{"paper_id":"29ebf930-0f9c-4a93-b2b4-7001029deab1","body_text":"1 \n \nAn expanded population of CD8dim T cells with features of mitochondrial 1 \ndysfunction and senescence is associated with persistent HIV-associated 2 \nKaposi’s sarcoma under ART 3 \nGenevieve T CLUTTON*¶, Ann Marie K WEIDEMAN†, Nilu P GOONETILLEKE*‡, 4 \nToby MAURER§ 5 \n*Department of Microbiology & Immunology, UNC Chapel Hill School of Medicine, 6 \nChapel Hill, North Carolina 27599, USA 7 \n†Department of Biostatistics and Center for AIDS Research, UNC Chapel Hill, Chapel 8 \nHill, North Carolina 27599, USA 9 \n ‡UNC HIV Cure Center, UNC Institute of Global Health and Infectious Diseases, Chapel 10 \nHill, North Carolina 27599, USA 11 \n§Department of Dermatology, Indiana University, Indiana, USA 12 \n¶Corresponding author. Email: gen_clutton@med.unc.edu 13 \nAbstract 14 \nHIV-associated Kaposi’s sarcoma (KS), which is caused by Kaposi’s sarcoma -associated 15 \nherpesvirus, usually arises in the context of uncontrolled HIV replication and 16 \nimmunosuppression. However, disease occasionally persists in individuals with durable HIV 17 \nviral suppression  and CD4 T cell recovery  under anti-retroviral therapy ( ART). The 18 \nunderlying mechanisms associated with this persistence are unclear. Suppression of viral 19 \ninfections can be mediated by CD8 T cells that detect infected cells via their T cell receptor 20 \nand the CD8 co -receptor. However, CD8 T cells  exhibit signs of functional exhaustion in 21 \nuntreated HIV infection that may not be fully reversed under ART. To investigate whether 22 \npersistent KS under ART was associated with phenotypic and functional perturbations of 23 \nCD8 T cells, we performed a cross-sectional study comparing HIV-infected individuals with 24 \npersistent KS under effective ART (HIV+ KS+) to HIV -infected individuals receiving 25 \neffective ART with no documented history of KS (HIV+ KS neg). A subset of T cells with 26 \nlow cell surface expression  of CD8 (“CD8 dim T cells”) was expanded in HIV+ KS+ 27 \ncompared with HIV+ KS neg participants. Relative to CD8 bright T cells, CD8 dim T cells 28 \nexhibited signs of senescence (CD57) and mitochondrial perturbations  (PGC-1α, 29 \nMitoTracker) ex vivo. Mitochondrial activity (MitoTracker) was also reduced in 30 \nproliferating CD8 dim T cells.  These findings indicate that a n expanded CD8 dim T cell 31 \npopulation displaying features of senescence and mitochondrial dysfunction  is associated 32 \nwith KS persistence under ART. CD8 co -receptor down-modulation may be symptomatic 33 \nof ongoing disease. 34 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \nRunning title: CD8dim T cells expanded in HIV-associated KS 35 \nKeywords: T cells; CD8 co-receptor; KSHV; Kaposi’s sarcoma; Tumor immunity; HIV; 36 \nMetabolism; Senescence; Mitochondria; MitoTracker; PGC-1α; Proliferation. 37 \n 38 \nFunding: This study was supported by a Young Investigator Pilot Award awarded to G.T.C. 39 \nby the AIDS and Cancer Specimen Resource, funded by the National Cancer Institute (UM1 40 \nCA181255). Initial sample collection was supported by NIH grants U01 AI117844, U01 41 \nAI095052, and R01 HL132791. The UNC Flow Cytometry Core Facility is supported in part 42 \nby P30 CA016086 Cancer Center Core Support Grant  to the UNC Lineberger 43 \nComprehensive Cancer Center and by the Center for AIDS Research award number 44 \n5P30AI050410. Statistical expertise was provided by the University of North Carolina at 45 \nChapel Hill Center for AIDS Research, an NIH funded program P30 AI050410.  The content 46 \nis solely the responsibility of the authors and does not necessarily represent the official views 47 \nof the National Institutes of Health. 48 \n 49 \nAbbreviations used in this article 50 \nART  (HIV) Anti-retroviral therapy 51 \nCFSE  carboxyfluorescein succinimidyl ester 52 \nKS  Kaposi’s sarcoma 53 \nKSHV  Kaposi’s sarcoma-associated herpesvirus, also known as HHV-8 54 \nMTDR  MitoTracker® Deep Red 55 \nPBMC  Peripheral blood mononuclear cells 56 \nPHA  Phytohaemagglutinin 57 \nTCR  T cell receptor 58 \n 59 \n 60 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n3 \n \nIntroduction 61 \nKaposi’s sarcoma ( KS), a cancer of epithelial and endothelial cells characterized by dark 62 \nplaques and nodules, is a common AIDS-related morbidity in individuals with untreated HIV 63 \ninfection [1]. However, while the etiologic agent of KS, Kaposi’s sarcoma -associated 64 \nherpesvirus ( KSHV), generates a  lifelong infection , it  rarely causes disease  in 65 \nimmunocompetent individuals. The introduction of HIV antiretroviral therapy (ART), and 66 \nresulting immune recovery in treated individuals, has been accompanied by  a steep decline 67 \nin HIV-associated KS cases [2, 3].  However, a minority of HIV-infected KSHV-seropositive 68 \nindividuals experience persist ent KS despi te durable HIV  suppression and CD4 T cell 69 \nrecovery under ART [4-6].  The underlying causes of this failure to achieve KS remission are 70 \nnot currently understood.  