Impaired Phenotype and Function of T Follicular Helper Cells in HIV-1-Infected Children Receiving ART

Abstract

T follicular helper (Tfh) cells are important components in development of specific humoral immune responses; whether the number and biology of Tfh cells is impaired in HIV-1-infected children is not yet studied.The frequency, phenotype, and function of Tfh cells and B cells were determined in blood of HIV-1-infected children receiving antiretroviral therapy (ART) and age-matched controls. Flow cytometry was used to characterize the frequency of Tfh cells and B cell subsets. Cytokine expression was measured after in vitro activation of Tfh cells.A reduced frequency of memory Tfh cells (P < 0.001) was identified in HIV-1-infected children and, on these cells, a reduced expression of programmed death-1 (PD-1) and inducible T cell costimulator (ICOS) (P < 0.001 and P < 0.01). Upon activation, the capacity of Tfh cells to express IL-4, an important cytokine for B cell function, was impaired in HIV-1-infected children.B cell subpopulations in HIV-1-infected children displayed significant differences from the control group: the frequency of resting memory (RM) B cells was reduced (P < 0.01) whereas the frequency of exhausted memory B cells increased (P < 0.001). Interestingly, the decline of RM cells correlated with the reduction of memory Tfh cells (P = 0.02).Our study shows that function and phenotype of Tfh cells, pivotal cells for establishment of adaptive B cell responses, are impaired during HIV-1 infection in children. A consistent reduction of memory Tfh cells is associated with declined frequencies of RM B cells, creating a novel link between dysfunctional features of these cell types, major players in establishment of humoral immunity.

FIGURE 1

Frequencies of CD4+, Tfh and memory Tfh cells in HIV-1-infected children and control individuals. The frequencies of CD4+ T cells (A), Tfh defined as CD4+CXCR5+ T cells (B) memory Tfh cells CD4+CD45RO+CXCR5+ (C) in control individuals (n = 40), HIV-1 infected (n = 38), aviremic (n = 25), and viremic (n = 13) children are shown. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

FIGURE 2

Expression of IFN-γ, IL-2, IL-4, and IL-21 in memory Tfh cells. The frequency of memory Tfh cells expressing the cytokines IFN-γ (A), IL-2 (B), IL-4 (C), and IL-21 (D) was determined after culture with PMA and Ionomycin in specimens from controls and HIV-1-infected children. The frequency of cytokine producing cells was determined after subtraction of values obtained in parallel control cultures without PMA and Ionomycin. In parenthesis the number of the analyzed specimens is shown. ∗P < 0.05; ∗∗P < 0.01.

FIGURE 3

Expression of ICOS and PD-1 in CXCR5+CD4+ and memory Tfh cells. The frequency of CXCR5+ PD-1+ cells (panels A and D), CXCR5+ICOS+ cells (panels B and E) and CXCR5+ICOS+PD-1+ cells (panels C and F) were determined among gated CD4+ cells (panels A–C) or CD4+CD45RO+ cells (panels D–F). The frequencies were compared between controls and aviremic and viremic HIV-1-infected children. In parenthesis the number of the analyzed specimens is shown. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

FIGURE 4

B cell subpopulations in HIV-1-infected children and controls. B cells and B cell subpopulations were characterized from whole blood of 55 controls and 45 HIV-1-infected children (26 aviremic and 19 viremic). (A) The whole peripheral B cell population was identified as CD19+ cells after exclusion of dead cells and (B) such gated cells were further delineated into 4 B cell subsets: CD19⁺CD10⁻CD21⁺CD27⁻ (naïve), CD19⁺CD10⁻CD21⁻CD27⁺ (activated memory, AM), CD19⁺CD10⁻CD21⁺CD27⁺ (resting memory, RM) and CD19⁺CD10⁻CD21⁻CD27⁻ (tissue like memory, TLM). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.