CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

(ART). CD8 T cells are important for control of KSHV, the etiologic agent of KS. Upon activation, CD8 T cells upregulate glycolysis, enabling rapid generation of ATP and biosynthetic precursors. However, CD8 T cells infiltrating the tumor microenvironment must operate under conditions of glucose restriction. We hypothesized that CD8 T cells from individuals with persistent KS under ART exhibit functional and metabolic abnormalities. Methods: Specimens were obtained from HIV-infected participants on ART with biopsy-confirmed KS (HIV KS; obtained from the AIDS and Cancer Specimen Resource; n = 8) and HIV-infected participants on ART with no known history of KS (HIV controls; n = 8). CD8 T cell differentiation (CD45RO, T-bet, Eomesodermin), metabolic phenotype (glucose transporter Glut1, mitochondrial master regulator PGC-1α), and senescence (CD57) were assessed by flow cytometry. Proliferation in response to PHA was measured by CFSE dilution, and mitochondrial activity using MitoTracker® Deep Red. Results: Relative to HIV controls, memory (CD45RO+) CD8 T cells from HIV KS participants were skewed toward a more terminally differentiated phenotype, with a lower frequency of T-bet low Eomes low CD57 low cells (p = 0.01). HIV KS participants displayed an expanded population of CD8 dim T cells (median 9.3% of CD3+ T cells vs 3.1% in controls; p = 0.001). This population exhibited reduced expression of Glut1 (p = 0.008) and PGC-1α (p = 0.04) and increased CD57 expression (p = 0.02) compared with CD8 bright T cells, suggesting impaired capacity to utilize glycolysis and proliferate. CD8 T cell proliferation and mitochondrial activity were compared in 10 mM and 5 mM glucose. Proliferation and mitochondrial activity were lower in 5 mM glucose in 2/4 HIV KS participants tested (replication index in 10 mM vs 5 mM glucose 11 vs 6.5 and 19 vs 16, respectively), indicating reduced metabolic flexibility when glucose is limiting. CD8 T cell proliferation and mitochondrial polarization were correlated (r = 0.73; p = 0.02). Conclusion: Our data suggest that metabolic and functional abnormalities in CD8 T cells may contribute to KS persistence in HIV-infected individuals receiving ART. Therapeutic strategies to normalize CD8 T cell metabolism represent a novel approach to the treatment of persistent KS under ART. 272 SINGLE CELL EVALUATION OF KAPOSI SARCOMA TUMORS REVEALS COMPLEX IMMUNE INFILTRATE Warren Phipps 1 , David Coffey 1 , Yuexin Xu 1 , James Kafeero 2 , Peter Mooka 2 , Lazarus Okoche 2 , Diana Basemera 2 , Britta Flach 1 , Andrea Towlerton 1 , Edus H. Warren 1 1 Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 2 Uganda Cancer Institute, Kampala, Uganda Background: Kaposi sarcoma (KS) is highly associated with immunosuppression, and evidence suggests that KS oncogenesis is associated with loss of T-cell mediated control of human herpesvirus-8 (HHV-8). KS is a complex tumor, characterized histologically by spindle-like tumor cells infected with HHV-8 and marked inflammatory infiltrate. Identifying the elements that comprise the KS tumor, the phenotypic and translational state of these cell types, and how these cellular components interact in vivo will advance our understanding of KS tumorigenesis and guide the development of new targeted therapies. Methods: We evaluated KS tumor and normal skin samples obtained from treatment-naïve HIV-positive and HIV-negative adults with KS enrolled in an ongoing study at the Uganda Cancer Institute in Kampala, Uganda. RNA was extracted from tissue that had been snap frozen or preserved in RNALater, and sequencing was performed on Illumina HiSeq 2500. Leukocyte composition within each biopsy was estimated using CIBERSORT, an analytic platform used to characterize cellular gene expression profiles. Single-cell suspensions of a subset of KS tumors were sorted and evaluated using targeted multiplex RT-PCR with primers specific for 24 genes relevant to immune cell lineage, function, proliferation, and exhaustion. Results: CIBERSORT analysis of 39 KS tumors revealed that CD4 and CD8 T cells, monocytes, and macrophages represent the majority of intratumoral hematopoietic cells. To date, 2 cryopreserved single-cell suspensions have been analyzed. Candidate KS tumor cells with a CD34+/VEGFR3+/LYVE-1+ surface phenotype comprised 1.54% and 0.35% of cells from HIV+KS and HIV-KS subjects, respectively. Flow cytometric sorting showed populations of immune cells, including CD4/CD8, monocytes, and macrophages. Targeted transcriptional profiling of the single CD8+ T cells revealed significant heterogeneity in the expression of various genes, but uniformly low expression of genes associated with proliferation and functional activation, such as Ki-67,

