Patterns and Cofactors of Polyfunctional Mycobacteria-Specific T-Cell Response Restoration Following 6-Month Antiretroviral Treatment in Children With HIV

Abstract

Background: Despite immune restoration after initiation of antiretroviral treatment (ART), the risk of tuberculosis (TB) persists in children with HIV (CHIV). We determined patterns of immune restoration of mycobacteria-specific T cells following ART in CHIV.

Methods: CD4 and CD8 T-cell activation and memory phenotype and functional profiles before and 6 months after ART were evaluated in peripheral blood mononuclear cells from CHIV enrolled in the PUSH study (NCT02063880) in Nairobi, Kenya. T-cell expression of cytokines and activation-induced markers were measured following stimulation of peripheral blood mononuclear cells with a pool of 300 peptides from TB (MTB300) or staphylococcal enterotoxin B.

Results: Among 47 CHIV (median age, 1.5 years), staphylococcal enterotoxin B-induced Th1 cytokine+ and activation-induced marker+ CD4 cell frequencies increased significantly after 6 months of ART. Although MTB300-specific CD4 and CD8 cell frequency did not increase after ART, polyfunctional capacity of MTB300-specific CD4 cells expressing combinations of Th1 cytokines with CD40L increased significantly after ART. Baseline age, immune activation, and effector memory CD4 levels were associated with less restoration of MTB300-specific polyfunctional CD4 cells, whereas CD4 percentage and levels of naive CD4 cells following ART were associated with improved MTB300-specific polyfunctional capacity.

Conclusions: Despite increases in Th1 cytokine production, deficits in mycobacteria-specific CD4 cells persisted 6 months after ART, with higher deficits in older CHIV with more immunosuppression, higher immune activation, and lower proportion of naive CD4 cells. These findings may explain persistent TB risk during early ART among CHIV and identify those at highest risk.

Keywords: CD4 T cell; HIV; antiretroviral therapy; immune reconstitution; tuberculosis.

Figure 1.

Initiation of ART is associated with increased Th1 cytokine production capacity in children with HIV. Peripheral blood mononuclear cells were stimulated with SEB and MTB300 before and 6 months after initiation of ART and analyzed by intracellular cytokine staining and flow cytometry. A, Representative flow cytometry plots of Th1 cytokine production by CD4 T cells before and 6 months after ART. Frequencies of cytokine⁺ and AIM⁺ CD4 T cells and cytokine⁺ and CD107a⁺ CD8 T cells following stimulation with (B) SEB and (C) MTB300. Frequencies of CD4 and CD8 T cells are shown after subtraction of background levels of each marker in the unstimulated control. Boxes represent the median and IQR; whiskers represent the 10th and 90th percentiles. Differences between pre-ART and 6 months post-ART were evaluated with a Wilcoxon matched-pairs signed rank test. AIM, activation-induced marker; ART, antiretroviral treatment; SEB, staphylococcal enterotoxin B.

Figure 2.

Polyfunctionality of MTB300-specific CD4 T cells increases in children with HIV after 6 months of ART. Peripheral blood mononuclear cells were stimulated with SEB and MTB300 and analyzed by flow cytometry, as described in Figure 1. Intracellular cytokine staining data were analyzed in COMPASS to calculate the functionality score (FS) and polyfunctionality score (PFS). A and B, FS and PFS of CD4 and CD8 T-cell responses to SEB and MTB300. C, Heat map indicates the posterior probabilities of antigen-specific responses to each subset of MTB300-specific CD4 and CD8 T cells before and after 6 months of ART. Each row represents an individual child; columns represent combinations of effector molecules. D, MTB300-specific CD4 T-cell subsets identified by COMPASS with significant changes in frequency after 6 months of ART. E, Proportion of TNF-α⁺ cells to the total MTB300-specific CD4 T-cell response. A, B, D, and E, Boxes represent the median and IQR. ART, antiretroviral treatment; COMPASS, combinatorial polyfunctionality analysis of antigen-specific T-cell subsets; SEB, staphylococcal enterotoxin B.

Figure 3.

Differential increases in functionality of MTB300-specific CD4 T cells in children with HIV according to TB disease classification at the time of ART initiation. A and B, FS and PFS for MTB300-specific CD4 and CD8 T-cell responses were calculated in COMPASS, as described in Figure 2. FS and PFS were compared before and 6 months after ART separately in children with TB (confirmed and unconfirmed) and in children who were unlikely to have TB at the time of study enrollment and initiation of ART. Differences in FS and PFS pre-ART and 6 months post-ART were evaluated by a Wilcoxon matched-pairs signed rank test. Boxes represent median and IQR; whiskers represent minimum and maximum values. ART, antiretroviral treatment; COMPASS, combinatorial polyfunctionality analysis of antigen-specific T-cell subsets; FS, functionality score; PFS, polyfunctionality score; TB, tuberculosis.