Effector and memory CD4+ T cell development during experimental blood-stage malaria

Abstract

Malaria remains a global health burden, inflicting millions of people worldwide. Both CD4+ T cell- and humoral-mediated immunity are crucial to help control parasites in vivo. The induction of a robust and long-lasting immunological memory response forms the basis of an effective vaccine, which is unfortunately lacking in the case of malaria. This necessitates the need to have a deeper understanding of how host immune responses are regulated during malaria. This thesis aimed to contribute to this knowledge gap by defining the molecular pathways underlying the development of effector and memory CD4+ T cell responses during Plasmodium infection in vivo.To assess how Plasmodium-specific CD4+ T cell responses are controlled in vivo, CD4+ T cell receptor transgenic PbTII cells were used in this thesis. PbTII cells were previously established in the lab to differentiate into two effector T helper subsets, T helper 1 (Th1) and T follicular helper (Tfh) cells. Two transcription factors, p53 and T cell factor 1 (TCF1) were also predicted previously to be associated with Tfh responses during Plasmodium infection in vivo, both of which have not been assessed thoroughly in malaria. By generating PbTII cells that lacked either p53 or TCF1, TCF1 was confirmed to be required for optimal Tfh responses in vivo, while the role of p53 remained inconclusive. P53-deficient PbTII cells were able to undergo initial proliferation, effector differentiation, and were retained long-term comparably to their p53- sufficient counterparts. While the absence of TCF1 in PbTII cells had no impact on the initial clonal expansion phase, PbTII cells that lacked TCF1 had reduced expression of several Tfh signature molecules.Next, the development of memory CD4+ T cell responses during Plasmodium infection in vivo was explored. PbTII cells were firstly confirmed to persist long-term in vivo and developed functional memory recall responses by the end of the first month of infection. PbTII cells spanning the first four weeks of infection were then subjected to single-cell RNA sequencing (scRNAseq) analysis to model the transcriptome dynamics underlying memory PbTII cell responses. An exploration of a previously published T central memory precursor (Tcmp) gene signature in PbTII cells was conducted to reveal that Plasmodium-specific CD4+ T cell memory responses developed in the absence of a Tcmp population. Memory responses instead were shown to be derived from effector cells along the same lineage, in which memory phenotypes were further enhanced in the absence of a persisting infection.In vivo testing of the transcriptomic framework then validated the proposed model. Th1 and Tfh effector cells persisted along the same lineage as they gradually transitioned into memory cells, with minimal plasticity observed. The relationships between memory Th1 and Tfh PbTII cells with other CD4+ T cell subsets such as type 1 regulatory T, germinal center Tfh, effector memory T, and central memory T cells were also characterised. Single-cell epigenomics data further confirmed that substantial heterogeneity in chromatin accessibility amongst effector cells was partially reset in memory cells, consistent with the transcriptomic model. Finally, all scRNAseq data from this thesis are provided on an online graphical user interface platform for easy public access for future exploratory work.In summary, work from this thesis provided novel molecular insights for how CD4+ T cell responses are regulated in vivo during experimental blood-stage malaria. This work also identified many previously unstudied molecular targets with the potential for improving long- term immunity against malaria.