The recent discovery that latent KSHV can be reactivated by 71 \nproteins from SARS-CoV-2 and some anti-COVID-19 drugs further underscores the need to 72 \nbetter understand the control and pathogenesis of KS [7]. 73 \nCD8 T cells are major mediators of anti -viral immunity, detecting virus-infected cells via 74 \nthe T cell receptor (TCR) and CD8-co-receptor. KSHV-infected individuals harbor CD8 T 75 \ncells capable of secreting antiviral cytokines and killing cells expressing KSHV antigens in 76 \nvitro [8-14]. These KSHV-specific CD8 T cells are detected at higher frequencies in KSHV -77 \nseropositive individuals who do not have KS compared with individuals with active disease 78 \n[14]. Collectively, these observations suggest that in immunocompetent individuals, CD8 T 79 \ncells may play a lifelong role in preventing KSHV from causing disease.  However, during 80 \nuntreated progressive HIV infection, T cells exhibit signs of functional exhaustion that may 81 \nnot be fully reversed by ART [15-19]. Notably, these defects include metabolic perturbations 82 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n4 \n \nsuch as impaired mitochondrial oxidative phosphorylation and increased reliance on 83 \nglycolysis [20, 21]. Mitochondrial metabolism is crucial for lasting CD8 T cell control of viral 84 \ninfections: while effector T cells upregulate glycolysis to rapidly generate ATP, memory T 85 \ncells use mitochondrial oxidative phosphorylation to support their long-term persistence [22-86 \n26]. Indeed, loss of mitochondrial mass is associated with senescence, a state where T cells 87 \nlose pro liferative capacity [27]. Defects in  CD8 T cell metaboli c fitness  in HIV -infected 88 \nindividuals on ART could be particularly detrimental in the tumor microenvironment, where 89 \nrapidly proliferating malignant cells create an environment of hypoxia and mitochondrial 90 \nstress [28-30].   91 \nWe investigated the possibility that altered CD8 T cell phenotype and metabolism could be 92 \nassociated with persistent  KS in HI V-infected individuals on ART by comparing HIV -93 \ninfected individuals with and without KS. We observed an elevated frequency of CD8 dim T 94 \ncells in individuals with HIV -associated KS. These cells expressed elevated levels of the 95 \nsenescence marker CD57, lower levels of the mitochondrial master-regulator PGC-1α, and 96 \nexhibited reduced mitochondrial activity. Persistent KS is therefore associated with the 97 \nexpansion of a subset of CD8 T cells with metabolic hallmarks of senescence. 98 \n  99 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n5 \n \nMaterials and Methods 100 \nStudy participants: 101 \nHIV-1-infected participants with biopsy-confirmed KS (“HIV+ KS+”) were recruited from 102 \nprimary care practices in San Francisco and the adjacent counties, UCLA-related primary 103 \ncare clinics, and the Study of the Consequences of the Protease Inhibitor Era (SCOPE) . 104 \nParticipants had received ART (including protease inhibitors, NNRTIs, and early integrase 105 \ninhibitors) and maintained plasma viral loads <75 copies/ml for ≥2 years. CD4 T cell counts 106 \nwere ≥340/µl. All participants had stage 1 tumors according to ACTG criteria  (tumor 107 \nconfined to skin and/or lymph nodes and/or minimal oral disease) [31]. The study was 108 \napproved by the Institutional Review Board of the University  of California, San Francisco 109 \n(approval no. 10 -02850). HIV-1-infected participants with  no documented history of KS  110 \n(“HIV+ KSneg”) were recruited from the UNC HIV Clinical Trials Unit.   Participants had 111 \nreceived ART for ≥2 years and maintained plasma viral loads <50 copies/ml and CD4 T cell 112 \ncounts >300/µl for ≥6 months. Participant characteristics are detailed in Table 1.  Initial 113 \ncollection of UNC samples was approved by the UNC Institutional Review Board  (ethics 114 \nnumbers 11-0228; 14-0741; and 15-1626). Retrospective use of all samples  was approved 115 \nby the UNC Institutional Review Board (ethics number 17-2415).   116 \nCD8 T cell phenotyping: 117 \nCryopreserved peripheral blood mononuclear cells ( PBMC) were rested  in R10 medium 118 \n(RPMI 1640 supplemented with 10% fetal bovine serum; penicillin/streptomycin; 2 mM L-119 \nglutamine; 10 mM sodium pyruvate;  and 10 mM HEPES)  at 37°C overnight. PBMC were 120 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n6 \n \nstained with Zomb ie NIR viability dye , then cell surface antibodies CD3 -PerCP-Cy5.5 121 \n(clone UCHT1); CD4 -PE-Cy5 (OKT4); CD8-Brilliant Violet 510  (SK1); CD14 (M5E2), 122 \nCD16 (3G8), CD19  (HIB19), and CD56  (HCD56)-Brilliant Violet 650 ; CD45RO -PE 123 \n(UCHL1); and CD57-PE-Dazzle 594 ( HNK-1) (all Biolegend). Cells were stained 124 \nintracellularly with T -bet-Brilliant Violet 421 ( 4B10; Biolegend); Eomes -eFluor 660 125 \n(WD1928; eBioscience); polyclonal anti-PGC-1α antibody (Santa Cruz Biotechnology); and 126 \nPE-Cy7 secondary antibo dy. Samples were acquired on an LSRII flow cytometer and  127 \nanalyzed using FlowJo 10 (BD Biosciences).  