T follicular helper cells (Tfh) provide crucial signals for germinal center (GC) formation, but Tfh populations are heterogeneous. While PD1hi Tfh are important in the GC response, the function of the PD1lo Tfh-like subset is unknown. We show that these cells, like the PD1hi GC–Tfh, depend upon B cells; however, their entry to follicles is independent of CXCR5 or cognate interactions with B cells. The differentiation into PD1hi Tfh is dependent on MHC class II interactions with B cells and requires CXCR5. Our data suggest a Tfh differentiation pathway that is initially B cell-independent, then dependent on non-cognate B cell interactions, and finally following cognate interaction with B cells and CXCR5-ligands allows the formation of GC–Tfh. The PD1lo Tfh-like cells make early cytokine responses and may represent precursors of CD4 memory cells.

No abstract

Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are believed to be vital for bacterial clearance due to their capacity to secrete IFN-γ, but an essential requirement for T-bet-expressing Th1 cells has yet to be demonstrated in the mouse model of Chlamydia infection. Here, we investigated the role of T-bet and IFN-γ in primary clearance of Chlamydia after FRT infection. Surprisingly, IFN-γ producing CD4 T cells from the FRT expressed low levels of T-bet throughout infection, suggesting that classical T-bet-expressing Th1 cells are inefficiently generated and therefore unlikely to participate in bacteria clearance. Furthermore, mice deficient in T-bet expression or with a CD4-specific T-bet deficiency cleared FRT infection similarly to wild-type controls. T-bet-deficient mice displayed significant skewing of FRT CD4 T cells towards Th17 responses, demonstrating that compensatory effector pathways are generated in the absence of Th1 cells. In marked contrast, IFN-γ-, and IFN-γR-deficient mice were able to reduce FRT bacterial burdens, but suffered systemic bacterial dissemination and 100% mortality. Together, these data demonstrate that IFN-γ signaling is essential to protect mice from fatal systemic disease, but that classical T-bet-expressing Th1 cells are non-essential for primary clearance within the FRT. Exploring the protective contribution of Th1 cells versus other CD4 effector lineages could provide important information for the generation of new Chlamydia vaccines.

Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time-consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia. Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.