Comparison of the transcriptomic profile of P. falciparum reactive polyfunctional and IFNg monofunctional human CD4 T cells

Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes but it is not known whether polyfunctional T cells are distinct from monofunctional cytokine producing T cells. In this study we compared the transcriptomic profile of P. falciparum reactive polyfunctional and IFNg monofunctional CD4 T cells by microarray analysis and show that polyfunctional CD4 T cells are associated with a unique transcriptomic signature. Whole blood samples collected from two independent cohorts (n=4 per cohort) of malaria-naïve humans 28 days after experimental infection with P. falciparum blood-stage parasite were in vitro stimulated overnight with P. falciparum parasite extract. Polyfunctional (IFNg/IL2/TNF), IFNg monofunctional CD4 T cells and IFNg negative CD4 T cells were sorted from each sample directly into RLT lysis buffer using a 3-colour polyfunctional cytokine secreting assay (Burel et al., 2016). mRNA was extracted using the RNEasy micro kit (Qiagen) and samples from each cohort were pooled for microarray analysis. Gene expression profiles for the two independent microarray experiments were determined using the low-input Affymetrix human gene 2.0 ST array at the Ramaciotti centre (University of NSW, Sydney, Australia). A polyfunctional gene signature was defined based on differential expression of genes which had an absolute fold change greater than 2 between IFNg triple positive and IFNg single positive cells in both of the independent microarray experiments.