At homeostasis filarial infections have expanded adaptive T regulatory but not classical Th2 cells

Abstract

Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4(+) and CD8(+) T cells in filaria-infected (Fil(+)) and -uninfected (Fil(-)) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4(+) lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil(+), as were those of IL-10(+)/IL-4(+) double-producing CD4(+) cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil(+) compared to Fil(-) individuals. Although the frequency of nTreg was increased in Fil(+), IL-10 was overwhelmingly produced by CD4(+)CD25(-) cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10-producing aTreg/Tr1s in Fil(+). Together, these data show that at steady state, IL-10-producing aTreg/Tr1 as well as nTreg and effector Th17 CD4(+) cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level.

FIGURE 1

Filarial infection is associated with higher frequency of CD3⁺CD4⁺ cells producing IL-4, IL-17A, and IL-10. Frequencies of cytokine-producing CD3⁺CD4⁺ cells are shown in Fil⁻ (white bars) and Fil⁺ (black bars) individuals with bars representing the GM and 95% confidence interval (CI) of the CD4⁺ cells producing the cytokines listed on the x-axis.

FIGURE 2

Filarial infection is associated with expansion of CD4⁺ cell populations producing IL-10 or IL-17A alone and CD4⁺ cells producing both IL-10 and IL-4. The frequency of multiple cytokine-producing CD4 cells was determined using Boolean gating. Bars represent the GM and 95% CI of CD4⁺ cells producing the combination of cytokines listed below each set of bars in Fil⁻ (white) and Fil⁺ (black) populations.

FIGURE 3

Filarial infection is associated with expansion of nTregs (A) and those expressing CTLA-4 or IL-10 (B). Each dot in A represents an individual’s frequency of CD3⁺CD4⁺CD25⁺Foxp3⁺CD127⁻ cells; the bar represents the GM for each group. In B, the bars represent the GM with the 95% CI of the frequencies of specific cells based on expression of markers listed below each bar.

FIGURE 4

CD4⁺CD25^−/low cells are the main source of IL-10 in filarial infection (A), and aTreg/Tr1s are the main source of IL-10 (B). A, Frequencies of CD4⁺CD25⁻IL-10⁺ (circles) and CD4⁺CD25⁺IL-10⁺ (squares) cells were calculated for each individual and plotted as individual lines for both Fil⁺ and Fil⁻ subjects. B, The iGMFI of IL-10–producing Th1, Th2, Th17, and aTreg/Tr1 cells was calculated, and the GM of each cell population was used to plot the pie chart. Each portion of the pie represents the relative contribution of each cell population to the total IL-10 pool in each group.

FIGURE 5

In patent filarial infection, the levels of spontaneously produced IL-10 correlate with the iGMFI of IL-10–producing CD4⁺CD25⁻ and aTreg/Tr1s. The iGMFI for IL-10–producing nTreg (A), single IL-10–producing CD4⁺ (aTreg/Tr1) (B), CD4⁺CD25⁻IL-10⁺ (C), and CD4⁺CD25⁺IL-10⁺ cells (D) for Fil⁻ (top row) and Fil⁺ (bottom row) individuals as a function of IL-10 produced spontaneously. Values of p and r derive from Spearman rank correlation analyses.

FIGURE 6

CD3/CD8⁺ cells also contribute to IL-10–producing populations in filaria-infected patients as total CD8⁺ T cells (A) or CD8⁺T cells producing only IL-10 (B). The frequency of single cytokine-producing CD8⁺ cells is shown in A with the bar representing the GM plus 95% CI for both Fil⁻ (white) and Fil⁺ (black) individuals for each cytokine listed below the bar. B shows the frequency of multiple cytokine-producing cells as determined by Boolean gating.