Live lymphocytes were defined by dim 128 \nstaining with Zombie viability dye, forward scatter height vs area (to identify single events), 129 \nand forward scatter versus side scatter. CD8 T lymphocytes were defined as CD3+ CD4- 130 \nCD14/16/19/56- and CD8 bright or dim. For phenotypic markers, positive events were gated 131 \nusing fluorescence minus one controls (Supplementary Figure 1).   132 \nTable 1: Participant Characteristics 133 \n HIV+ KS+ (n = 8) HIV+ KSneg (n = 12) \nAge in years; median (range) 57 (35 – 65) 48 (33 – 66)  \nMale sex 8/8 9/12 \nViral load (copies/ml) <70 (<70 - 97) <50 \nCD4 count (cells/µl) 677 (340 – 1331) 769 (491 – 1289) \nKS stage ACTG stage 1 N/A \n 134 \nCD8 T cell proliferation:  135 \nCryopreserved PBM C were rested overnight , then pulsed under rotation with 5 µM 136 \ncarboxyfluorescein succinimidyl ester (CFSE, Biolegend). Staining was quenched with ice-137 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n7 \n \ncold R10. Cells were stimulated with vehicle (0.5% DMSO) or 3 µg/ml phytohaemagglutinin 138 \n(PHA, Sigma) for 5 days at 37° C. PBMC were stained with  Zombie NIR; CD3-PE-Cy7; 139 \nCD4-BV421; CD8-BV510; CD14, CD16, CD19, and CD56 -BV650 (clones as previously; 140 \nBiolegend). To assess mitochondrial polarization,  cells were  stained with 25 nM 141 \nMitoTracker® Deep Red (MTDR, Molecular Probes) at 37°C. Cells were acquired on an 142 \nLSR Fortessa and a nalyzed using FlowJo 10  and Modfit LT 4 (Verity Software House). 143 \nProliferation was assessed using prol iferation index , defined as the mean number of 144 \nproliferative cycles underg one by each proliferating cell [32].  MTDR high cells were gated 145 \nusing a previously described method [33]. Briefly, after excluding outliers (the brightest and 146 \ndimmest 0.1% of events), the fluorescence intensities of the brightest and dimmest cells were 147 \nused to calculate the fluorescence range (brightest – dimmest). Cells that fell within the top 148 \n90% of this range were considered MTDRhigh (Supplementary Figure 2).   149 \nStatistical analysis: 150 \nData were analyzed using GraphPad Prism  version 8. Between-group differences were 151 \nanalyzed using a n exact, two -sided Mann-Whitney U test. Within-individual differences 152 \nbetween CD8bright and CD8dim T cells were analyzed using an exact, two -sided Wilcoxon 153 \nsigned-rank test. The monotonic (strictly increasing or decreasing)  relationship between 154 \nvariables such as CD8dim percentage and proliferation index was assessed using Spearman’s 155 \nrank correlation coefficient.  156 \n  157 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n8 \n \nResults 158 \nCD8dim T cells are expanded in HIV+ individuals with persistent KS, and exhibit features of 159 \nsenescence 160 \nWe compared  the phenotype of CD8 T cells betwe en HIV -infected indivi duals with 161 \npersistent KS under ART (HIV+ KS+) and HIV-infected individuals receiving ART with no 162 \ndocumented history of KS (HIV+ KS neg). CD8 T cells were defined as CD3+ CD8+ CD4 - 163 \nCD14/CD16/CD56- to exclude NKT cel ls (Supplementary Figure 1). W e observed two 164 \npopulations of CD8 T cells: a CD8bright subset with high cell surface expression of CD8 and 165 \na CD8 dim subset with lower surface expression  (Fig. 1A). CD3ε, which forms part of the  166 \nTCR complex, was also expressed at a lower level on the surface of CD8dim cells compared 167 \nwith CD8 bright cells ( median of differences  = -4358 MFI; Wilcoxon signed -rank test p = 168 \n0.007; Supplementary Figure 3 ). The CD8 dim subset was significantly expanded, as a 169 \npercentage of total CD8 T cells, in HIV+ KS+ compared wi th HIV+ KS neg participants 170 \n(difference of medians = 12. 28%; Mann-Whitney test p = 0.0006; Fig. 1B).  Since highly 171 \ndifferentiated T cells accumulate during chro nic untreated infections [34, 35] , we next 172 \ncompared the differentiation state of CD8 bright and CD8dim cells within participants. CD57 173 \nexpression was higher on CD8 dim than CD8 bright T cells  (median of differences = 8%; 174 \nWilcoxon signed-rank test p = 0.008) , indicating that late -differentiated or senescent cells 175 \nwere overrepresented in the CD8 dim population (Fig. 1C and Supplementary Figure 3 ). 176 \nSupporting this observation, Eomes odermin (Eomes) , a transcription factor expressed in 177 \nterminal memory cells, was expressed in a higher percentage of CD8 dim than CD8bright cells 178 \n(median of differences = 13.5%; Wilcoxon signed -rank test p = 0.008, Fig . 1D and 179 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n9 \n \nSupplementary Figure 3) [36, 37]. T-bet, a transcription factor expressed by effector cells, was 180 \nexpressed at similar levels in CD8bright and CD8dim cells (Supplementary Figure 3).   