CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Background: T-cell exhaustion is not reverse by effective ART. T-cell exhausted cells have been associated with HIV persistence during ART. The plasmacytoid dendritic cells (pDCs) sense viral and bacterial products through Toll-like receptors (TLR)-7 and -9 and translate this sensing in IFN-α production and T-cell polarization. It is unknown whether pDCs can reverse T-cell exhaustion in HIV-infected patient on long-term suppressive ART. The aim of the present study was to analyze, through a pDC/T-cell co-culture, whether pDCs after stimulation with different TLR agonist were able to reverse T-cell exhaustion. Methods: Patients on suppressive ART (ART, n=5) were compared with healthy donors (HD, n=5) and viremic patients naïve for ART (VIR, n=4). pDCs, CD4+ and CD8+ T-cells were isolated from 450mL of whole blood using by negative selection. After pDC overnight stimulation with HIV inactivated with aldritiol (AT-2-HIV), CpGA, CpGC, and GS9620 or no stimuli, stimulated pDCs were cocultured for 6h with autologous CD4+ or CD8+ T-cells. The expression of PD-1, TIGIT, TIM-3 and LAG-3 in different T-cell subsets was quantified ex vivo and in vitro by multiparametric flow cytometry. Results: Ex vivo the expression of PD1, TIGIT, TIM3 or LAG3 were increased in several CD4+ T-cell memory subsets from ART compared to HD (e.g.:PD1+TIGIT+TIM3+LAG3-CD4+CD45RA-CD27+,p=0.002). After the coculture, we observed a trend to decrease in the expression of these markers after AT-2 and CpG-A pDC stimulation (p=0.047 and p=0.06, respectively). This reversion in CD4+ T-cell exhaustion phenotype was specially patent after CpG-C and GS9620 pDC stimulation with normalization compared to HD (p=0.117,p=0.144, respectively). This decrease of CD4 T cells exhaustion markers occurs at the same time of an increase in the polyfunctionality of different CD4+ T-cell subsets in terms of cytokine production (e.g.: CD107a+IL2- IL17a+INFg+TNFαCD4+CD45RA+CD27- expression were significantly increased in ART respect HD after CpG-C and GS9620 stimulation p=0.047,p=0.037; respectively). Conclusion: The modulation of the pDCs through TLR agonists reverses CD4+ T-cell exhaustion in HIV-infected patients on ART. These results may have important implications in the reduction of deleterious effect of pDCs and T-cells causing non AIDS events and may decrease HIV-reservoir levels. 302 CD8+ SUBSET-DEPENDENT OVEREXPRESSION OF TIGIT AND TIGIT+TIM3 BY HIV DESPITE ART Oscar Blanch-Lombarte 1 , Esther Jimenez-Moyano 1 , Dan Ouchi 1 , Adam Pelletier 2 , Aarthi Talla 2 , Ashish Sharma 2 , Ruth Penya 1 , Judith Dalmau 1 , José R. Santos 3 , Rafick-Pierre Sekaly 2 , Bonaventura Clotet 1 , Julia G Prado 1 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 2 Case Western Reserve University, Cleveland, OH, USA, 3 Fundació Lluita Contra la Sida, Badalona, Spain Background: The expression of inhibitory Receptors (iRs) blocks CD8+ T-cell activity in HIV-1 infection. Consequently, the control of iRs is critical for recovering CD8+ T-cell function. The alterations of iR expression by HIV-1 infection are not fully delineated but are essential to identify future immunotherapeutic targets. With this aim, we performed a high-dimensional cytofluorimetrics of iRs, CD39, and CD8+ lineage markers in early and chronically suppressed HIV-infected individuals. Methods: We selected PBMCs from early (Ei=24) and chronically HIV-infected individuals with longitudinal samples in a median of 3 (CiS1) and 10 years (CiS2) on suppressive cART (n=24). For comparisons, we selected PBMCs from healthy seronegative individuals (HC, n=24). We stained PBMCs using antibodies for iRs (TIGIT, PD1, LAG3, and TIM3), CD39, and CD8+ T-cell lineage (CD3, CD8, CD45RA, CCR7, and CD27). We analyzed multivariate datasets by FlowJo, SPICE, and R package. Also, we performed an unsupervised KNN algorithm for cell clustering and tSNE for visualizing single-cell data. Results: Based on the expression levels of iRs and lineage markers, we identified 23 cellular clusters. From this analysis, we observed a remarkable heterogeneity of CD8+ T cells and detected four clusters with significant differences across CiS1 and CiS2 individuals (p<0.05). These four clusters were high on TIGIT expression and one of themwas also high on TIM3 expression. Moreover, differentiated clusters had additional lineage markers indicative of memory or effector-like features. We confirmed the overexpression of TIGIT at a single level or combined with TIM3, LAG3, and CD39 in CiS1 and CiS2 by combinatorial profiling with SPICE. Single TIGIT was elevated on CM and TM (p<0.05) and TIGIT+TIM3 on CM and E (p<0.05). The combinations of four iRs, including TIGIT+TIM3 with LAG3 or CD39, were upregulated on CM or E (p<0.05). Also, we found a correlation between CD4 counts and the absence of iR expression in CiS2 (r=0.51, p<0.05).

