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The local microenvironment shapes macrophage differentiation in each tissue. We hypothesized that in the peritoneum, local factors in addition to retinoic acid can support GATA6-driven differentiation and function of peritoneal large cavity macrophages (LCMs). We found that soluble proteins produced by mesothelial cells lining the peritoneal cavity maintained GATA6 expression in cultured LCMs. Analysis of global gene expression of isolated mesothelial cells highlighted mesothelin (Msln) and its binding partner mucin 16 (Muc16) as candidate secreted ligands that potentially regulate GATA6 expression in peritoneal LCMs. Mice deficient for either of these molecules showed diminished GATA6 expression in peritoneal and pleural LCMs that was most prominent in aged mice. The more robust phenotype in older mice suggested that monocyte-derived macrophages were the target of Msln and Muc16. Cell transfer and bone marrow chimera experiments supported this hypothesis. We found that lethally irradiated Msln−/− and Muc16−/− mice reconstituted with wild-type bone marrow had lower levels of GATA6 expression in peritoneal and pleural LCMs. Similarly, during the resolution of zymosan-induced inflammation, repopulated peritoneal LCMs lacking expression of Msln or Muc16 expressed diminished GATA6. These data support a role for mesothelial cell–produced Msln and Muc16 in local macrophage differentiation within large cavity spaces such as the peritoneum. The effect appears to be most prominent on monocyte-derived macrophages that enter into this location as the host ages and also in response to infection.

Intricate processes in the thymus and periphery help curb the development and activation of autoreactive T cells. The subtle signals that govern these processes are an area of great interest, but tuning TCR sensitivity for the purpose of affecting T cell behavior remains technically challenging. Previously, our lab described the derivation of two TCR-transgenic CD4 T cell mouse lines, LLO56 and LLO118, which recognize the same cognate Listeria epitope with the same affinity. Despite the similarity of the two TCRs, LLO56 cells respond poorly in a primary infection while LLO118 cells respond robustly. Phenotypic examination of both lines revealed a substantial difference in their surface of expression of CD5, which serves as a dependable readout of the self-reactivity of a cell. We hypothesized that the increased interaction with self by the CD5-high LLO56 was mediated through TCR signaling, and was involved in the characteristic weak primary response of LLO56 to infection. To explore this issue, we generated an inducible knockin mouse expressing the self-sensitizing voltage-gated sodium channel Scn5a. Overexpression of Scn5a in peripheral T cells via the CD4-Cre promoter resulted in increased TCR-proximal signaling. Further, Scn5a-expressing LLO118 cells, after transfer into BL6 recipient mice, displayed an impaired response during infection relative to “wild-type” LLO118 cells. In this way, we were able to demonstrate that tuning of TCR sensitivity to self can be used to alter in vivo immune responses. Overall, these studies highlight the critical relationship between TCR:self-pMHC interaction and an immune response to infection.

CD11c+ cells increase greatly with islet inflammation in non-obese diabetic mice and contribute to autoimmune destruction of pancreatic beta cells. In this study, we investigated their origin and mechanism of recruitment. CD11c+ cells in inflamed islets resembled classical dendritic cells (DC) based on their transcriptional profile. However, the majority of these cells were not from the Zbtb46-dependent DC lineage. Instead, monocyte precursors could give rise to CD11c+ cells in inflamed islets. Chemokines Ccl5 and Ccl8 were persistently elevated in inflamed islets and the influx of CD11c+ cells was partially dependent on their receptor Ccr5. Treatment with islet antigen-specific regulatory T cells (Tregs) led to a marked decrease of Ccl5 and Ccl8 and a reduction of monocyte recruitment. These results implicate a monocytic origin of CD11c+ cells in inflamed islets and suggest that therapeutic Tregs directly or indirectly regulate their influx by altering the chemotactic milieu in the islets.

Th cell subsets have unique calcium (Ca2+) signals when activated with identical stimuli. The regulation of these Ca2+ signals and their correlation to the biological function of each T cell subset remains unclear. Trpm4 is a Ca2+-activated cation channel that we found is expressed at higher levels in Th2 cells compared with Th1 cells. Inhibition of Trpm4 expression increased Ca2+ influx and oscillatory levels in Th2 cells and decreased influx and oscillations in Th1 cells. This inhibition of Trpm4 expression also significantly altered T cell cytokine production and motility. Our experiments revealed that decreasing Trpm4 levels divergently regulates nuclear localization of NFATc1. Consistent with this, gene profiling did not show Trpm4-dependent transcriptional regulation, and T-bet and GATA-3 levels remain identical. Thus, Trpm4 is expressed at different levels in Th cells and plays a distinctive role in T cell function by differentially regulating Ca2+ signaling and NFATc1 localization.

The initiation, intensity, and duration of T cell-directed inflammatory responses are dependent upon the coordination of both activating and inhibitory signals mediated by specific receptors on the T lymphocyte. The recently described receptor, B and T lymphocyte attenuator (BTLA), has been demonstrated to have an important role in limiting the duration of inflammation in a murine model of allergic asthma. In this study, we have examined the role of BTLA on the proliferation, recruitment, and survival of T cells in response to inhaled allergen. We find that there is decreased cell death in T cells from BTLA-deficient mice, whereas proliferation and recruitment to the lungs are unchanged. Thus, the regulation of cell death through BTLA signaling is a key determinant of the inflammatory response in the lung.

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.

The role of type I IFN in Th1 development, STAT4 activation, and IFN-γ production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-α induced Th1 development and IFN-γ production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-α also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-α actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-α to induce Th1 differentiation, STAT4 phosphorylation, and IFN-γ production in murine T cells. First, we show that IFN-α, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-α can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-γ production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-α-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-γ production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-α-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-α may act similarly in human and murine T cells.

T cell activation is regulated by coordinate interaction of the T cell Ag receptor and costimulatory signals. Although there is considerable insight into processes that regulate the initiation of inflammation, less is known about the signals that terminate immune responses. We have examined the role of the inhibitory receptors programmed death receptor-1 and B and T lymphocyte attenuator in the regulation of allergic airway inflammation. Our results demonstrate that there is a temporally regulated expression of both the receptors and their ligands during the course of allergic airway inflammation. Following a single inhaled challenge, sensitized wild-type mice exhibit peak inflammation on day 3, which resolves by day 10. In contrast, mice deficient in the expression of programmed death receptor-1 or B and T lymphocyte attenuator have persistent inflammation out to 15 days following challenge. Thus, these receptors are critical determinants of the duration of allergic airway inflammation.

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4+ Th1 development in both murine and human T cells, IFN-αβ drives Th1 development only in human cells. This IFN-αβ-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4+ T cell responses to IFN-αβ. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4+ T cells and was activated by murine IFN-α signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-α-driven Th1 development as defined by IFN-γ expression. Furthermore, IL-12, but not IFN-α, promoted acute IFN-γ secretion in collaboration with IL-18 stimulation in both CD4+ and CD8+ T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-α. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-αβR), it is not sufficient to restore this process through the murine IFNAR complex.