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Phenotypic, cytolytic and transcriptional differences between LN and blood CD4+ T cells in HIV-infected and -uninfected individuals.

(A) Flow cytometry plots (HIV-infected CP) and scatter plots for naïve and memory subsets of LN and peripheral blood (PB) CD4+ T cells in HIV-infected and -uninfected subjects. (B) Flow cytometry plots (HIV-infected CP) showing the lack of T-bethiEomes+ CD4+ T cells in LNs. Corresponding scatter plots demonstrating the frequency of T-bethi cells of memory (non-naïve) CD4+ T cells (top) and frequency of Eomes+ cells of T-bethi CD4+ T cells (bottom) for matched LN and PB. (C) Flow plots (HIV-infected CP) showing the lack of Granzyme B+perforin+ CD4+ T cells in LNs and scatter plots with the frequency of LN and PB perforin+ cells of memory CD4+ T cells (top). Frequencies of Granzyme B+ cells of perforin+ CD4+ T cells (bottom) for matched LN and PB. (D) Flow plots (HIV-infected CP) and scatter plots showing the distribution of CD27+ cells within the Granzyme B+ CD4+ T cell compartment for matched LN and PB. (E) The distribution of different populations in the tSNE space is based on 30.000 live CD4+ T cells that were merged from LN and PB from a HIV-infected CP with detectable levels of cytolytic cells in the PB and LN Tfh cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme B, perforin, CXCR5 and PD-1 expression on gated bulk CD4+ T cells. The naïve cluster (green) is based on high CCR7 and low CD45RO intensity; the Tfh cluster (red) on high intensity of PD-1 and CXCR5; and the effector cluster (orange) on high T-bet and perforin expression intensity. After separating out the merged LN and PB single CD4+ T cell data, a lack of Tfh cells was apparent in PB and effector CD4+ T cells in the LN (lower right tSNE plots). Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between two unmatched groups, and Wilcoxon matched-pairs single rank tests between matched samples; *P < 0.05, **P < 0.01 and ***P < 0.001. All data-points are derived from the North-American and Mexico cohort.</p

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Cytolytic CD4+ T cells express high levels of T-bet and Eomes in blood.

(A) Representative flow cytometry plots of Granzyme B and perforin expression in CD4+ T cells for an HIV-infected and–uninfected subject. The distribution of Granzyme B+perforin+ (red) and Granzyme B-perforin- (blue) CD4+ T cells are shown for T-bet and Eomes expression. (B) Frequency of perforin+ CD4+ T cells within the T-bethiEomes+ and T-betdim/- population (left) and T-bethiEomes+ within the perforin+ or perforin- population for HIV-infected and–uninfected subjects. (C) Correlation between the frequency of perforin+ and T-bethi CD4+ T cells. (D) Imagestream analysis on T-bethi and T-betdim CD4+ T cells. Overlays of fluorescent channels for DAPI (nuclear) and T-bet, showing where in the cells T-bet are localized. The frequency of nuclear, nuclear/cytoplasmic and cytoplasmic localization for T-bethi and T-betdim CD4+ T cells are shown in the before-after graphs. (E) tSNE plots based on 30,000 live CD4+ T cells that were merged from three HIV-uninfected subjects with detectable cytolytic CD4+ T cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme A, Granzyme B and perforin expression intensity. The red gate indicates the identified “effector” cluster with overlapped expression of cytolytic markers as well as T-bet and Eomes. (F) Flow plots of MIP-1α production using media (NC) and aCD3-CD28 stimulations for T-bethi and Eomes+ CD4+ T cells, as well as correlation between the frequency of T-bethiEomes+ and MIP-1α+ CD4+ T cells following aCD3-CD28 stimulations. Median and IQR are shown for all scatter plots and Mann-Whitney tests were performed to compare differences between groups; ***P < 0.001. A non-parametric Spearman test was used for the correlations analysis. All data are derived from the North-American cohort.</p

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Functional characteristics of polyclonal and virus-specific effector CD4+ T cell responses in HIV-infected LNs and blood.

