ABSTRACT
Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.
Subject(s)
CD4-Positive T-Lymphocytes , Cell Differentiation , Malaria , Animals , Mice , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Malaria/immunology , Malaria/parasitology , Mice, Inbred C57BL , Phenotype , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR5/metabolism , Receptors, CCR5/genetics , Spleen/immunology , Th1 Cells/immunologyABSTRACT
The anti-Xa activities of unfractionated heparin (UFH) and nine low molecular weight heparins (LMWH) have been measured in the presence and absence of 3 mM CaCl2, using bovine Factor Xa, purified human ATIII and an amidolytic assay. The addition of CaCl2 increased the activity of UFH by 93%, but the effect on LMWH was less, ranging from -20% to +55%. Studies of gel filtration fractions of UFH showed marked Mr dependence of the CaCl2 effect in the range 4,000-12,000. The differences among the various LMW heparins with respect to the effect of CaCl2 were closely correlated with the amount of polysaccharide above an Mr of 6,500. Kinetic studies confirmed the potentiation of the activity of UFH with bovine Xa and showed an even more marked effect using human Xa.