TL1A-DR3 Plasma Levels Are Predictive of HIV-1 Disease Control, and DR3 Costimulation Boosts HIV-1-Specific T Cell Responses

Abstract

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm³, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm³, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm³) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR⁺CD8⁺ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.

Figure 1:. Plasma soluble factors in chronic untreated HIV groups.

“Communicome” arrays performed in 96 HIV-infected individuals with HIV-High (n=47) and HIV-Low (n=49) ((8), Table S1) were analyzed by applying a random forest model. A) Frequency plot indicating the relevance of the top 25 scoring soluble factors with frequency ≥100 obtained after CART analysis. B) Pie chart representing the gene ontology (GO) biological processes gene enrichment analysis percentages for each category represented among the top 25 candidates. C) KEGG enrichment analysis showing the fold enrichment value of each pathway among the top 25 selected factors.

Figure 2:. sTL1A and sDR3 plasma levels and PBMC gene expression in chronic HIV-infected individuals.

sTL1A (A) and sDR3 (B) relative plasma levels (Z-score “communicome” values) in HIV-High (n=47) and HIV-Low (n=49) (Table S1). C-D) Gene expression (Table S1) of TL1A (C) and DR3 (D) in HIV-High (n=16) and HIV-Low (n=30). E-F) Unrelated validation cohorts for gene expression of TL1A (E) and DR3 (F) in dry pellet PBMC samples from seronegatives (SN, n=6) and HIV-infected individuals one year before and after initiation of treatment (untreated [n=11] and treated [n=5]) and in LTNP (n=23) (Table S1). G) Absolute DR3 quantifications in plasma (ELISA) in a confirmatory cohort including seronegatives (SN, n=8) and samples from time points one year before and after initiation of treatment (untreated [n=15] and treated [n=10]) and controllers (n=31), Table S1). The Mann-Whitney test was applied for group comparisons, and p-values < 0.05 were considered significant.

Figure 3:. DR3 correlates with effector CD8 T-cells and TL1A, with regulatory CD4 and CD8 phenotypes.

A–B) Correlation between plasma DR3 protein levels and the percentage of effector CD8 T cells (A) and HLA-DR+ CD45RO+ CD8 T cells (B) in a cohort of treated individuals (n=26, Table I). C) Correlation between ELISA-determined plasma TL1A levels and the percentage of CD25^high FOXP3₊ T cells (circles CD4 T cells and squares CD8 T cells) in treated individuals including immune-concordant and -discordant individuals (n=26, Table I). The Spearman rank test was used for the correlations, and p-values <0.05 were considered statistically significant.

Figure 4:. TL1A/DR3 axis role in HIV-specific T-cell responses.

A-B) sDR3 relative plasma levels (Z-score communicome) correlate with T-cell breadth (A) and magnitude (B) of HIV-High (n=46) and HIV-Low (n=41) individuals (Table S1). C) IFN-g-ELISPOT (percentage increase in the magnitude of T-cell) after HIV-specific peptide pool stimulation in the presence of recombinant TL1A and DR3 and specific anti-DR3 mAb in PBMCs from untreated PLWH (Table S1). D) Percentage increase in the magnitude of CEF-specific responses upon co-stimulation with anti-DR3 mAb. E) Increase in magnitude of DNA.HTI-specific T cell responses in isolated splenocytes from HTI-vaccinated mice after stimulation with the mouse DR3 specific antibody 4C12 (agonist DR3). The Spearman rank test was used for correlation analysis and the Wilcoxon signed rank test for group comparisons. p-values <0.05 were considered statistically significant.

Figure 5.. Model of TL1A-DR3 axis in HIV infection.

A) Failure of TL1A-DR3 axis during uncontrolled HIV infection. Upon HIV-specific activation, there is no upregulation of DR3 in surface membrane of CD8 T cells (A.1). The reduced levels of sTL1A do not allow the signaling through DR3 receptor (A.2) that would weaken the IFNg production (A.3). B) HIV control. Upon HIV-specific activation, DR3 protein is upregulated in cell membrane (B.1) that together with higher plasma levels of sTL1A and/or the presence of a DR3-agonist would ensure the signaling of the TL1A-DR3 axis (B.2). That co-stimulatory effect would enhance the HIV-specific IFNg responses (B.3)