gp120 proteins. IgG was then tested for breath and potency of neutralization against a global HIV panel as well as autologous outgrowth viruses derived from each individual. Results: Binding against gp41 was highly correlated with gp120, and these binding titers were correlated with neutralization potency against the global panel. On average, participants exhibited low-potency neutralization of 8 of 12 viruses on the panel. Interestingly we did not observe potent autologous neutralization of outgrowth virus, and in fact 2 of 8 people harbored completely resistant virus at the highest level of IgG tested. 5 of the 8 individuals had a documented HIV-negative date, and therefore antibody functionality could be correlated to estimated length of infection before ART and duration of ART. We observe that length of infection is not correlated with autologous neutralization, but we do observe a trend toward more potent neutralization of the global panel by individuals infected for longer periods of time. Conclusion: Our findings agree with published studies of untreated individuals that length of infection is related to neutralization breadth. By contrast, we found that duration of ART treatment was not associated with differences in neutralization – either heterologous or autologous. Overall, these data suggest that the inducible reservoir is relatively resistant to autologous antibodies whether the individuals are ART-suppressed early or late after diagnosis. 300 NEAR NORMALIZATION OF IMMUNE ACTIVATION IN PLWH ON LONG- TERM SUPPRESSIVE ART Óscar Brochado Kith 1 , Isidoro Martínez 1 , Juan Berenguer 2 , Luz M. Medrano 1 , Juan González-García 3 , Pilar Garcia-Broncano 4 , María Á. Jimenez-Sousa 1 , Ana Carrero 2 , Victor Hontanon 3 , M Ángeles Muñoz-Fernández 2 , Juan Carlos López- Bernaldo 2 , Amanda Fernández-Rodríguez 1 , Salvador Resino 1 1 Institute of Health Carlos III, Madrid, Spain, 2 University Hospital Gregorio Marañon, Madrid, Spain, 3 La Paz University Hospital, Madrid, Spain, 4 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Background: In HIV-infected patients, chronic immune activation and inflammation persist after suppressive combination antiretroviral therapy (cART). We compared gene expression and biomarkers in peripheral blood between HIV-infected patients on long-term suppressive cART (HIV-group) and age-matched healthy controls (HC-group). Methods: Cross-sectional study of 22 subjects in HIV-group with HIV-RNA <50 copies/ml and CD4+ T-cells ≥500 cells/mm 3 for more than one year before sampling, and CD4/CD8 ratio ≥1 at sampling. RNA-sequencing of polyA-RNAs was performed from peripheral blood mononuclear cells (PBMCs). Thirteen T-cell subsets were evaluated by flow-cytometry and 32 plasma biomarkers by immunoassays. All p-values were corrected by the false discovery rate (q-values). Results: Only the serine/arginine repetitive matrix 4 (SRRM4) gene was differentially expressed between HIV and HC groups (q-value≤0.05 and fold- change≥2). However, 147 differentially expressed genes were found with a more relaxed threshold (p-value≤0.05 and fold-change≥1.5). Sixty-seven of them, with values of variable importance in projection (VIP)≥1, were selected for pathway analysis. Significant Ribosome-related pathways were represented by six ribosomal genes (RPs): S27 (RPS27), L18a (RPL18A), L8 (RPL8), L26 (RPL26), L4 (RPL4), and S21 (RPS21), all of them downregulated in the HIV-group. T-cells subset and plasma biomarkers were also analyzed, but none of themwere significant (q-value>0.05). However, non-corrected p-values showed higher values of CD4+ Treg cells (CD4+CD25+CD127-/low), MCP-1, and sVEGF-R1 in the HIV-group (p-value≤0.05). Correlation patterns between RNA-seq expression and peripheral biomarkers (T-cells and plasma) were different between HC and HIV groups. Conclusion: Immune activation and inflammatory biomarkers were close to normalization in HIV-infected patients on long-term suppressive cART, compared to HC-group. However, residual alterations remained at the gene expression of PBMCs, which still reveal the impact of HIV infection in these patients. 301 REVERSION OF CD4+ T-CELL EXHAUSTION MEDIATED BY PLASMACYTOID DENDRITIC CELLS M Reyes Jimenez-Leon 1 , M. Carmen Gasca-Capote 1 , Macarena López- Verdugo 1 , Laura Tarancon-Diez 1 , María Trujillo Rodríguez 1 , Cristina Roca 1 , Nuria Espinosa 1 , Alicia Gutiérrez-Valencia 1 , Pompeyo Viciana 1 , Luis López-Cortés 1 , Ezequiel Ruiz-Mateos 1 1 Institute of Biomedicine of Seville, Sevilla, Spain

Poster Abstracts

CROI 2020 103