SummaryInflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c−MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3−CD11c−MHCII−PU.1lo monocytes differentiated into FcγRIII+PD-L2−CD209a−iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/− mice had reduced Flt3+CD11c−MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)–cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKCKD) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKCKD NK cells exhibit defective killing of MHC-I–deficient, but otherwise normal, target cells, resulting in defective rejection by NKCKD mice of transplants from various types of MHC-I–deficient mice. Self–MHC-I immunosurveillance by NK cells in NKCKD mice can be rescued by self–MHC-I–specific Ly49 transgenes. Although NKCKD mice display defective recognition of MHC-I–deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity–induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self–MHC-I molecules and that the absence of these receptors leads to loss of MHC-I–dependent “missing-self” immunosurveillance by NK cells.

Material

We evaluated the impact of premature cell death of antigen-presenting cells (APCs) by Caspase-1- and RipK3-signaling pathways on CD8 T-cell priming during infection of mice with Salmonella typhimurium (ST). Our results indicate that Caspase1 and RipK3 synergize to rapidly eliminate infected APCs, which does not influence the initial activation of CD8 T cells. However, the maintenance of primed CD8 T cells was greatly compromised when both these pathways were disabled. Caspase-1- and RipK3-signaling did not influence NF-κB signaling in APCs, but synergized to promote processing of IL-1 and IL-18. Combined deficiency of Caspase1 and RipK3 resulted in compromised innate immunity and accelerated host fatality due to poor processing of IL-18. In contrast, synergism in cell death by Caspase-1- and RipK3 resulted in restriction of PD-1 and TIM3 expression on primed CD8 T cells, which promoted the survival of activated CD8 T cells.

Salmonella enterica serovar Typhimurium (ST) is a virulent intracellular bacterium that conceals itself in the phagosomes of infected cells. Although CD8(+) T cells promote protection against various intracellular pathogens, the role of CD8(+) T cells against virulent ST has been unclear due to early fatality of susceptible (B6) mice. Herein, we generated MHC I-deficient mice on the resistant (129SvJ) and susceptible (Nramp1 transgenic B6) background to evaluate the role of CD8(+) T cells against virulent ST. Our results indicate that CD8(+) T cells have a critical protective role in host survival during infection with virulent ST. As antigen presentation and CD8(+) T-cell activation against phagosomal antigens are considered to operate through the cross-presentation pathway, we have evaluated CD8(+) T-cell response against ST in Batf3-deficient mice that lack CD8α dendritic cells (DCs). Using a recombinant of ST that expresses antigen (ST-OVA) mainly in the phagosomes of infected cells, we show that CD8(+) T-cell response is compromised throughout the duration of infection in Batf3-deficient mice. In contrast, when ST delivers antigen to the cytosol of infected cells (ST-OVA-C), CD8(+) T-cell response against the cytosolic antigen was compromised only in the short term in the absence of CD8α DCs, with wild-type and Batf3-deficient mice generating similar CD8(+) T-cell response in the long term. Thus, Batf3 has an important role in CD8(+) T-cell priming regardless of antigenic location; however, its role is redundant at later time intervals against cytosolic antigen.