Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection

Abstract

Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis.

Fig 1. Functional characteristics of C. albicans- and CMV-specific CD4 T cells in healthy subjects.

(A) Representative flow cytometric plots are shown for different cytokine expression in CFSE-low CD4+ T cells in PBMC after cognate antigen C. albicans (top) or CMV (bottom) stimulation. CFSE-labeled healthy donor PBMCs were stimulated with antigens for 6 days, followed by re-stimulation with PMA for de novo cytokine synthesis. CD3+CD4+ T lymphocytes are gated for analysis and the number in each plot indicates the percentage of CFSE-low Ag-specific CD4 T cells positive for each cytokine. (B) Comparison for percentages of Ag-specific CD4 T cells positive for each cytokine (cytokine+ CFSE-low%) between C. albicans- and CMV-specific CD4 T cells from multiple healthy subjects (n = 6). (C) Gene expression of Th1 (T-bet and EOMES) and Th17 (RORC) lineage-specific transcription factors between C. albicans- and CMV-specific CD4 T cells. Ag-specific CD4 T cells were sorted from PBMCs based on CFSE-low and subjected to RNA extraction and real-time PCR quantification. The data is shown as fold change for mRNA levels in C. albicans-specific CD4 T cells relative to CMV-specific CD4 T cells.

Fig 2. HIV infectivity in functional subsets of C. albicans-specific CD4 T cells.

(A) Overall in vitro HIV susceptibility of C. albicans- and CMV-specific CD4 T cells in the same healthy donor PBMCs. CFSE-labeled PBMCs were stimulated with C. albicans (left) or CMV (right) for 3 days, and exposed to HIV for infection. HIV infection of Ag-specific CD4 T cells was examined as intracellular p24+ rates in CFSE-low CD4 T cells (number in top-left quadrant). Representative flow cytometric data (left) and cumulative results (right) are shown. (B) Flow cytometric analysis of HIV infection in each cytokine+ subset of C. albicans-specific CD4 T cells. CFSE-low, C. albicans-specific CD4 T cells were gated (left) and co-expression of intracellular p24 and cytokine within the gated population was analyzed (right). Number in top-right quadrant shows p24+ percentage in each cytokine+ population. (C) Analysis of HIV infectivity (p24+, red dot) in IFN-γ- or MIP-1β-producing, C. albicans-specific CD4 T cells with or without co-expression with IL-2, IL-17 or IL-22 (blue background). Number in each quadrant shows p24+% in each functional subset of C. albicans-specific CD4 T cells and calculated using FlowJo program. Comparison of HIV infectivity (p24+%) in different functional subsets of C. albicans-specific CD4 T cells is shown (n = 6). (D) Boolean gating and spice analysis for HIV infectivity (p24+%) in all different functional subsets of C. albicans-specific CD4 T cells. (E) Analysis of CD25 expression and HIV infectivity in C. albicans and CMV-specific CD4 T cells with (top) and without (bottom) exogenous rIL-2. Number in each quadrant shows p24+% in CD25- or CD25+ subset of CFSE-low, Ag-specific CD4 T-cell population. (F) Cumulative data comparing p24+% in CD25- and CD25+ subset for multiple subjects (n = 6). (G) Analysis of CD25 and cytokine co-expression in C. albicans-specific CD4 T cells. Two-tailed p values are denoted.

Fig 3. Functional profiles of C. albicans- and CMV-specific CD4 T-cell responses in healthy donors and HIV-infected subjects.

(A) Functional profile of C. albicans-CD4 T cell responses. Representative flow cytometry data showing expression of each cytokine in CFSE-low, C. albicans-specific CD4 T cells in PBMCs of un-infected healthy (top) or HIV-infected (bottom) subjects. PBMCs of HIV-infected subjects used here were collected during early infection before massive CD4 depletion occurred. Shown are the gated CFSE-low CD4 T cells and the number in each plot shows cytokine+ % in CFSE-low C. albicans-specific CD4 T cells. Comparison for percentage of C. albicans-specific CD4 T cells positive for each cytokine from multiple un-infected and HIV-infected subjects (n = 7) is shown. (B) Functional profile of CMV-specific CD4 T cell responses. Representative flow cytometry data showing expression of each cytokine in CFSE-low, CMV-specific CD4 T cells in PBMCs of un-infected healthy (top) or HIV-infected (bottom) subjects. Comparison for multiple un-infected (n = 7) and infected subjects (n = 6) is also shown. Two-tailed p values are denoted. N.S. represents non-significant.

