Cytotoxic Signature and IFN-γ Production Dominate CD4+ T-Cell Response During Human Toxoplasmosis

Abstract

Toxoplasma gondii is a highly versatile parasite that infects most warm-blooded animals and is a major cause of retinochoroiditis and uveitis in humans. The pathophysiology of these conditions remains poorly understood. Both parasite virulence and host inflammatory response contribute to the development of ocular disease. While CD4⁺ T cells play a critical role in host resistance to Toxoplasma infection, their kinetics and effector functions, as well as their contribution to the clinical outcome of the infection, including ocular involvement, remain poorly understood. To address this question, we investigated the immune response during acute and convalescent toxoplasmosis and stratified patients further based on the presence or absence of ocular disease. We found that T. gondii infection leads to decreased and increased proportions of central and effector memory CD4⁺ T cells, respectively. Applying unsupervised analysis, distinct CD4⁺ T-cell subsets were determined. Among 50 clusters, 10 produced cytotoxic proteins (granzyme B and perforin) and one produced cytokines upon antigen-specific stimulation. We observed that proportions of five CD4⁺ T-cell clusters out of 50 were different during acute disease between T. gondii-infected patients with and without ocular lesions. Interestingly, three of the five displayed a cytotoxic signature indicating their possible involvement in ocular immunopathology. Taken together, our results reveal that during T. gondii infection, CD4⁺ T cells not only develop a Th1 cytokine profile, but also acquire previously unappreciated cytotoxic capacity/function. These results, while underscoring the complexity of the CD4⁺ T-cell response to T. gondii, suggest that specific subsets may be involved in the development of pathology and provide possible targets for therapeutic intervention.

Keywords: toxoplasma gondii; CD4+ T cells; cytotoxic T cells; human toxoplasmosis.

FIGURE 1

Toxoplasma gondii infection induces increased frequency of effector memory CD4⁺ T cells in humans. (A) UMAP, normalised to represent the same number of events in each experimental group, depicts proportions of naïve (Nv, CD45RO⁻CCR7⁺), effector (Eff, CD45RO⁻CCR7⁻), effector memory (EM, CD45RO⁺CCR7⁻), central memory (CM, CD45RO⁺CCR7⁺) and regulatory T cells (T_reg, CD25⁺FoxP3⁺) among CD4⁺ T cells in blood samples from T. gondii‐negative (CTL) and T. gondii‐infected individuals during acute phase (AC), and 6 months (6mo) and 12 months (12mo) later evaluated by flow cytometry and manually analysed. (B and C) Boxplots depict (B) longitudinal changes in the abundance of naïve and memory CD4⁺ T‐lymphocyte subsets during T. gondii infection or (C) based on the presence or the absence of ocular lesions. Box and whiskers indicate minimum and maximum values, median and interquartile range with superimposed symbols representing single individual data points. Coloured asterisks represent differences between the assigned group and the group represented by the same colour. *p < 0.05, **p < 0.01, ***p < 0.001 by Dunn's test adjusted by Bonferroni.

FIGURE 2

Characterisation of CD4⁺ T‐cell metaclusters with naïve phenotype. (A) Normalised UMAP overlayed with MC1, 2, 3, 4 and 8 populations (from top to bottom) is depicted for T. gondii‐negative (control) and T. gondii‐infected patients at acute stage (AC) as well as 6 (6mo) and 12 months (12mo) after infection. (B) Minimum spanning trees (MST) display levels of expression for CD45RO, CD28, CCR7, CD27 and CD69. Naïve clusters are encircled in black. (C) Heatmap indicates the per‐measurement normalised expression of measured markers for the naïve clusters MC1, 2, 3, 4 and 8. (D) Box and whiskers with minimum and maximum values, median and interquartile range and superimposed symbols representing individual values in each experimental group. Coloured asterisks represent differences between the assigned group and the group represented by the same colour. *p < 0.05, **p < 0.01, ***p < 0.001 by Dunn's test adjusted by Bonferroni.

