CD4 T cells are required to fight malaria infection by promoting both phagocytic activity and B cell responses for parasite clearance. In Plasmodium chabaudi infection, one specific CD4 T cell subset generates anti-parasitic IFN-γ and the antibody-promoting cytokine, IL-21. To determine the lineage of these multifunctional T cells, we followed IFN-γ+ effector T cells (Teff) into the memory phase using Ifng-reporter mice. While Ifng + Teff expanded, the level of the Th1 lineage-determining transcription factor T-bet only peaked briefly. Ifng + Teff also co-express ICOS, the B cell area homing molecule CXCR5, and other Tfh lineage-associated molecules including Bcl6, the transcription factor required for germinal center (GC) T follicular helper cells (Tfh) differentiation. Because Bcl6 and T-bet co-localize to the nucleus of Ifng + Teff, we hypothesized that Bcl6 controls the Tfh-like phenotype of Ifng + Teff cells in P. chabaudi infection. We first transferred Bcl6-deficient T cells into wildtype hosts. Bcl6-deficient T cells did not develop into GC Tfh, but they still generated CXCR5+IFN-γ+IL-21+IL-10+ Teff, suggesting that this predominant population is not of the Tfh-lineage. IL-10 deficient mice, which have increased IFN-γ and T-bet expression, demonstrated expansion of both IFN-γ+IL-21+CXCR5+ cells and IFN-γ+ GC Tfh cells, suggesting a Th1 lineage for the former. In the memory phase, all Ifng + T cells produced IL-21, but only a small percentage of highly proliferative Ifng + T cells maintained a T-bethi phenotype. In chronic malaria infection, serum IFN-γ correlates with increased protection, and our observation suggests Ifng + T cells are maintained by cellular division. In summary, we found that Ifng + T cells are not strictly Tfh derived during malaria infection. T cells provide the host with a survival advantage when facing this well-equipped pathogen, therefore, understanding the lineage of pivotal T cell players will aid in the rational design of an effective malaria vaccine.

Chronic exposure to the malaria parasite is required for full clinical protection, possibly due to improved survival versus maintenance of effector function of CD4 memory T cells (Tmem). IFN-γ is required for protection; therefore, we investigated the development of Th1 memory in chronic Plasmodium chabaudi malaria infection using ifng-reporter mice. We observed that the expression of Th1 markers (T-bet, CXCR3) peaked well before T cell contraction, suggesting poor long-term commitment to IFN- γ production. In addition, markers of T follicular helper cells (Tfh), such as CXCR5 and Bcl6, were on most Th1 cells, and some co-produced IL-21 and IL-10. While Th1 effector cells differentiate into CXCR3+ Tmem in malaria, they are Tbetlo and do not maintain ifng expression, unless they are dividing, though they do express Bcl6 and IL-21. Interestingly, Bcl6-deficient Th1 cells produce more IL-21, while infection of IL-10-deficient mice increased T cell expression of both T-bet and Bcl6, generating more mixed phenotype cells. P. chabaudi infected IL-10-/- mice die of T cell-mediated pathological inflammation, suggesting that the dominant Th1/Tfh phenotype is pathogenic if not regulated. Expression of Bcl6 and IL-21 in Th1 cells has been described as an early and transient event before full commitment; however, these results suggest that this intermediate stage can be maintained in chronic infection, potentially to prevent both exhaustion and pathology, via IL-10.

There is an error in the ninth sentence of the second paragraph of the "Bcl6 T cell deficiency abolishes CXCR5+ Germinal Center T follicular helper cells, but not CXCR5+IL-21+IFN-γ+ T cells" section of the Results. The correct sentence is: However, only a fraction of the CXCR5+ Teff are Bcl6-dependent, though we observed a decrease in the MFI of CXCR5 on the Bcl6 cKO T cells ( Fig 5D).There is an error in the third sentence of the third paragraph of the Discussion section. The correct sentence is: While IL-21 and CXCR5, widely considered Tfh-related molecules, are both predominantly expressed by IFN-γ+ cells in this infection, this population is only slightly affected by deficiency of Bcl6.There is an error in the ninth sentence of the fifth paragraph of the Discussion section. The correct sentence is: In agreement with this data, we found that primarily the IFN-γ+CXCR5+PD-1 hi GC Tfh population was regulated by Bcl6.There is an error in the second sentence of the Acknowledgements section. The correct sentence is: We are grateful for the kind advice of Jean Langhorne and Ken Murphy, and the provision of Ifng /Thy1.1 animals from Casey Weaver and Laurie Harrington.

In Fig 5, the graphs of Fig 5C lack percentages for individual recipients of effector T cell subsets. Please see the updated Fig 5 here.