Expression of inhibitory receptors and polyfunctional responses of T cells are linked to the risk of congenital transmission of T. cruzi

Abstract

Congenital T. cruzi infections involve multiple factors in which complex interactions between the parasite and the immune system of pregnant women play important roles. In this study, we used an experimental murine model of chronic infection with T. cruzi to evaluate the changes in the expression of inhibitory receptors and the polyfunctionality of T cells during gestation and their association with congenital transmission rate of T. cruzi infection. The results showed that pregnant naïve mice had a higher percentage of CD4+ and CD8+ T cells that expressed inhibitory receptors than cells from non-pregnant naïve mice. However, in mice chronically infected with T. cruzi, gestation induced a significant decrease in the frequency of T cells that expressed or co-expressed inhibitory receptors, as well as an increase in the frequency of polyfunctional CD4+ and CD8+ T cells. This different behavior may be due to the breakdown in the infected mice of the gestation-induced immune homeostasis, probably to control the parasite load. Remarkably, it was observed that the mothers that transmitted the parasite had a higher frequency of T cells that expressed and co-expressed inhibitory receptors as well as a lower frequency of polyfunctional parasite-specific T cells than those that did not transmit it, even though the parasitemia load was similar in both groups. All together these data suggest that the maternal immune profile of the CD4+ and CD8+ T cells could be a determining factor in the congenital transmission of T. cruzi.

Fig 1. Inhibitory receptor expression in the CD4⁺ and CD8⁺ T cells from non-pregnant or pregnant uninfected mice and non-pregnant or pregnant T. cruzi infected mice.

The frequencies of the CD4⁺ (A) and CD8⁺ (C) T cells that expressed PD-1, 2B4, CD160, or CTLA-4 in non-pregnant (Wt) (white box) or pregnant (U-p) (light grey box) uninfected mice and in infected pregnant (I-p) (dark grey box) or infected non-pregnant (I-np) (striped box) mice are shown. The lower and upper limits of the boxes indicate the 25th and 75th percentiles, respectively. The lines within the boxes depict the medians, and the whiskers indicate the lower and upper adjacent values (5th and 95th, respectively). The co-expression of PD-1, 2B4, CD160, and CTLA-4 in the CD4⁺ (B) and CD8⁺ (D) T cells from non-pregnant or pregnant uninfected mice and pregnant or non-pregnant T. cruzi-infected mice is shown. Increasing or decreasing frequencies of CD4⁺ (B) and CD8⁺ (D) T cells that co-express inhibitory receptors are indicated with the upward or downward arrowheads, respectively. The colors in the pie charts depict the co-expression levels of the inhibitory receptors: one (grey), two (light grey) and three (red) inhibitory receptors. The p values were calculated using the Mann–Whitney U test (A and C). The p values of the permutation test in the co-expression analysis (B and D) are shown in the pie charts (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig 2. Polyfunctional profiles of the CD4⁺ and CD8⁺ T cells from infected mice.

The frequency of the CD4⁺ (A1) and CD8⁺ (B1) T cells that expressed IFN-γ, TNF-α, IL-2, perforin and granzyme B in infected non-pregnant (white box) and pregnant mice (grey box) is shown. Panels A2 and B2 show the percentages of polyfunctional activity of the T cells from infected pregnant and non-pregnant mice, determined by the simultaneous measurements of the granzyme B, IFN-γ, IL-2, perforin and TNF-α expression after STcA stimulation. Panels A2 and B2 also show the frequency of the 16 most prevalent combinations of these functional molecules in CD4⁺ (A2) and CD8⁺ T cells (B2) from infected non-pregnant (white box) and pregnant mice (grey box) after stimulation with STcA. The functional profiles are grouped and color-coded according to the number of functions, as shown in the pie charts. A positive cytokine response was defined as a frequency of 0.05%, determined as the average frequency of the CD4⁺ or CD8⁺ T cell response obtained from non-infected mice after stimulation with STcA following subtraction of the values for cells cultured without antigen.

Fig 3. Inhibitory receptor expression from CD4⁺ and CD8⁺ T cells in infected pregnant mice with detectable parasitemia by PCR.

The frequencies of the T cells that expressed PD-1, 2B4, CD160, or CTLA-4 from mothers that did not transmit (black triangle) or did transmit the parasite (black circle) (A1 and B1) are shown. The co-expression of the inhibitory receptors in T cells from mothers that did not transmit and did transmit the parasite is shown. The colors in the pie charts indicate the co-expression of inhibitory receptors (A2 and B2). Increasing and decreasing frequencies of inhibitory receptor co-expression are indicated, respectively, with upward and downward arrowheads and with color-coded according to the number of inhibitory receptors that co-express. The p values were calculated using the Mann–Whitney U test (A1 and B1). The p values of the permutation test in the co-expression analysis (A2 and B2) are shown in the pie charts (*p<0.05).

Fig 4. Polyfunctional profile of the T. cruzi- CD4⁺ and CD8⁺ T cells response from infected pregnant mice with detectable parasitemia by PCR.

The functional activity of CD4⁺ (A1) and CD8⁺ (B1) T cells from non-transmitting and transmitting mice, determined using a five-function assay to simultaneously measure the expression of granzyme B, IFN-γ, IL-2, perforin and TNF-α after stimulation with STcA is shown. The frequency of the parasite-specific CD4⁺ (A2) and C8⁺ (B2) T cells that produced 16 distinct combinations of five functions after stimulation with STcA in non-transmitting (white bars) and transmitting mice (grey bars) is shown. The functional profiles are grouped and color-coded according to the number of functions, as shown in the pie charts. Increasing or decreasing frequencies of polyfunctional T cells are indicated with the upward or downward arrowheads, respectively. The p values of the permutation test in the co-expression analysis (A1 and B1) are shown in the pie charts. The p values in A2 and B2 panels were calculated using the Mann–Whitney U test (***p<0.001, ****p<0.0001).