granzyme B, and TNFa (Figure). Analysis of additional KS tumor single cell suspensions is ongoing. Conclusion: Our findings to date indicate that the immune infiltrate in KS tumors is dominated by T-cells and macrophages. Initial analyses suggest that the transcriptional profile of immune cells in KS tumors is consistent with an “exhausted” profile, which may have implications for the use of anti-PD1 or other immunotherapies targeting T-cell exhaustion in the treatment of KS.

Poster Abstracts

273 NEW VARIANT OF KAPOSI SARCOMA–ASSOCIATED HERPESVIRUS IN MEN WHO HAVE SEX WITH MEN Aude Jary 1 , Valentin Leducq 1 , Nathalie Desiré 1 , Romain Palich 1 , Veronique Joly 2 , Ana Canestri 3 , Adélie Gothland 1 , Sidonie Lambert 4 , Pierre-Marie Girard 4 , Corinne Amiel 3 , Diane Descamps 2 , Jean-Philippe Spano 1 , Christine Katlama 1 , Vincent Calvez 1 , Anne-Geneviève Marcelin 1 1 AP–HP, Hôpitaux Universitaires Pitié Salpêtrière, Paris, France, 2 AP–HP, Hôpital Bichat-Claude Bernard, Paris, France, 3 AP–HP, Hôpital Tenon, Paris, France, 4 AP–HP, Hôpital Saint-Antoine, Paris, France Background: Kaposi sarcoma-associated herpesvirus (KSHV) is involved in the development of Kaposi sarcoma (KS) and lymphoid malignancies: primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Molecular epidemiology led to the identification of 7 distinct subtypes whose worldwide distribution was mainly related to geographic region and more rarely to the clinical presentation severity. To assess KSHV diversity, we conducted a retrospective study including 57 HIV-infected men who have sex with men (MSM) with KSHV-associated diseases and 8 MSM KSHV-seropositive on HIV preexposure prophylaxis (PrEP). Methods: Whole blood sample of 41 KS, 12 MCM and 4 PEL patients and 8 oral swabs (PrEP users) were analyzed for (i) KSHV-DNA quantification, (ii) KSHV typing by ORF-K1 (or VR1) Sanger sequencing. A maximum of likelihood phylogenetic tree (PhyML) was reconstructed; pairwise genetic distances (GD) between amino-acid ORF-K1 sequences were calculated (Mega) and GraphPad was used to perform non-parametric tests. Results: KSHV typing was contributive in 34/57 patients (19 KS, 11 MCD and 4 PEL) and 5/8 PrEP users. All pathologies combined, subtype C was the most prevalent (18/34) followed by subtype A (11/34). Most of subtype C fell in genotype variant C3 (15/18). Among KS patients, variant C3 was more associated with cutaneous and/or oral mucosa lesions than others subtypes (Odd ratio=11.7, IC95% 1.1-214.2, p=0.023) regardless of the immuno- virological status (CD4 count cells p=0.97; HIV VL p=0.89) and KSHV-DNA viral load (VL) in subtype A tend to be higher than those of subtype C(p=0.055). Among PrEP users, 2 fell in variant C3 and 2 others in variant A4. Viruses of 5 patients (2 visceral KS, 1 MCD, 1 PEL and 1 PrEP user) were identified as “subtype F”. However, phylogenetic analysis showed that theirs sequences differed from 11% at amino-acid level of subtype F already described in Uganda (AY953882) as well as epidemiological context (MSM Caucasian versus African subtype). Moreover, ORF-K1 sequence was closed (GD=10-6) to that of KSHV described in a French MSM HIV+ patient with PEL in 2000 (AF178810).


CROI 2019

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