181 \n 182 \nFigure 1. CD8dim cells with low mitochondrial activity are expanded in individuals 183 \nwith persistent KS. A) Representative plots showing CD8bright and CD8dim T cells in a 184 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n10 \n \nHIV+ KSneg and a HIV+ KS+ participant. B) The frequency of CD8dim T cells (as a 185 \npercentage of total CD8 T cells) is significantly elevated in HIV+ individuals with 186 \npersistent KS under ART (KS+; n = 7) compared with the KSneg group (n = 8)  (difference 187 \nof medians= 12.28%; Mann-Whitney test). C) A significantly higher percentage of CD8dim 188 \nT cells express CD57 compared with CD8bright T cells (median of differences = 8%; 189 \nWilcoxon signed-rank test).  D) A significantly higher percentage of CD8dim T cells 190 \nexpress Eomes compared with CD8bright T cells (median of differences = 13.5%; Wilcoxon 191 \nsigned-rank test). E) Expression of the mitochondrial master regulator PGC-1α is 192 \nsignificantly reduced in CD8dim T cells (median of differences = -789 MFI; Wilcoxon 193 \nsigned-rank test).  F) The frequency of MitoTracker Deep Red high cells is significantly 194 \nlower for CD8dim T cells compared with CD8bright T cells (median of differences = -8.99%; 195 \nWilcoxon signed-rank test). Gray open squares, HIV+ KS+ participants; black circles, 196 \nHIV+ KSneg participants.  197 \n  198 \nCD8dim T cells have an altered mitochondrial phenotype 199 \nThe development, persistence, and recall function of memory CD8 T cells is highly 200 \ndependent on mitochondrial metabolism [24, 38]. Compared with CD8bright T cells, expression 201 \nof the mitochondrial master regulator PGC-1α was significantly reduced in CD8 dim T cells 202 \n(median of differences = -789 MFI; Wilcoxon signed -rank test p = 0.001 ; Fig. 1E).  To 203 \nfurther investigate mitochondrial phenotype  in CD8 bright vs CD8 dim T cells , we used  204 \nMitoTracker® Deep Red (MTDR), which selectively binds actively respiring mitochondria 205 \n[39].  MTDRhigh cells were defined using a previously described objective gating strategy ([33]; 206 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n11 \n \nSupplementary Figure 2).  The frequency of MTDRhigh cells was lower in the CD8dim T cell 207 \npopulation than CD8 bright T cells (median of differences = -8.99%; Wilcoxon signed -rank 208 \ntest p = 0.002; Fig. 1F).  These observations indicate that individuals with HIV-associated 209 \nKS have an expanded population of CD8 dim T cells with a highly  differentiated/senescent 210 \nphenotype and reduced mitochondrial activity. 211 \n 212 \nMitochondrial activity is reduced in CD8dim proliferating cells 213 \nWe next examined whether cell surface expression of the CD8 co-receptor was related to 214 \nreplicative capacity and mitochondrial activity in proliferating cells. PBMC were stimulated 215 \nwith the polyclonal stimulus PHA  for five days  and proliferation was measured by CFSE 216 \ndilution (Fig. 2A) . Proliferative capacity was reported as  proliferation index ( the mean 217 \nnumber of proliferative cycles undergone by each responding cell).   218 \nCD8 T cells that had proliferated (CFSElow) exhibited greater mitochondrial activity ( % of 219 \ncells MTDRhigh) than non-proliferating (CFSEhigh) cells (median of differences = 23.25%; 220 \nWilcoxon signed -rank test p = 0.002; Fig. 2B) . T here was a strong positive correl ation 221 \nbetween mitochondrial activity  of all CD8 T cells  and proliferation index  at day five , 222 \ndemonstrating the importance of mitochondrial respiration to CD8 T cell proliferation (rs = 223 \n0.75, p = 0.017; Fig. 2C) [26]. Conversely, there was a strong negative correlation between 224 \nmitochondrial activity of all CD8 T cells and the percentage of CD8dim T cells in the culture 225 \nat five days (rs = -0.75, p = 0.017; Fig. 2D), suggesting that low CD8 expression is associated 226 \nwith reduced mitochondrial activity . Further supporting this hypothesis, CD8 dim cells that 227 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n12 \n \nhad proliferated exhibited lower mitochondrial activit y (% MTDR high) than CD8 bright cells 228 \nthat had proliferated (median of differences = -23.35%; Wilcoxon signed-rank test p = 0.002; 229 \nFig. 2E). There was also evidence of a moderate negative correlation between the percentage 230 \nof CD8 dim T cells in the culture and proliferation index (rs = -0.61, p = 0.067 ; Fig. 2F). 231 \nCollectively these results indicate that low surface expression of CD8 is associated with 232 \nreduced mitochondrial respiration and replicative capacity of proliferating CD8 T cells.   233 \n 234 \nFigure 2. Mitochondrial activity is reduced in CD8dim proliferating cells.  235 \nA) Histograms showing CFSE dilution in unstimulated and PHA-stimulated CD8 T cells.  236 \nB) MitoTracker Deep Red (MTDR) fluorescence in PHA-stimulated proliferating 237 \n(CFSElow) cells.  A significantly higher percentage of proliferating (CFSElow) CD8 T cells 238 \nare MTDRhigh compared with non-proliferating (CFSEhigh) CD8 T cells, indicating that 239 \nproliferating cells have higher mitochondrial activity (n = 10; median of differences = 240 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n13 \n \n23.25%; Wilcoxon signed-rank test). C) Positive association between mitochondrial 241 \nactivity (% MTDRhigh) of all CD8 T cells in the culture and the proliferation index of 242 \nproliferating cells in response to PHA stimulation (Spearman correlation). D) Negative 243 \nassociation between mitochondrial activity and the frequency of CD8dim T cells following 244 \nstimulation with PHA (Spearman correlation).  