Fig 1.
Phenotype of CD101-expressing CD4 T cells in healthy rhesus macaques.
A) Representative staining of CD101 on CD4 subsets in healthy RM PBMC. B) Representative histograms of CD101 on detailed CD4 subsets in healthy RM PBMC. C) Expression of CD101 within detailed CD4 subsets in PBMC and LN from healthy RM (top); total contribution of CD101+ cells to the overall CD4 pool from each CD4 subset in PBMC and LN. D) UMAP plot of flow cytometry data showing overall Phenograph cluster location and expression intensity of markers of interest. E) Hierarchical clustering of expression (z-score) for selected markers from flow cytometry data in each Phenograph cluster. F) Heatmap of the top 50 differentially expressed genes between CD101+ and CD101- Tregs (CD95+ CD25+ CD127-) by scRNA-seq analysis of healthy RM PBMC. G) Violin plots showing RNA expression levels of 4 genes of interest. H) Cytokine expression levels in CD101- and CD101+ memory CD4 after 3hr PMA/ionomycin stimulation of total PBMC from healthy individuals.
Fig 2.
Dynamics of CD101-expressing CD4 T cells after SIV infection.
A) Representative staining of CD101 on PBMC memory CD4 pre- and post-SIV infection. B) Expression levels of CD101 on PBMC memory CD4 pre- and post-SIV infection. C) Total loss of CD101+ and CD101- cells within CD4 subsets calculated based on absolute counts in blood pre- and post-SIV infection (mean+SEM). D) Representative staining of CD101 on PBMC CD4 Tregs pre- and post-SIV infection. E) Expression levels of CD101 on PBMC CD4 Tregs pre- and post-SIV infection. F) Expression levels of CD101 on PBMC CD4 Tregs from HIV-infected individuals during active viremia and during suppressive antiretroviral therapy (ART). G) Representative staining of CD101 on rectal biopsy (RB) memory CD4 pre- and post-SIV infection. H) Expression levels of CD101 on RB memory CD4 pre- and post-SIV infection. I) Expression levels of α4β7 and CCR5 on RB memory CD4 CD101- and CD101+ cells from healthy RM. J) Association between depletion of CD101+ CD4 T cells in rectal biopsies at day 14 p.i. (calculated from baseline to acute infection as % of live lymphocytes) and SIV DNA levels in rectal biopsies at day 14 p.i. (spearman correlation, n = 9). K) CD101 expression within PBMC CD4 subsets during longitudinal SIV infection (mean±SEM). L) Overall contribution of CD101+ cells to the overall CD4 pool from each PBMC CD4 subset during longitudinal SIV infection (mean±SEM). Lines designate mean values.
Table 1.
Correlations of CD101 dynamics with plasma viral load, immune activation and plasma cytokine levels.
Analyses were done as Spearman correlations with r and p value displayed. All values are from the D14 p.i. or D42 p.i for 28 SIV-infected RMs. CD4 loss was calculated based on absolute counts of CD101+ CD4 pre- and post-infection. Statistically significant findings are highlighted in yellow (n = 28).
Fig 3.
Phenotypic profile of CD101+ CD4 during long-term antiretroviral therapy.
A) Representative staining of PD-1 and CTLA-4 within naïve, memory CD101- and CD101+ CD4 T cells in lymph node (LN) from ART-suppressed SIV-infected RM. B) Quadrant distribution of PD-1 and CTLA-4 expression within CD101- and CD101+ LN memory CD4 from ART-suppressed SIV-infected RM. C) Representative histogram of CD101 expression across PD-1 and CTLA-4 expression quadrants in LN memory CD4 from ART-suppressed SIV-infected RM. D) CD101 expression across PD-1 and CTLA-4 quadrants in LN memory CD4 from ART-suppressed SIV-infected RM. E) CD101 expression across PD-1 and CTLA-4 quadrants in PBMC memory CD4 from ART-suppressed HIV-infected individuals. F,G) Expression levels of CD39 (F) and TIGIT (G) within CD101- and CD101+ LN memory CD4. H) Pie chart representing the frequency of cells expressing PD-1 and/or CTLA-4 and/or CD39 and/or TIGIT within CD101- and CD101+ LN memory CD4. I,J) Expression levels of Ki-67 within CD101- and CD101+ memory CD4 from LN from ART-suppressed SIV-infected RM (I) and PBMC from ART-suppressed HIV-infected individuals (J). K) Cytokine expression levels in CD101- and CD101+ memory CD4 after 3hr PMA/ionomycin stimulation of total PBMC from ART-suppressed PLWH. Lines designate mean values.
Fig 4.
HIV DNA levels in sorted memory CD4 T cells.
A) Sorting strategy for memory CD101- and CD101+ memory CD4 T cells. B-D) Total HIV proviruses (B), defective HIV DNA copies (C) and intact HIV DNA copies (D) measured by IPDA in sorted CD101- and CD101+ memory CD4 T cells from PBMC from ART-suppressed individuals.