) . T here was a strong positive correl ation 221 between mitochondrial activity of all CD8 T cells and proliferation index at day five , 222 demonstrating the importance of mitochondrial respiration to CD8 T cell proliferation (rs = 223 0.75, p = 0.017; Fig. 2C) [26]. Conversely, there was a strong negative correlation between 224 mitochondrial activity of all CD8 T cells and the percentage of CD8dim T cells in the culture 225 at five days (rs = -0.75, p = 0.017; Fig. 2D), suggesting that low CD8 expression is associated 226 with reduced mitochondrial activity . Further supporting this hypothesis, CD8 dim cells that 227 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 12 had proliferated exhibited lower mitochondrial activit y (% MTDR high) than CD8 bright cells 228 that had proliferated (median of differences = -23.35%; Wilcoxon signed-rank test p = 0.002; 229 Fig. 2E). There was also evidence of a moderate negative correlation between the percentage 230 of CD8 dim T cells in the culture and proliferation index (rs = -0.61, p = 0.067 ; Fig. 2F). 231 Collectively these results indicate that low surface expression of CD8 is associated with 232 reduced mitochondrial respiration and replicative capacity of proliferating CD8 T cells. 233 234 Figure 2. Mitochondrial activity is reduced in CD8dim proliferating cells. 235 A) Histograms showing CFSE dilution in unstimulated and PHA-stimulated CD8 T cells. 236 B) MitoTracker Deep Red (MTDR) fluorescence in PHA-stimulated proliferating 237 (CFSElow) cells. A significantly higher percentage of proliferating (CFSElow) CD8 T cells 238 are MTDRhigh compared with non-proliferating (CFSEhigh) CD8 T cells, indicating that 239 proliferating cells have higher mitochondrial activity (n = 10; median of differences = 240 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 13 23.25%; Wilcoxon signed-rank test). C) Positive association between mitochondrial 241 activity (% MTDRhigh) of all CD8 T cells in the culture and the proliferation index of 242 proliferating cells in response to PHA stimulation (Spearman correlation). D) Negative 243 association between mitochondrial activity and the frequency of CD8dim T cells following 244 stimulation with PHA (Spearman correlation). E) The frequency of MTDRhigh cells, 245 comparing CD8bright and CD8dim proliferating (CFSElow) cells. CD8dim proliferating cells 246 are significantly less likely to be MTDRhigh, indicating lower mitochondrial activity 247 (median of differences = -23.35%; Wilcoxon signed-rank test). F) Proliferation index of 248 PHA-stimulated CD8 T cells versus the frequency of CD8dim T cells in the culture 249 (Spearman correlation). Gray open squares, HIV+ KS+ participants; black circles, HIV+ 250 KSneg participants. 251 252 . 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253 The underlying causes of persistent KS in a minority of HIV -infected individuals with 254 durable viral suppression and CD4 T cell recovery under ART are unknown. Here, we have 255 identified an expanded population of CD8 dim T cells with phenotypic characteristics of 256 senescence and mitochondrial dysfunction in these individuals. 257 The CD8 co-receptor amplifies signals through the TCR [40, 41] . Following antigenic 258 stimulation (e.g. by a virus-infected cell), CD8, along with the TCR, is downregulated from 259 the cell surface, possibly to limit the str ength or duration of signaling [42-44]. This suggests 260 that the expansion of CD8 dim T cells may be a response to high and/or persistent antigen 261 stimulation. Supporting this hypothesis, elevated frequencies of CD8 dim T cells have been 262 reported during acute HIV infection, and in children exposed to a high cumulative pathogen 263 burden during the first years of life [45, 46]. Our observation that CD8 dim T cells are also 264 expanded in individuals with per sistent KS under ART supports the notion that CD8 265 downregulation is a general phenomenon in settings of unresolved infection. 266 Chronic viral infections are also associated with CD8 T cell terminal differentiation and/or 267 senescence[47]. Senescent CD8 T cells exhibit reduced expression of PGC -1α, the master -268 regulator of mitochondrial biogenesis, and lower mitochondrial activity [27, 48]. Conversely, 269 forced expression of PGC -1α promotes robust CD8 T cell memory responses [49]. These 270 observations underscore the importance of mitochondrial respiration for long -term CD8 T 271 cell anti-viral function. In the setting of persistent KS, we observed that the expanded CD8dim 272 T cell population expressed high levels of Eomesodermin and CD57, proteins respectively 273 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 15 associated with terminal differentiation and senescence, and had reduced expression of PGC-274 1α. CD8 dim cells also cont ained fewer respiring mitochondria both ex vivo and when 275 proliferating. Our data suggest that mitochondrial dysfunction may underlie the 276 accumulation of senescent CD8 T cells that has previously been reported in KS [34]; however, 277 this hypothesis must be tested in future studies. It is unclear from our current data whether 278 CD8 expression directly regulates mitochondrial activity in CD8 T cells. 279 A key question is whether the CD8dim T cells we observed are specific for KSHV, for HIV, 280 or for other persistent viral infections such as CMV , which is highly seroprevalent in HIV-281 infected individuals [50]. It is possible that CD8 down-modulation could be driven by multiple 282 concurrent infections, by ongoing immune activation, or by a combination of factors. In this 283 initial study, we were unable to determine whether the CD8 dim T cells we observed were 284 KSHV-specific, as KSHV is a large virus, and immunoprevalent epitopes eliciting responses 285 in a high percentage of seropositive individuals have not yet been identified. The question 286 of the antigen specificity of CD8dim T cells will be the subject of subsequent investigations. 287 Our work has some limitations. As this was an observational study, we were unable to 288 determine whether the expansion of CD8 dim T cells plays a causative role in the failure to 289 control KSHV under ART or is a consequence of this lack of suppression. Due to lack of 290 available tissue , we were unable to assess whether CD8 T cells infiltrating the tumor 291 microenvironment also exhibit a CD8dim phenotype. This question, together with the antigen 292 specificity of CD8 dim T cells and a direct examination of their functional profile, is the 293 subject of ongoi ng investigations. If KSHV -specific CD8 T cells infiltrating the tumor 294 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 16 microenvironment express low levels of CD8 and exhibit reduced mitochondrial activity, 295 this will have important implications for immunotherapeutic approaches to KS treatment. 296 297 298

299 Author contributions: The manuscript was written by G.T.C., A.M.W., N. P.G., and T.M. 300 G.T.C. and N.P.G. contributed to study design. G.T.C. performed experimentation and 301 collection of data. G.T.C. and A.M.W. performed the statistical analyses. G.T.C. acquired 302 funding for the study . T.M. facilitated participant recruitment and sampl e collection. All 303 authors provided review of the final manuscript. We thank Joann Kuruc and Cynthia Gay 304 for their work recruiting participants to UNC cohorts that were retrospectively utilized for 305 this study. 306 Conflicts of interest: There are no conflicts of interest. 307 308 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint 17

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