E) The frequency of MTDRhigh cells, 245 \ncomparing CD8bright and CD8dim proliferating (CFSElow) cells.  CD8dim proliferating cells 246 \nare significantly less likely to be MTDRhigh, indicating lower mitochondrial activity 247 \n(median of differences = -23.35%; Wilcoxon signed-rank test).  F) Proliferation index of 248 \nPHA-stimulated CD8 T cells versus the frequency of CD8dim T cells in the culture 249 \n(Spearman correlation). Gray open squares, HIV+ KS+ participants; black circles, HIV+ 250 \nKSneg participants.   251 \n  252 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n14 \n \nDiscussion 253 \nThe underlying causes of persistent KS in a minority of HIV -infected individuals with 254 \ndurable viral suppression and CD4 T cell recovery under ART are unknown. Here, we have 255 \nidentified an expanded population of CD8 dim T cells with phenotypic characteristics of 256 \nsenescence and mitochondrial dysfunction in these individuals.  257 \nThe CD8 co-receptor amplifies signals through the TCR [40, 41] . Following antigenic 258 \nstimulation (e.g. by a virus-infected cell), CD8, along with the TCR, is downregulated from 259 \nthe cell surface, possibly to limit the str ength or duration of signaling [42-44]. This suggests 260 \nthat the expansion of CD8 dim T cells may be a response to high and/or persistent antigen 261 \nstimulation. Supporting this hypothesis, elevated frequencies of CD8 dim T cells have been 262 \nreported during acute HIV infection, and in children exposed to a high cumulative pathogen 263 \nburden during the first years of  life [45, 46]. Our observation that CD8 dim T cells are also 264 \nexpanded in individuals with per sistent KS under ART supports  the notion that CD8 265 \ndownregulation is a general phenomenon in settings of unresolved infection.  266 \nChronic viral infections are also associated with CD8 T cell terminal differentiation and/or 267 \nsenescence[47]. Senescent CD8 T cells exhibit reduced expression of PGC -1α, the master -268 \nregulator of mitochondrial biogenesis, and lower mitochondrial activity [27, 48]. Conversely, 269 \nforced expression of PGC -1α promotes robust CD8 T cell memory responses [49]. These 270 \nobservations underscore the importance of  mitochondrial respiration for long -term CD8 T 271 \ncell anti-viral function. In the setting of persistent KS, we observed that the expanded CD8dim 272 \nT cell population expressed high levels of Eomesodermin and CD57, proteins  respectively 273 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n15 \n \nassociated with terminal differentiation and senescence, and had reduced expression of PGC-274 \n1α. CD8 dim cells also cont ained fewer respiring mitochondria both ex vivo  and when 275 \nproliferating. Our data suggest that mitochondrial dysfunction may underlie the 276 \naccumulation of senescent CD8 T cells that has previously been reported in KS [34]; however, 277 \nthis hypothesis must be tested in future studies.  It is unclear from our current data  whether 278 \nCD8 expression directly regulates mitochondrial activity in CD8 T cells.   279 \nA key question is whether the CD8dim T cells we observed are specific for KSHV, for HIV, 280 \nor for other persistent viral infections such as CMV , which is highly seroprevalent in HIV-281 \ninfected individuals [50]. It is possible that CD8 down-modulation could be driven by multiple 282 \nconcurrent infections, by ongoing immune activation, or by a combination of factors. In this 283 \ninitial study, we were unable to determine whether the CD8 dim T cells we observed were 284 \nKSHV-specific, as KSHV is a large virus, and immunoprevalent epitopes eliciting responses 285 \nin a high percentage of seropositive individuals have not yet been identified. The question 286 \nof the antigen specificity of CD8dim T cells will be the subject of subsequent investigations.  287 \nOur work has some limitations. As this  was an observational study, we were unable to 288 \ndetermine whether the expansion of CD8 dim T cells plays a causative role in the failure to 289 \ncontrol KSHV under ART or is a consequence of this lack of suppression. Due to lack of 290 \navailable tissue , we were unable to assess whether CD8 T cells infiltrating the tumor 291 \nmicroenvironment also exhibit a CD8dim phenotype. This question, together with the antigen 292 \nspecificity of CD8 dim T cells and a direct examination of their functional profile, is the 293 \nsubject of ongoi ng investigations. If KSHV -specific CD8 T cells infiltrating the tumor 294 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n16 \n \nmicroenvironment express low levels of CD8 and exhibit reduced  mitochondrial activity, 295 \nthis will have important implications for immunotherapeutic approaches to KS treatment. 