CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

247 INCREASED GUT AND BLOOD CD4+ T-CELL EXHAUSTION IN IMMUNOLOGICAL NONRESPONDERS Kristina B. Lorvik 1 , Malin H. Meyer-Myklestad 1 , Asle W. Medhus 1 , Marius Lund- Iversen 1 , Birgitte Stiksrud 1 , Dag Kvale 2 , Anne Ma D. Riise 1 , Dag Henrik Reikvam 1 , Kjetil Taskén 1 1 Oslo University Hospital, Oslo, Norway, 2 University of Oslo, Oslo, Norway Background: Immunological non-responders (INR) have increased inflammation and non-AIDS related morbidity. We hypothesized that their insufficient immune recovery is associated with a gut-induced exhausted T cell phenotype. Methods: Blood samples and mucosal biopsies from terminal ileum and sigmoid colon were collected from Caucasian men; 19 INR (ART>4 years with HIV RNA <50 copies/ml and CD4 count <400 cells/µL for >3.5 years); 20 immunological responders (IR) (ART>4 years with HIV RNA <50 copies/ mL and CD4 count >600 cells/µL for >3.5 years) matched on nadir CD4 count and age; and 20 age-matched healthy HIV-negative controls (HC). Peripheral blood and lamina propria mononuclear cells were analyzed with a multi- color flow cytometry panel to investigate the expression of the exhaustion markers PD1 and TIGIT in addition to T cell surface markers (CD3, CD4, CD8, CD25,, CD38, CD45RA, CD127, HLA DR) and the gut homing marker integrin β7. Immunohistochemistry was applied to detect PD1 ligand 1 (PD-L1). Results: INR had increased fractions of PD1+ and TIGIT+ CD4+ T cells compared with IR and HC both in blood (p<0.01) and gut (p<0.05). PD1 and TIGIT expression in blood and gut both correlated negatively with systemic CD4:CD8 ratio. In the blood, but not in the gut, INR had more activated (def.: CD45RAneg) gut-homing β7high CD4+ T cells than both IR and HC (p<0.05), but these cells did not display more exhaustion markers than activated non-gut homing CD4+ T cells. Immunohistochemistry staining of gut biopsies showed that neither INR nor IR expressed PD-L1. Conclusion: INR have a more exhausted CD4+ T cell pool than IR, both in blood and gut, supporting the hypothesis that T cell exhaustion may be a contributor to insufficient immunological response to ART. The higher prevalence of blood activated gut-homing CD4+ T cells in INR implies an enhanced stimulation and activation CD4+ T cells in the gut of in INR compared with IR, but this feature is not associated with differential expression of PD-L1. 248 NOVEL MECHANISM OF HIV-1 ELITE CONTROL BY ENRICHING GUT DIPEPTIDES AS CCR5-ANTAGONIST Ujjwal Neogi 1 , Maike Sperk 1 , Flora Mikaeloff 1 , Ashokkumar Manickam 1 , Anoop T. Ambikan 1 , Kamlendra Singh 2 , Jan Vesterbacka 3 , Anders Sönnerborg 1 1 Karolinska Institute, Stockholm, Sweden, 2 University of Missouri St Louis, St Louis, MO, USA, 3 Karolinska University Hospital, Stockholm, Sweden Background: A small subset (<0.5%) of HIV-1 positive individuals, the “Elite Controllers” (EC), controls viral replication for a long duration of time without receiving antiretroviral therapy. Due to the lack of data fromwell-controlled clinical EC cohorts, the mechanisms by which EC can control the virus remain mostly unknown. Transcriptomics analysis of blood cells has shown that CCR5 was downregulated in EC compared to viremic progressors (VP). Here we used untargeted plasma and fecal metabolomics to identify the metabolomic signature in EC followed by in vitro and ex vivo mechanistic studies. Methods: Blood and fecal material were collected from EC (n=14), matched HIV-negative control (HC, n=12) and VP (n=16). Untargeted metabolomics was performed by Ultra-High-Performance Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (UHPLC/MS/MS). Microscale thermophoresis (MST) was performed to describe the peptide-protein interactions. Viral infection and release assays were performed in TZMBl-cell lines and primary CD4+ T-cells, respectively. The significance was considered p<0.05 and false discovery rate <0.1. Results: In total 825 biochemicals were identified in feces and 950 biochemicals in plasma of which 485 and 294 biochemicals had group effects identified by ANOVA in fecal and plasma samples respectively. The top 30 metabolites important for group separation identified by random forest analysis were part of lipid metabolism, nucleotide metabolism, and amino acid metabolism. However, among the 19 identified peptides 79% (15/19) were significantly enriched in EC compared to HC and VP in feces. Of these, 47% (7/15) were significantly enriched in the plasma of EC compared to VP. We synthesized these seven dipeptides in amide forms (DP-am) and performed MST with protease and gp120 proteins. The DP-am binds to gp120 but not to the protease. This was further supported by infection assays in TZMbl cells lines which gave an