FIGURE 3

Characterisation of CD4⁺ T‐cell metaclusters with T_reg phenotype. (A) Representative flow cytometry contour plots show the expression of CD25 by FoxP3 among all CD4⁺ T cells (grey) while MC5, 6, 7 and 10 populations (red dots) are overlayed for each experimental group. (B) MC5, 6, 7 and 10 are encircled on the MST, showing expression of CD25, FoxP3, CD39 and CD95. (C) Heatmap shows the relative per‐measurement normalised expression of markers in T_reg MC clusters. (D) Box and whiskers show minimum and maximum values, median and interquartile range and superimposed symbols for individual values for each indicated experimental groups. Coloured asterisks colours represent differences between the assigned group and the group represented by the same colour. *p < 0.05, **p < 0.01 by Dunn's test adjusted by Bonferroni.

FIGURE 4

Metacluster 48 is comprised of Toxoplasma gondii responsive CD4⁺ T cells. (A) CD4⁺ T‐cell MC48 is overlaid on total CD4⁺ T‐cell‐derived UMAP for each experimental group. (B) Box and whiskers bar graphs superimposed with symbols representing individual values in each experimental group. Responders represent the number of individuals per group that produce MC48 population. (C) Heatmap depicts per‐measurement normalised expression of MC48 for each infection time point. (D) MC48 is encircled on the MST, showing expression of IFN‐γ, TNF and granzyme B. (E–F) Representative dot plots show the production of IFN‐γ and TNF (E) or granzyme B and perforin (F) in total CD4⁺ T cells (grey) and MC48 population (red) from T. gondii‐infected individuals at different time points. Flow cytometry plots are normalised to display 4000 events after culture in the absence (NS, top panels) or presence of soluble T. gondii antigens (STAg, bottom panels). Coloured asterisks represent differences between the assigned group and the group represented by the same colour. *p < 0.05 by Dunn's test adjusted by Bonferroni.

FIGURE 5

Expression of granzyme B and perforin by EM CD4⁺ T cells in T. gondii‐infected individuals. (A) Heatmap of the per‐measurement normalised relative expression. FlowSOM clusters are grouped in three groups (mesocluster 37 and 40; mesocluster 39, 45 and 46; mesocluster 29, 36, 41, 42 and 47) based on the level of expression of granzyme B and perforin. (B) Representative dot plots show the expression of granzyme B (x‐axis) and perforin (y‐axis) based on the three groups (red): Mesocluster 37 and 40; mesocluster 39, 45 and 46; and mesocluster 29, 36, 41, 42 and 47, from top to bottom, overlayed on total CD4⁺ T cells (grey) in differential experimental groups. (C and D) Box and whiskers indicate minimum and maximum values, median and interquartile range and superimposed symbols representing individual values for three mesocluster sets in each experimental group depending on the time point after infection (C) or on the presence or the absence of ocular lesions (D). Coloured asterisks represent differences between the assigned group and the group represented by the same colour. *p < 0.05,  **p < 0.01, ***p < 0.001 by Dunn's test adjusted by Bonferroni.

FIGURE 6

Acute ocular toxoplasmosis is associated with alteration of effector and EM CD4⁺ T‐cell pool. (A) Heatmap shows the per‐measurement normalised relative expression of the markers for MC18, 29, 41, 47 and 44 for each experimental group. (B) Representative contour plots show the expression of CCR7 (x‐axis) and CD45RO (y‐axis) among CD4⁺ T cells (grey) and MC18, 29, 41, 47 or 44 population (red dots) from Toxoplasma‐negative and T. gondii‐infected individuals at different time points without (top panels) or with ocular lesions (bottom panels). (C) Box and whiskers graphs with superimposed symbols representing individual values indicate frequencies of MC18, 29, 41, 47 and 44 in CD4⁺ T cells from acutely T. gondii‐infected with or without ocular lesions. Coloured asterisks represent differences between groups. *p < 0.05 by Dunn's test adjusted by Bonferroni.