296 \n 297 \n 298 \nAcknowledgements 299 \nAuthor contributions: The manuscript was written by G.T.C., A.M.W., N. P.G., and T.M. 300 \nG.T.C. and N.P.G. contributed to study design. G.T.C. performed experimentation and 301 \ncollection of data. G.T.C. and A.M.W. performed the statistical analyses. G.T.C. acquired 302 \nfunding for the study . T.M. facilitated participant recruitment and sampl e collection. All 303 \nauthors provided review of the final manuscript.  We thank Joann Kuruc and Cynthia Gay 304 \nfor their work recruiting participants to  UNC cohorts that were retrospectively utilized for 305 \nthis study.   306 \nConflicts of interest: There are no conflicts of interest. 307 \n  308 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n17 \n \nReferences 309 \n1. Shiels MS, Pfeiffer RM, Gail MH, Hall HI, Li J, Chaturvedi AK, Bhatia K, Uldrick TS, Yarchoan R, 310 \nGoedert JJ, Engels EA. Cancer burden in the HIV-infected population in the United States. J Natl 311 \nCancer Inst 2011; 103(9):753-762. 312 \n2. Eltom MA, Jemal A, Mbulaiteye SM, Devesa SS, Biggar RJ. Trends in Kaposi's sarcoma and non-313 \nHodgkin's lymphoma incidence in the United States from 1973 through 1998. J Natl Cancer Inst 314 \n2002; 94(16):1204-1210. 315 \n3. Franceschi S, Maso LD, Rickenbach M, Polesel J, Hirschel B, Cavassini M, Bordoni A, Elzi L, Ess S, 316 \nJundt G, Mueller N, Clifford GM. Kaposi sarcoma incidence in the Swiss HIV Cohort Study before 317 \nand after highly active antiretroviral therapy. Br J Cancer 2008; 99(5):800-804. 318 \n4. Maurer T, Ponte M, Leslie K. HIV-associated Kaposi's sarcoma with a high CD4 count and a low 319 \nviral load. N Engl J Med 2007; 357(13):1352-1353. 320 \n5. Mani D, Neil N, Israel R, Aboulafia DM. A retrospective analysis of AIDS-associated Kaposi's 321 \nsarcoma in patients with undetectable HIV viral loads and CD4 counts greater than 300 322 \ncells/mm(3). Journal of the International Association of Physicians in AIDS Care (Chicago, Ill : 323 \n2002) 2009; 8(5):279-285. 324 \n6. von Braun A, Braun DL, Kamarachev J, Gunthard HF. New onset of kaposi sarcoma in a human 325 \nimmunodeficiency virus-1-infected homosexual man, despite early antiretroviral treatment, 326 \nsustained viral suppression, and immune restoration. Open forum infectious diseases 2014; 327 \n1(1):ofu005. 328 \n7. Chen J, Dai L, Barrett L, James J, Plaisance-Bonstaff K, Post SR, Qin Z. SARS-CoV-2 proteins and 329 \nanti-COVID-19 drugs induce lytic reactivation of an oncogenic virus. Communications biology 330 \n2021; 4(1):682. 331 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n18 \n \n8. Osman M, Kubo T, Gill J, Neipel F, Becker M, Smith G, Weiss R, Gazzard B, Boshoff C, Gotch F. 332 \nIdentification of human herpesvirus 8-specific cytotoxic T-cell responses. J Virol 1999; 333 \n73(7):6136-6140. 334 \n9. Bihl F, Narayan M, Chisholm JV, 3rd, Henry LM, Suscovich TJ, Brown EE, Welzel TM, Kaufmann 335 \nDE, Zaman TM, Dollard S, Martin JN, Wang F, Scadden DT, Kaye KM, Brander C. Lytic and latent 336 \nantigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and 337 \nEpstein-Barr virus induce T-cell responses with similar functional properties and memory 338 \nphenotypes. J Virol 2007; 81(9):4904-4908. 339 \n10. Bihl F, Berger C, Chisholm JV, 3rd, Henry LM, Bertisch B, Trojan A, Nadal D, Speck RF, Flepp M, 340 \nBrander C, Mueller NJ, Swiss HIVCS. Cellular immune responses and disease control in acute 341 \nAIDS-associated Kaposi's sarcoma. AIDS 2009; 23(14):1918-1922. 342 \n11. Robey RC, Lagos D, Gratrix F, Henderson S, Matthews NC, Vart RJ, Bower M, Boshoff C, Gotch 343 \nFM. The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is 344 \nskewed towards early and late lytic antigens. PLoS One 2009; 4(6):e5890. 345 \n12. Lepone L, Rappocciolo G, Knowlton E, Jais M, Piazza P, Jenkins FJ, Rinaldo CR. Monofunctional 346 \nand polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins. 347 \nClinical and vaccine immunology : CVI 2010; 17(10):1507-1516. 348 \n13. Lepone LM, Rappocciolo G, Piazza PA, Campbell DM, Jenkins FJ, Rinaldo CR. Regulatory T Cell 349 \nEffect on CD8(+) T Cell Responses to Human Herpesvirus 8 Infection and Development of 350 \nKaposi's Sarcoma. AIDS Res Hum Retroviruses 2017; 33(7):668-674. 351 \n14. Lambert M, Gannage M, Karras A, Abel M, Legendre C, Kerob D, Agbalika F, Girard PM, Lebbe 352 \nC, Caillat-Zucman S. Differences in the frequency and function of HHV8-specific CD8 T cells 353 \nbetween asymptomatic HHV8 infection and Kaposi sarcoma. Blood 2006; 108(12):3871-3880. 354 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n19 \n \n15. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie 355 \nAJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, 356 \nGoulder PJ, Klenerman P, Ahmed R, Freeman GJ, Walker BD. PD-1 expression on HIV-specific T 357 \ncells is associated with T-cell exhaustion and disease progression. Nature 2006; 443(7109):350-358 \n354. 359 \n16. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart 360 \nE, Sepulveda H, Balderas RS, Routy JP, Haddad EK, Sekaly RP. Upregulation of PD-1 expression on 361 \nHIV-specific CD8+ T cells leads to reversible immune dysfunction. Nature medicine 2006; 362 \n12(10):1198-1202. 