HDACi down-regulate HLA class I expression in patient samples.

<p>Fresh CD4 T cells from eight ART treated patients were treated for 24 hours with vorinostat (1uM, 333nm and 50nM), panobinostat (100nM, 20nM, and 5nM), romidepsin (100nM, 10nM, and 5nM) or 300nM prostratin. A) Median fluorescent intensity (MFI) values after treatment with clinically relevant doses are shown. A repeated measures one-way ANOVA with a Greenhouse-Geisser correction with Dunnett’s multiple comparison test was performed. B-D) MFI values reported as a percent reduction compared to untreated levels for vorinostat, panobinostat, and romidepsin dilution series, respectively.</p

HDACi treatment of CD4 T cells induces NK cell degranulation.

<p>Uninfected CD4 T cells were treated or not with HDACi for 24h and then cultured with NK cells for 5h and stained for extracellular CD107a. A) Representative plot showing CD107a expression in NK cells alone, NK cells treated with 100nM panobinostat for 5h, untreated NK cells co-cultured with CD4 T cells at a 1:1 ratio and untreated NK cells co-cultured with 100nM panobinostat treated CD4 T cells at a 1:1 ratio. B) Mean of four experiments. A Mann-Whitney U test was used. C) CD4 T cells were treated with 20nM panobinostat and 10nM romidepsin and co-cultured with NK cells as in A (n = 9). A Friedman’s test with Dunn’s test for multiple comparisons was performed. D) NK cells were co-cultured with untreated CD4 T or CD4 T cells treated with 333nM vorinostat, 20nM panobinostat, or 10nM romidepsin in the presence of 10μg/mL brefeldin A and cells were intracellularly stained with antibodies against IFN-γ.</p

HDACi treatment of CD4 T cells increases NK mediated killing.

<p>HIV-1 LAI infected CD4 T cells +/- HDACi (targets) were cultured overnight with uninfected CD4 T cells (non-targets) with or without NK cells (effectors) at an E:T:NT ratio of 10:1:1. A) CD4 T cells were treated with 100nM panobinostat and co-cultured with NK cells overnight. The percent p24 reduction with NK co-culture is shown (n = 5). A Mann-Whitney U test was used. B) A representative FATAL assay with Cell Trace Violet (CTV) stained infected CD4 T cells (targets), CFSE stained uninfected CD4 T cells (non-targets) +/- NK cells. C) Mean of four experiments using the FATAL assay. A Mann-Whitney U test used. D) CD4 T cells were treated with 333nM vorinostat (n = 7), 20nM panobinostat (n = 10) and 10nM romidepsin (n = 8) before culture with NK cells. Percent p24 reduction is shown. A Kruskal-Wallis test with Dunn’s test for multiple comparisons was performed. E-G) Uninfected CD4 T cells and infected cells 48 hours post infection were treated for 24 hours with 20nM panobinostat or 10nM romidepsin. Cells were then stained for levels of E) MICA/B, F) ULBP1, and G) ULBP2 (n = 3).</p

HDACi down-regulate HLA class I in uninfected primary CD4 T cells.

<p>A) Unstimulated CD4 T cells from eight HIV negative donors were treated with 1uM vorinostat, 100nm panobinostat or 10nM romidepsin for 24h. Cells were stained for HLA class I. Median fluorescent intensity (MFI) is shown (n = 8). A Friedman test with Dunn’s test for multiple comparisons was performed. B) Data from A was normalized to the MFI of the untreated control and shown as a percentage. C) Culture of primary CD4 T cells from seven new donors was repeated using 333nM vorinostat and 20nM panobinostat (n = 7). A Friedman test with Dunn’s test for multiple comparisons was performed. D) Data from C was normalized to the MFI of the untreated control and shown as a percent.</p

Mechanism of HLA class I down-regulation.

<p>(A, B) Fold up-regulation of HLA class I and β₂ microglobulin RNA, respectively, by qPCR of DMSO and 1uM vorinostat treated samples normalized to untreated controls. All amounts were normalized to 18S copies (n = 3). A Mann-Whitney U test used to compare levels of mRNA expression. C) MFI of total levels (after cell permeabilization) of HLA class I after 24h of culture with HDACi-free media (n = 7), 1uM vorinostat (n = 7), 100nM panobinostat (n = 3) and 10nM romidepsin (n = 3). A Kruskal Wallis test with Dunn’s test for multiple comparisons was performed. D) Kinetics of extracellular and total levels of HLA class I are shown. Extracellular and total values were calculated as a percentage of their respective 0h timepoint MFI (n = 3). E) Cells were treated with 1uM vorinostat or 100nM panobinostat for 24h. Cells were then either washed 3 times or left alone and cultured until HLA class I levels matched untreated controls. Times shown are post wash. Percent down-regulation of HLA class I compared to untreated controls is shown for vorinostat (n = 3) and panobinostat (n = 5).</p

Effects of treating both CD4 T cells and NK cells with HDACi depends on the chosen HDACi.

<p>A) An NK co-culture assay was performed overnight at a 10:1:1 E:T:NT ratio with either infected, untreated CD4 T (targets) and untreated NK cells (effectors) or with 20nM panobinostat treated targets and HDACi treated effectors. Intracellular p24 was measured and compared between the samples with and without NK cells. The difference in p24 expression was then converted to percent killing as described. A Wilcoxon matched-pairs signed rank test was performed (n = 5). In B, a similar co-culture assay was performed using 2 doses of vorinostat (333nM and 50nM), 2 doses of panobinostat (20nM and 5nM), 2 doses of romidepsin (10nM and 5nM) and 300nM prostratin. A Friedman test with Dunn’s multiple comparison test was performed (n = 5). Different colors represent different donors.</p

HDACi do not inhibit CD4 T cell susceptibility to CTL killing.

<p>HIV-1 LAI infected CD4 T cells with or without HDACi treatment (targets) were cultured overnight with uninfected CD4 T cells with or without HDACi treatment (non-targets) and SL9 transduced CD8 T cells (effectors) at an E:T:NT ratio of 1:1:1. HLA class I A*02 negative donors (n = 3) were used as a control. A) Representative plots of infected CD4 T cells in the absence (left) or presence (right) of CD8 T cells. B) Percent p24 reduction was calculated based on the percentage of p24 positive target cells before and after CD8 T cell co-culture, using 100nM panobinostat treated targets (n = 7) and 10nM romidepsin treated targets (n = 6). Different colors represent different donors. Separate Mann-Whitney U tests were performed.</p

Cox proportional hazards models on disease progression and PD-1, Tim-3 and Lag-3 expression.

<p>Cox proportional hazards models on disease progression and PD-1, Tim-3 and Lag-3 expression.</p

Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection

<div><p>The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches.</p></div

data_sheet_1_CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA.docx

<p>Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3⁺CD4⁺ cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32^low, CD32⁺CD14⁺, and CD32^high). Of note, CD4 negative enrichment kits remove the majority of CD4⁺CD32⁺ T cells, potentially skewing subsequent analyses if used. CD32^high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαβ, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32^low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3⁺CD4⁺CD32⁺ phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32⁺ T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.</p