• The impact of the non-immune chemiome on T cell activation

      Rosenberg, Kenneth; Singh, Nevil; 0000-0002-1231-7836 (2020)
      T cells are critical organizers of the immune response and rigid control over their activation is necessary for balancing host defense and immunopathology. It takes 3 signals provided by dendritic cells (DC) to fully activate a T cell response – T cell receptor (TCR) engagement of antigen on MHC (Signal 1), co-stimulatory signals (Signal 2) and cytokines (Signal 3). Yet, even before activation T cells are typically exposed to a universe of chemicals (a “chemiome”) including drugs, metabolites, hormones etc. which are not typically ascribed an immunological role. In this thesis, we hypothesized that members of this non-immune chemiome acting on T cells, prior to antigen encounter, flavor specific signaling pathways to differentially influence subsequent T cell activation and fate. Unraveling these signals, which we termed “Signal 0”, could help us understand and manipulate tissue and time specific flavoring of immunity. In this thesis we first developed a pharmacological model for signal 0, by treating T cells with drugs that activate only subsets of the TCR-signaling network prior to full antigen exposure. We found that pharmacological pre-activation of the PKCƟ/ERK pathways modulates long time survival of T cells without changing proliferation or cytokine production. Next, we examined receptors for the non-immune chemiome that resting T cells express and identified neurotransmitter receptors (NR) as a major family. All T cells expressed a core NR signature, but very few NR were also modulated in a T cell lineage-specific fashion. Of these, we focused on VPAC1, the receptor for vasoactive intestinal peptide (VIP). We found that VIP signaling attenuates ERK phosphorylation, but paradoxically drives increased differentiation towards IL-17 and IL-22 secretion. In addition ERK signaling induced by drugs (phorbol esters) versus the TCR followed differential kinetics and recruited non-overlapping negative feedback mechanisms, suggesting that even the same branch of TCR signaling is subject to different localization and temporal controls. Taken together, our data suggest that the branches of the TCR-signaling network integrate pre-existing signals (Signal 0) into the activation program of T cells, allowing localized cues, including neurotransmitter levels, to modify the long-term trajectory of the immune response.
    • Monomeric IL-12p40 binds partner proteins to modulate immune cell function

      Gerber, Allison; Singh, Nevil; 0000-0001-9442-3255 (2021)
      Cytokines are critical mediators used by immune cells to communicate as well as protect. The IL-12 family of cytokines are made up of  and  subunits typically assembled within one cell and secreted as a heterodimer. IL-12p40 is the shared β-subunit for both IL-12 (paring with IL-12p35) and IL-23 (with IL-23p19). However, the IL-12p40 monomer is often secreted in excess during infections, but its biological role was not known. In this thesis we investigated the function of secreted IL-12p40 monomer in vivo with the central hypothesis that the monomer combines with multiple α-subunits in vivo to generate IL-12 as well as other heterodimeric cytokines. Consistent with this hypothesis, in chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, we found that functionally active IL-12 was generated. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism was sufficient to influence local T cell differentiation in sites distal to the initial infection and helped control systemic dissemination of a pathogen although not parasite burden at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival. In addition to this role in generating IL-12 through two-cell assembly, we found that IL-12p40 has a novel partner protein, CD5L. This novel heterodimer was present in the serum of uninfected mice, with differences in the basal levels between B6 and Balb/c animals, with Balb/c having higher amounts of p40-CD5L. Functionally, we found that treatment with p40-CD5L leads to IL-4 and IL-10 production by T cells. Taken together, this thesis offers at least two major fundamental advances in cytokine biology – one the concept of a two-cell assembled cytokine and second the identity of a novel TH2-promoting heterodimeric cytokine. The first has significance in immunotherapy and understanding immunity to tissue-specific modulation of immune responses. The second is expected to drive significant research on allergy, responses to parasites and immune deviation.
    • Regulation of intrinsic activation thresholds of T cells

      Matson, Courtney; Singh, Nevil; 0000-0002-9817-4986 (2020)
      T cells are activated when their T cell receptor (TCR) senses peptide-MHC (pMHC) molecules from pathogens and tumors. A network of signaling molecules downstream of the TCR drives the extent and nature of subsequent cellular responses. The activation threshold (AT) of these signaling pathways is a critical checkpoint for T cell responses. Here, we examined mechanisms by which the AT of a T cell is first determined and how it changes during the course of responding to antigen. The initial AT of a T cell is set during development by calibrating to how strongly it senses pMHC in the thymus. This calibration affects the surface levels of a receptor CD5, whose subsequent role is poorly defined. We found that CD5, independent of the TCR, sets basal levels of IκBα in T cells. Since IκBα critically modulates the transcription factor NFκB, which regulates multiple T cell functions including cell-survival, we hypothesized that variations in basal AT of T cells stem from varying NFκB depots maintained by CD5. Indeed, blocking NFκB abolished differences in cell-survival of thymocytes with different CD5 levels. The initial heterogeneities are further modified when peripheral T cells encounter antigen. Resulting memory T cells acquired higher CD5 levels and continuously required CD5 expression to maintain higher IκBα expression. If the stimulating antigen was not cleared efficiently, peripheral T cells further ‘tuned’ their AT in the opposite direction and resulted in loss of sensitivity to antigen (as seen in exhausted T cells). Importantly, this AT tuning involved additional regulation of TCR-proximal kinases such as Zap70 and was reversible in vivo, but not in vitro. We also found that rather than just the duration of antigen exposure, AT-tuning was potentially influenced by the rate at which antigen changes in vivo. This can help us understand how different persistent pathogens or tumors affect T cell responses, potentially based on the rates at which they replicate in the host. Finally, we characterized compounds that can target a T cell’s AT, identified in a high-throughput pharmacological screen, to potentially isolate drugs for altering T cell function during these physiological contexts.