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Recent reports have highlighted greater complexity, plasticity, and functional diversity of mononuclear phagocytes (MPCs), including monocytes, macrophages, and dendritic cells (DCs), in our organs than previously understood. The functions and origins of MPCs resident within healthy organs, especially in the kidney, are less well understood, whereas studies suggest they play roles in disease states distinct from recruited monocytes. We developed an unbiased approach using flow cytometry to analyze MPCs residing in the normal mouse kidney, and identified five discrete subpopulations according to CD11b/CD11c expression as well as F4/80, CD103, CD14, CD16, and CD64 expression. In addition to distinct marker profiles, these subpopulations have different lineages and expression of genes involved in tissue homeostasis, including angiogenesis. Among them, the CD11b(int)CD11c(int) F4/80(high) subpopulation notably exhibited high capacity to produce a representative anti-inflammatory cytokine, IL-10. Each subpopulation had different degrees of both macrophage (phagocytosis) and DC (Ag presentation) capacities, with a tendency to promote differentiation of regulatory T cells, whereas two of these showed expression of transcription factors reported to be highly expressed by classical DCs, and proclivity to exit the kidney following stimulation with LPS. In summary, resident kidney MPCs comprise discrete subpopulations, which cannot be simply classified into the conventional entities, and they produce anti-inflammatory and tissue-homeostatic factors to differing degrees.

Naive CD4 T cells can differentiate into a number of functional subsets in response to Ag, including Foxp3(+) induced regulatory T cells (iTregs). The in vivo development and function of iTregs has been primarily demonstrated in systems involving Ag encountered systemically or delivered via the intestinal mucosa. In this study, we demonstrate that de novo Foxp3 expression in naive CD4 T cells is a critical mechanism for establishing tolerance for a tissue-restricted neo-self Ag. Naive CD4 T cells lacking a functional Foxp3 gene cannot achieve tolerance, but can be suppressed in vivo in the presence of wild type naive CD4 T cells. Exposure to nonspecific inflammation during priming undermines tolerance through impaired Foxp3 induction, suggesting that the microenvironment also has a role. These data show that de novo Foxp3 expression is an integral component of establishing and maintaining tolerance among naive peripheral CD4 T cells.

Type-I IFNs (IFN-I) provide direct survival signals to T cells during Ag-driven proliferation. Because IFN-I production differs depending on the pathogen, we assessed CD8 T cell requirement for direct IFN-I signals during responses to vaccinia virus (VV), vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), and Listeria monocytogenes (LM) immunizations in vivo. IFN-I-receptor-deficient (IFN-IR(o)) CD8 T cells expanded 3- to 5-fold less and formed a diminished memory pool compared with wild-type (WT) CD8 T cells in response to VV, VSV, or LM. WT CD8 T cells expanded more robustly in response to LCMV-encoded Ags than to Ags encoded by the other three pathogens, and under these conditions the lack of direct IFN-I signals inhibited their expansion by approximately 100-fold. To test whether the high antigenic-load provided by LCMV caused greater expansion and greater IFN-I dependency, we primed WT and IFN-IR(o) OVA-specific OT-1 CD8 T cells with a fixed-number of OVA-peptide-pulsed dendritic cells along with adjuvant effect provided by LCMV, VV, VSV, or LM. Both WT and IFN-IR(o) OT-1 cells were recruited, proliferated, and differentiated into effectors in all the four cases. However, WT OT-1 cells expanded similarly in all four cases. IFN-IR(o) OT-1 cells expanded approximately 20-fold less than the WT OT-1 CD8 T cells when LCMV was used as adjuvant, whereas their expansion was affected only marginally when VV, VSV, or LM were used as adjuvants. Thus, innate/inflammatory signals induced by different pathogens contribute to CD8 T cell expansion and memory formation via distinct levels of IFN-I dependence.

TGF-beta opposes proliferative signaling by IL-2 through mechanisms that remain incompletely defined. In a well-characterized CD8(+) T cell model using wild-type and mutated IL-2 receptors, we examined the effects of TGF-beta on distinct IL-2 signaling events in CD8(+) T cells. IL-2 induces c-myc, cyclin D2, and cyclin E in a redundant manner through the Shc and STAT5 pathways. TGF-beta inhibited the ability of either the Shc or STAT5 pathway to induce these genes, as well as T cell proliferation. The inhibitory effects of TGF-beta were reversed by expression of a dominant-negative form of Smad3. TGF-beta did not impair proximal signaling by Shc or STAT5, and induction of some downstream genes, including cytokine-inducible Src homology-2-containing protein (CIS), bcl-x(L), and bcl-2, was spared. Experiments with c-fos, cyclin D2, and CIS reporter genes revealed that promoter-proximal regulatory elements dictate the sensitivity of IL-2 target genes to inhibition by TGF-beta. By leaving the Shc and STAT5 pathways functional while inhibiting their target genes selectively, TGF-beta was found to uncouple the proliferative and antiapoptotic functions of IL-2. Thus, TGF-beta is not a simple antagonist of IL-2, but rather serves to qualitatively modify the IL-2 signal to create a unique pattern of gene expression that neither cytokine can induce independently.