Fig 4. Quantification of cell-associated HIV DNA in sorted Ag-specific CD4 T cells of HIV-infected subjects.

(A) PBMCs of untreated HIV-infected subjects (early during infection before massive CD4 depletion occurred) were CFSE-labeled and stimulated with C. albicans or CMV antigen in the presence of AZT, to prevent de novo HIV replication, for 5 days. C. albicans- and CMV-specific CD4 T cells were sorted from PBMCs based on CFSE-low. (B) Cell-associated HIV DNA in sorted CD4 T cells was quantified by real-time PCR and compared between C. albicans and CMV antigens (n = 5). (C) Comparison of cell-associated HIV DNA in C. albicans-specific CD4 T cells with that in CMV-specific (red), VZV-specific (green), HIV Env-specific (purple) and CFSE-Hi non-specific (gray) CD4 T cells within the same HIV-infected individuals.

Fig 5. Analysis of mucosal homing markers of C. albicans-specific and CMV-specific CD4 T cells.

(A) Representative flow cytometry dot plots (left) and histogram comparison (middle) for expression of α4β7 and CCR6 between C. albicans- and CMV-specific CD4 T cells are shown. Cumulative results for comparing mean fluorescence intensity (MFI) of α4β7 and CCR6 expression between C. albicans- and CMV-specific CD4 T cells is also shown (right) (n = 6). (B) Gene expression of mucosal homing chemokines CCL-20 and CCL-25 in sorted C. albicans- and CMV-specific CD4 T cells. The data is shown as fold change for C. albicans-specific CD4 T cells relative to CMV-specific CD4 T cells. (C) Comparison of cytokine expression in α4β7+ (blue) and α4β7- subsets (red) of C. albicans-specific CD4 T cells. Representative histogram (top) and cumulative results for comparing cytokine+% between α4β7+ and α4β7- subset (bottom) are shown. (D) Comparison of HIV infectivity (p24+%) in α4β7+ and α4β7- subsets of C. albicans-specific CD4 T cells. Both representative and cumulative results are shown. (E) Impact of α4β7 blocking by ACT-1 antibody on HIV infectivity in C. albicans-specific CD4 T cells.

Fig 6. Preferential and progressive depletion of C. albicans-specific CD4 T-cell response in HIV-infected subjects.

(A) Representative flow cytometry data (2 subjects) showing C. albicans- (top) and CMV-specific (bottom) CD4 T-cell proliferative responses within the same PBMCs from HIV-infected subjects who manifested ongoing CD4 depletion. PBMCs collected at early infection (high CD4 count) and chronic infection (low CD4 count) from the same subjects are shown (gated on CD3+ T cells). (B) Longitudinal magnitudes of C. albicans- (left) and CMV- (right) CD4 T-cell proliferative responses from multiple HIV-infected subjects (n = 4) are shown. (C) Short-term ex vivo stimulation of PBMCs with peptide pools (C. albicans MP65 and CMV pp65) and intracellular cytokine staining for measuring frequencies of Ag-specific CD4 T cells in PBMCs. Representative data from 2 subjects show cytokine expression in C. albicans- (IL-17 and IL-2) and CMV-specific (IFN-γ and MIP-1β) CD4 T cells. The number in each plot shows total percentage of CD4 T cells expressing one and both cytokines. (D) Longitudinal frequencies of C. albicans- (left) and CMV- (right) CD4 T cells from multiple HIV-infected subjects (n = 5) are shown. Two-tailed p values are denoted.