Abstract
IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.
Highlights
Effector CD8ϩ T cells were generated in vivo by i.p. vaccination of C57BL/6 mice with live, uracil auxotrophic T. gondii parasites termed carbamoyl phosphate synthase (CPS) that were further rendered nonreplicative by irradiation
This strategy is advantageous for assessing the role of innate cytokines during CD8ϩ T cell activation because there is no risk of host mortality, especially under cytokine-deficient conditions
Following CPS vaccination we find that T-bet-deficient mice exhibit impaired generation of the KLRG1ϩ CTL subpopulations (FIII and most notably FIV) similar to but less severe than that exhibited by the IL-12 signaling-deficient mice in both peritoneal exudate cells (PECs) (Fig. 8A) and the spleen (Fig. 8B)
Summary
LCMV, lymphocytic choriomeningitis virus; CPS, carbamoyl phosphate synthetase; F (plus roman numeral), fraction; GrB, granzyme B; ICS, intracellular staining; KLRG1, killer cell lectin-like receptor G1; MPEC, memory precursor effector cell; PEC, peritoneal exudate cell; SLEC, short-lived effector cell; WT, wild type. We initially used the presence of intracellular granzyme B (GrB) as a marker to identify putative parasite-reactive CTLs, given that cytotoxic granules are prestored within armed effector cells [24]. Subsequent correlative mapping of effector molecule (GrB and IFN-␥) expression with cell surface activation markers (CD62L and killer cell lectin-like receptor G1 (KLRG1)) was conducted on putative parasite-reactive CTLs harvested from a peripheral effector site (peritoneum) of Toxoplasmainfected mice. Using this multiparametric phenotyping approach, we were able to define four distinct CTL subpopulations exhibiting varying effector profiles and differential IL-12 dependencies. Our observation that effector site CTLs present such a highly heterogeneous activation profile supports current models of effector differentiation [25, 26] whereby CD8ϩ T cells initially develop along divergent pathways for the generation of acute effector or memory precursor cells
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