(A) Flow cytometry plots (HIV ART+ subject) and plots for matched HIV-Gag or–Env-specific CD4+ T cell responses in HIV-infected LN and PB. (B) Flow cytometry plots (HIV-infected CP) showing the negative control (NC) and Gag-specific response of LN CD4+ T cell response. The high abundance of CD107a (red) that is not co-expressed with IFNγ or TNF is illustrated in this example. SPICE analysis of functional combination between LN (red) and PB (red-gray) Gag-specific CD4+ T cell responses for HIV-infected CPs and ART+ subjects. (C) Flow plots (HIV-infected CP) of Gag-specific CD4+ T cell response (red) from LN and PB in relation to CD27 and perforin expression. Graphs represent the frequency of (C) perforin+ and (D) T-bethi cells between LN and PB Gag-specific CD4+ T cells. (E) Flow plots (HIV-infected CP) of MIP-1α versus IFNγ and TNF production for LN and PB SEB stimulated CD4+ T cells. Corresponding plots showing the frequency of MIP-1α+ SEB stimulated CD4+ T cells (top) and MIP-1α+ of IFNγ/TNF/CD107a/MIP-1α+ SEB stimulated CD4+ T cells (bottom). (F) Flow plots (HIV-infected CP) of MIP-1α versus IFNγ and TNF production for LN and PB CMV-specific CD4+ T cells and corresponding graphs showing the frequency of MIP-1α+ CMV-specific CD4+ T cells (top) and MIP-1α+ of IFNγ/TNF/CD107a/MIP-1α+ CMV-specific CD4+ T cells (bottom). Median and IQR are shown for all bar plots. Permutation test was performed between the pie charts. Wilcoxon matched-pairs single rank tests were performed to compare differences between two matched groups; *P < 0.05, **P < 0.01 and ***P < 0.001. All data-points are derived from the Mexico cohort.</p

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Temporal dynamics of T-bethi expression and effector HIV-specific CD4+ T cell responses following HIV infection in blood.

(A) Frequencies of T-bethi CD4+ T cells before and first sample taken 1 year after HIV infection (n = 10). (B) Longitudinal changes of T-bethi CD4+ T cells before and subsequently after HIV infection. Every individual is depicted with black connecting lines and red line indicate the estimated mean value (linear regression) over time (n = 10). (C) Frequency of T-bethi expression on memory CD4+ T cells in HIV- and HIV+ chronic progressors (CP) (D) T-bet, (E) perforin and (F) MIP-1α expression by IFNγ+ Gag-specific CD4+ T cells before and subsequently following HIV infection (n = 10). The colored lines represent each subject and their frequencies of T-bethi, perforin+ and MIP-1α+ Gag-specific CD4+ T cells over time. A Wilcoxon or Mann-Whitney test was performed to compare the difference between groups; *P < 0.05. Longitudinal data-points are derived from the RV217 cohort and cross-sectional data from the European cohort.</p

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Functional and transcriptional differences between degranulating cells in LN and blood.

(A) Flow cytometry plots (HIV-uninfected subject) of matched LN and PB CD107a+ SEB stimulated CD4+ T cell responses. Graphs are showing the frequencies of LN (top) and PB (bottom) CD107a+ SEB stimulated CD4+ T cell responses for CXCR5-, CXCR5+ and CXCR5hi cells. (B) Flow plots (HIV-infected CP) illustrating the expression of CD107a+ (red) Gag-specific CD4+ T cells in relation to CXCR5 between LN (top) and PB (bottom). (C) Corresponding plots from the same subject showing the expression of CD107a+ (red) Gag-specific CD4+ T cells in relation to CXCR5 and perforin for LN (top) and PB (bottom). Graphs represent the frequency of CD107a+ cells within the CXCR5+, CXCR5hi and perforin+ compartment for LN (top) and PB (bottom) Gag-specific CD4+ T cells. (D) Biomark analysis illustrating the tSNE distribution of single SEB stimulated CD107+ CD4+ T cells from LN (black) and PB (gray). Individual graphs represent the relative Log2 expression of different markers being significantly different (P<0.05) between blood and LN CD107+ cells. Non-parametric Kruskal Wallis test with Dunn’s multiple comparison test was performed to determine significant differences between groups; *P < 0.05, **P < 0.01 and ***P < 0.001. All data-points are derived from the North-American and Mexico cohort.</p

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Expression of effector molecules by Gag-specific CD4+ T cells from HIV elite controller LNs and blood.

(A) Flow cytometry plots (HIV elite controllers) illustrating the distribution of LN (red) and PB (blue) IFNγ+ Gag-specific CD4+ T cell responses between the T-betdimEomes- and T-bethiEomes+ compartment. Corresponding scatterplots showing the frequencies of T-betdimEomes- (left) and T-bethiEomes+ (right) cells of Gag-specific CD4+ T cells between LN and PB for HIV elite controllers. Flow plots (HIV elite controllers) of IFNγ+ Gag-specific CD4+ T cell response from LN and PB in relation to (B) perforin and (C) MIP-1α expression. Graphs represent the frequency of (B) perforin+ and (C) MIP-1α+ Gag-specific CD4+ T cells between LN and PB. (D) Flow plots and graphs demonstrating the expression pattern of perforin+Granzyme B+ memory CD4+ T cells in acute/early seroconverters (left graph). Right graphs show the frequency perforin+Granzyme B+ or T-bet expression out of total memory Ki-67+ CD4+ T cells. Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between groups; *P < 0.05, **P < 0.01 and ***P < 0.001. All data-points are derived from the North-American cohort.</p

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