363 \n17. Day CL, Kiepiela P, Leslie AJ, van der Stok M, Nair K, Ismail N, Honeyborne I, Crawford H, 364 \nCoovadia HM, Goulder PJ, Walker BD, Klenerman P. Proliferative capacity of epitope-specific CD8 365 \nT-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 366 \n1 infection. J Virol 2007; 81(1):434-438. 367 \n18. Migueles SA, Weeks KA, Nou E, Berkley AM, Rood JE, Osborne CM, Hallahan CW, Cogliano-368 \nShutta NA, Metcalf JA, McLaughlin M, Kwan R, Mican JM, Davey RT, Jr., Connors M. Defective 369 \nhuman immunodeficiency virus-specific CD8+ T-cell polyfunctionality, proliferation, and 370 \ncytotoxicity are not restored by antiretroviral therapy. J Virol 2009; 83(22):11876-11889. 371 \n19. Gaiha GD, McKim KJ, Woods M, Pertel T, Rohrbach J, Barteneva N, Chin CR, Liu D, Soghoian 372 \nDZ, Cesa K, Wilton S, Waring MT, Chicoine A, Doering T, Wherry EJ, Kaufmann DE, Lichterfeld M, 373 \nBrass AL, Walker BD. Dysfunctional HIV-Specific CD8(+) T Cell Proliferation Is Associated with 374 \nIncreased Caspase-8 Activity and Mediated by Necroptosis. Immunity 2014; 41(6):1001-1012. 375 \n20. Korencak M, Byrne M, Richter E, Schultz BT, Juszczak P, Ake JA, Ganesan A, Okulicz JF, Robb 376 \nML, de Los Reyes B, Winning S, Fandrey J, Burgess TH, Esser S, Michael NL, Agan BK, Streeck H. 377 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n20 \n \nEffect of HIV infection and antiretroviral therapy on immune cellular functions. JCI Insight 2019; 378 \n4(12). 379 \n21. Angin M, Volant S, Passaes C, Lecuroux C, Monceaux V, Dillies M-A, Valle-Casuso JC, Pancino 380 \nG, Vaslin B, Le Grand R, Weiss L, Goujard C, Meyer L, Boufassa F, Müller-Trutwin M, Lambotte O, 381 \nSáez-Cirión A. Metabolic plasticity of HIV-specific CD8+ T cells is associated with enhanced 382 \nantiviral potential and natural control of HIV-1 infection. Nature Metabolism 2019; 1(7):704-716. 383 \n22. Cham CM, Gajewski TF. Glucose availability regulates IFN-gamma production and p70S6 384 \nkinase activation in CD8+ effector T cells. J Immunol 2005; 174(8):4670-4677. 385 \n23. Jacobs SR, Herman CE, Maciver NJ, Wofford JA, Wieman HL, Hammen JJ, Rathmell JC. Glucose 386 \nuptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and 387 \nindependent pathways. J Immunol 2008; 180(7):4476-4486. 388 \n24. van der Windt GJ, Everts B, Chang CH, Curtis JD, Freitas TC, Amiel E, Pearce EJ, Pearce EL. 389 \nMitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. 390 \nImmunity 2012; 36(1):68-78. 391 \n25. van der Windt GJ, O'Sullivan D, Everts B, Huang SC, Buck MD, Curtis JD, Chang CH, Smith AM, 392 \nAi T, Faubert B, Jones RG, Pearce EJ, Pearce EL. CD8 memory T cells have a bioenergetic 393 \nadvantage that underlies their rapid recall ability. Proc Natl Acad Sci U S A 2013. 394 \n26. Chang CH, Curtis JD, Maggi LB, Jr., Faubert B, Villarino AV, O'Sullivan D, Huang SC, van der 395 \nWindt GJ, Blagih J, Qiu J, Weber JD, Pearce EJ, Jones RG, Pearce EL. Posttranscriptional control of 396 \nT cell effector function by aerobic glycolysis. Cell 2013; 153(6):1239-1251. 397 \n27. Callender LA, Carroll EC, Bober EA, Akbar AN, Solito E, Henson SM. Mitochondrial mass 398 \ngoverns the extent of human T cell senescence. Aging cell 2020; 19(2):e13067. 399 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n21 \n \n28. Kim JW, Gao P, Liu YC, Semenza GL, Dang CV. Hypoxia-inducible factor 1 and dysregulated c-400 \nMyc cooperatively induce vascular endothelial growth factor and metabolic switches 401 \nhexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol 2007; 27(21):7381-7393. 402 \n29. Delgado T, Carroll PA, Punjabi AS, Margineantu D, Hockenbery DM, Lagunoff M. Induction of 403 \nthe Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently 404 \ninfected endothelial cells. Proc Natl Acad Sci U S A 2010; 107(23):10696-10701. 405 \n30. Siska PJ, Beckermann KE, Mason FM, Andrejeva G, Greenplate AR, Sendor AB, Chiang YJ, 406 \nCorona AL, Gemta LF, Vincent BG, Wang RC, Kim B, Hong J, Chen CL, Bullock TN, Irish JM, Rathmell 407 \nWK, Rathmell JC. Mitochondrial dysregulation and glycolytic insufficiency functionally impair 408 \nCD8 T cells infiltrating human renal cell carcinoma. JCI Insight 2017; 2(12). 409 \n31. Krown SE, Metroka C, Wernz JC. Kaposi's sarcoma in the acquired immune deficiency 410 \nsyndrome: a proposal for uniform evaluation, response, and staging criteria. AIDS Clinical Trials 411 \nGroup Oncology Committee. Journal of clinical oncology : official journal of the American Society 412 \nof Clinical Oncology 1989; 7(9):1201-1207. 413 \n32. Roederer M. Interpretation of cellular proliferation data: avoid the panglossian. Cytometry 414 \nPart A : the journal of the International Society for Analytical Cytology 2011; 79(2):95-101. 415 \n33. Clutton G, Mollan K, Hudgens M, Goonetilleke N. A Reproducible, Objective Method Using 416 \nMitoTracker(R) Fluorescent Dyes to Assess Mitochondrial Mass in T Cells by Flow Cytometry. 417 \nCytometry Part A : the journal of the International Society for Analytical Cytology 2019; 95(4):450-418 \n456. 