Dissecting the phenotype and functionality of different malaria-induced regulatory T cell subsets

Abstract

In infectious diseases like malaria, maintaining a balance between pro-inflammatory and anti-inflammatory immune reactions is crucial to ensure successful control of the pathogen without causing immunopathology. Pro-inflammatory-acting cytotoxic CD8+GrzB+ T cells have been shown to be a main contributor to experimental cerebral malaria. However, their role in patients with acute Plasmodium falciparum (Pf) malaria remains incompletely understood. The induction of co-inhibitory molecules on T cells is one mechanism, which might counteract this T cell-mediated immunopathology. Regulatory CD4+FoxP3-PD-1+ T cells with characteristics of type 1 regulatory T cells (Tr1 cells) have shown to be induced in patients suffering from Pf malaria. However, whether they have an influence on the disease outcome of malaria remains unclear. Thus, this study aimed to understand the role of cytotoxic CD8+ T cells and potentially counteracting regulatory T cell subsets induced during Pf malaria. To this end, T cells of adults and Ghanaian children suffering from malaria, as well as corresponding healthy donors were analyzed via flow cytometry. Furthermore, different subsets of malaria-induced regulatory CD4+FoxP3-PD-1+ T cells were isolated from peripheral blood of malaria patients and healthy donors and incubated with autologous responder cells to elucidate their suppressive function. Here, we showed that the proportion of CD8+GrzB+ T cells correlates significantly with disease severity in Ghanaian children suffering from Pf malaria, underlining their contribution to the development of severe malaria in humans. Furthermore, when analyzing different subsets of regulatory CD4+FoxP3-PD-1+ T cells, we identified a malaria-induced subset that co-expresses LAG-3 and CD49b, which are markers for type 1 regulatory T cells (Tr1 cells). While their frequency did not differ between Ghanaian children suffering from severe or mild malaria, phenotypic analyses revealed a significant upregulation of TIGIT and TIM-3 in children with severe malaria compared to children with mild malaria. Besides PD-1, we identified CCR5 as an additional surface marker of malaria-induced CD4+LAG-3+CD49b+ T cells. Because of the low amount of CD4+LAG-3+CD49b+ T cells in the peripheral blood of malaria patients, we instead analyzed the suppressive function of different subsets of CD4+FoxP3-PD-1+ T cells, which contained the CD4+LAG-3+CD49b+ T cell population. Interestingly, the CCR5+ subset showed a more activated phenotype and exerted a stronger suppressive function than the CCR5- population. In contrast, expression of TIGIT led to diminished suppressive capacities and a less activated phenotype. Strikingly, the frequency of CD4+PD-1+TIGIT+ T cells correlated with disease severity in Ghanaian children suffering from malaria, indicating that higher numbers of those dysfunctional regulatory T cells favor immunopathology in malaria patients. In conclusion, this study further emphasizes the critical role of GrzB-producing CD8+ T cells in the development of severe clinical malaria and indicates that also a failure of regulatory T cell functions might contribute to immunopathology. Further analysis of the interplay between cytotoxic CD8+ T cells and malaria-induced regulatory T cells and the mechanisms regulating their functionality could be of great importance to treat malaria and improve the clinical outcome of severe cases in the future.