Defining cellular and molecular determinants of CD4+ T helper cell differentiation during blood-stage Plasmodium infection

Abstract

Malaria is a disease resulting from infection with protozoan parasites of the genus, Plasmodium. Malaria remains a leading cause of global deaths, particularly of children living in sub-Saharan Africa. Despite significant research efforts, an effective strategy for inducing robust Plasmodium-specific immunity remains elusive. CD4+ T helper (Th) cells are critical components of the adaptive immune response to blood-stage Plasmodium parasites. During experimental malaria in mice two major Th cell types arise, T helper 1 (Th1) and T follicular helper (Tfh) cells, both of which are associated with improved infection outcomes. Immune mechanisms controlling the emergence of Th1 and Tfh cells during Plasmodium infection remain poorly characterised, due in part to the absence of appropriate in vivo reagents. To elucidate mechanisms involved in the emergence of parasite-specific Th cells, recently developed Plasmodium-specific CD4+ T cell receptor (TCR) transgenic T cells (termed PbTII cells) were assessed for their in vivo response in mouse models of malaria. Adoptively-transferred PbTII cells proliferated during infection in a manner dependent on MHCII-expressing cells and conventional dendritic cells. By the peak of infection, PbTII cells had differentiated into Th1 and Tfh cells, mirroring the endogenous polyclonal CD4+ T cell response, and emphasising the utility of this reagent for further studies of in vivo Th responses. Next, a relatively new technology, single-cell RNA sequencing (scRNAseq), was applied to PbTII cells to explore at a molecular level the process of Th differentiation in vivo. After initial priming events, emerging Th cells entered a state of high cell cycling, in which they expressed genes associated with both Th1 and Tfh subsets. This occurred prior to their commitment to a differentiated, and possibly terminal, Th1 or Tfh effector state. In this pre-Th state, cells remained 'impressionable' to external signals. Using scRNAseq, inflammatory monocytes were identified as likely providing one such signal. Furthermore, in vivo depletion of monocytes after a period of initial priming resulted in a reduction in Th1 but not Tfh cell responses. Although numerous innate immune sensors have been implicated in the detection of blood-stage Plasmodium infection, whether such molecules influence Th responses remains unclear. Therefore, PbTII cells were employed to determine T-cell extrinsic signals that might drive Th responses, and modest roles for individual innate immune sensing molecules were observed. However, interferon regulatory factor 3 (IRF3), which sits downstream of several innate sensors, played a strong role in the clonal expansion of PbTII cells during infection. Importantly, using Irf3-/-mice, IRF3 was shown to support T cell clonal expansion and promote Th1 differentiation. IRF3 was also required for inflammatory monocytes to express MHCII, suggesting a possible contributory mechanism. Interestingly, IRF3 exerted a suppressive effect on humoral responses, by limiting Tfh cell and germinal centre (GC) B cell responses. Notably, IRF3-deficiency improved GC B cell responses, promoted stronger and longer-lasting Plasmodium-specific serum antibody levels, and accelerated the resolution of infection. Finally, in mixed bone marrow chimeric mice, IRF3-deficent B cells responded more effectively than WT counterparts, suggesting a B-cell intrinsic suppressive role for IRF3. In summary, PbTII cells have been demonstrated to be an effective tool for studying Th1/Tfh differentiation in vivo. During experimental blood-stage malaria PbTII cells do not commit to an effector state until several days after initial priming. Prior to this point, pre-Th cells remain receptive to external inputs, such as those supplied by myeloid cells including inflammatory monocytes. Finally, an innate immune transcription factor, IRF3, exhibits roles in sustaining Th1 differentiation, and suppressing Tfh cell and B cell responses. Therefore, IRF3 represents a potential target for improving immunological protection against blood-stage Plasmodium parasites.