419 \n34. Unemori P, Leslie KS, Hunt PW, Sinclair E, Epling L, Mitsuyasu R, Effros RB, Dock J, Dollard SG, 420 \nDeeks SG, Martin JN, Maurer TA. Immunosenescence is associated with presence of Kaposi's 421 \nsarcoma in antiretroviral treated HIV infection. AIDS 2013; 27(11):1735-1742. 422 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n22 \n \n35. Papagno L, Spina CA, Marchant A, Salio M, Rufer N, Little S, Dong T, Chesney G, Waters A, 423 \nEasterbrook P, Dunbar PR, Shepherd D, Cerundolo V, Emery V, Griffiths P, Conlon C, McMichael 424 \nAJ, Richman DD, Rowland-Jones SL, Appay V. Immune activation and CD8+ T-cell differentiation 425 \ntowards senescence in HIV-1 infection. PLoS Biol 2004; 2(2):E20. 426 \n36. Paley MA, Kroy DC, Odorizzi PM, Johnnidis JB, Dolfi DV, Barnett BE, Bikoff EK, Robertson EJ, 427 \nLauer GM, Reiner SL, Wherry EJ. Progenitor and terminal subsets of CD8+ T cells cooperate to 428 \ncontain chronic viral infection. Science 2012; 338(6111):1220-1225. 429 \n37. Hasley RB, Hong C, Li W, Friesen T, Nakamura Y, Kim GY, Park JH, Hixon JA, Durum S, Hu Z, 430 \nSneller MC, Oguariri R, Imamichi T, Lane HC, Catalfamo M. HIV immune activation drives 431 \nincreased Eomes expression in memory CD8 T cells in association with transcriptional 432 \ndownregulation of CD127. Aids 2013; 27(12):1867-1877. 433 \n38. van der Windt GJ, O'Sullivan D, Everts B, Huang SC, Buck MD, Curtis JD, Chang CH, Smith AM, 434 \nAi T, Faubert B, Jones RG, Pearce EJ, Pearce EL. CD8 memory T cells have a bioenergetic 435 \nadvantage that underlies their rapid recall ability. Proc Natl Acad Sci U S A 2013; 110(35):14336-436 \n14341. 437 \n39. Bengsch B, Johnson AL, Kurachi M, Odorizzi PM, Pauken KE, Attanasio J, Stelekati E, McLane 438 \nLM, Paley MA, Delgoffe GM, Wherry EJ. Bioenergetic Insufficiencies Due to Metabolic Alterations 439 \nRegulated by the Inhibitory Receptor PD-1 Are an Early Driver of CD8+ T Cell Exhaustion. 440 \nImmunity 2016. 441 \n40. van den Berg HA, Wooldridge L, Laugel B, Sewell AK. Coreceptor CD8-driven modulation of T 442 \ncell antigen receptor specificity. J Theor Biol 2007; 249(2):395-408. 443 \n41. Wooldridge L, Laugel B, Ekeruche J, Clement M, van den Berg HA, Price DA, Sewell AK. CD8 444 \ncontrols T cell cross-reactivity. J Immunol 2010; 185(8):4625-4632. 445 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n23 \n \n42. Park JH, Adoro S, Lucas PJ, Sarafova SD, Alag AS, Doan LL, Erman B, Liu X, Ellmeier W, Bosselut 446 \nR, Feigenbaum L, Singer A. 'Coreceptor tuning': cytokine signals transcriptionally tailor CD8 447 \ncoreceptor expression to the self-specificity of the TCR. Nat Immunol 2007; 8(10):1049-1059. 448 \n43. Lachmann R, Bajwa M, Vita S, Smith H, Cheek E, Akbar A, Kern F. Polyfunctional T cells 449 \naccumulate in large human cytomegalovirus-specific T cell responses. J Virol 2012; 86(2):1001-450 \n1009. 451 \n44. Balyan R, Gund R, Chawla AS, Khare SP, Pradhan SJ, Rane S, Galande S, Durdik JM, George A, 452 \nBal V, Rath S. Correlation of Cell-Surface CD8 Levels with Function, Phenotype and 453 \nTranscriptome of Naive CD8 T Cells. Immunology 2018. 454 \n45. Eller MA, Goonetilleke N, Tassaneetrithep B, Eller LA, Costanzo MC, Johnson S, Betts MR, 455 \nKrebs SJ, Slike BM, Nitayaphan S, Rono K, Tovanabutra S, Maganga L, Kibuuka H, Jagodzinski L, 456 \nPeel S, Rolland M, Marovich MA, Kim JH, Michael NL, Robb ML, Streeck H. Expansion of Inefficient 457 \nHIV-Specific CD8 T Cells during Acute Infection. J Virol 2016; 90(8):4005-4016. 458 \n46. Falanga YT, Frascoli M, Kaymaz Y, Forconi C, Ong'echa JM, Bailey JA, Berg LJ, Moormann AM. 459 \nHigh pathogen burden in childhood promotes the development of unconventional innate-like 460 \nCD8+ T cells. JCI Insight 2017; 2(15). 461 \n47. Koch S, Larbi A, Ozcelik D, Solana R, Gouttefangeas C, Attig S, Wikby A, Strindhall J, Franceschi 462 \nC, Pawelec G. Cytomegalovirus infection: a driving force in human T cell immunosenescence. 463 \nAnn N Y Acad Sci 2007; 1114:23-35. 464 \n48. Henson SM, Lanna A, Riddell NE, Franzese O, Macaulay R, Griffiths SJ, Puleston DJ, Watson AS, 465 \nSimon AK, Tooze SA, Akbar AN. p38 signaling inhibits mTORC1-independent autophagy in 466 \nsenescent human CD8(+) T cells. J Clin Invest 2014; 124(9):4004-4016. 467 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint \n\n24 \n \n49. Dumauthioz N, Tschumi B, Wenes M, Marti B, Wang H, Franco F, Li W, Lopez-Mejia IC, Fajas L, 468 \nHo PC, Donda A, Romero P, Zhang L. Enforced PGC-1alpha expression promotes CD8 T cell 469 \nfitness, memory formation and antitumor immunity. Cellular & molecular immunology 2020. 470 \n50. Compston LI, Li C, Sarkodie F, Owusu-Ofori S, Opare-Sem O, Allain JP. Prevalence of persistent 471 \nand latent viruses in untreated patients infected with HIV-1 from Ghana, West Africa. Journal of 472 \nmedical virology 2009; 81(11):1860-1868. 473 \n 474 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 25, 2022. ; https://doi.org/10.1101/2022.02.23.22271244doi: medRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}