246 GUT MUCOSAL IL22+ T CELLS ARE RELATED WITH INCOMPLETE CD4 RESTORATION DESPITE cART Isaac Rosado-Sánchez 1 , Ines Herrero-Fernández 1 , Salvador Sobrino 2 , Ana E. Carvajal 3 , Miguel Genebat 2 , Rocío M. De Pablo 3 , Rocio Ruiz 3 , Jorge Sanchez- Villegas 2 , Manuel Leal 2 , Yolanda M. Pacheco 1 1 Institute of Biomedicine of Seville, Sevilla, Spain, 2 Hospital Universitario Virgen del Rocio, Sevilla, Spain, 3 University of Sevilla, Sevilla, Spain Background: Gut mucosal immunity plays a central role in the HIV pathogenesis but large gaps of knowledge remain, particularly in the scenario of the incomplete CD4-recovery. Impaired gut junctional complexes and increased markers of intestinal permeability and damage have been described in such scenario. Our aimwas to explore gut mucosal T-cell function and its potential relationship with the mucosal damage and immune reconstitution. Methods: Biopsies of both, caecum (CA) and terminal ileum (TI), in parallel of peripheral samples, of non-HIV subjects and treated, virally-suppressed HIV-infected subjects were obtained: subjects with CD4 T-cell counts bellow 250 cell/ul after two years of suppressive-treatment (INR) and control subjects overcoming such threshold (IR). Histological assessment of mucosal damage was performed using a semi-quantitative scale of five physical parameters. MMCs were digested, isolated and stimulated (PMA/Iono) to quantify the T-cell production of different homeostatic cytokines by flow-cytometry. Th22, Th17, Th1 and Treg, as well as their production of combined cytokines, were analyzed. The expression of mucosal caspase-3, gal-3, Zo-1 and mucin was analyzed by immunofluorescence. LBP levels, as a marker of intestinal permeability, was measured by ELISA in peripheral samples. Potential correlations were explored using Spearman rank test. Results: The highest mucosal damage was observed in both types of biopsies from INR subjects. The production of IL22 by T-cells correlated with peripheral CD4 T-cell counts and CD4/CD8 T-cell ratio (stronger with mucosal than peripheral T-cells; P<0.005 for both locations). INR showed the lowest frequencies of IL22+CD4+mucosal T-cells, whereas the highest IL17+/ IL22+CD4 T-cells ratios, independent of the location. INR showed increased frequencies of FoxP3+CD4+mucosal T-cells, particularly at TI, and reduced Th17/Treg and Th22/Treg ratios at both locations. IL22+CD4+/Treg ratios negatively correlated with mucosal damage (p<0.001 at both locations). At TI, the % IL22+CD4+ T-cells correlated with the mucosal expression of caspase-3 and Zo-1 (negatively), as well as with the expression of gal-3 (positively), and showed a negative correlation with LBP levels (p<0.05). Conclusion: Subjects with incomplete CD4-recovery show reduced capacity of gut mucosal T-cells to produce IL22. This cytokine, with a dual “inflammatory- protective” role during tissue responses to inflammation, could have a protective-regenerative potential on the gut potentially necessary for the normal CD4-recovery.

Poster Abstracts

84

CROI 2020