T cell receptor (TCR) engagement of peptide-major histocompatibility complex (MHC) is essential to adaptive immunity, but it is unknown if TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed peptide-MHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3-dimensional (3D) and 2D affinities, using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling, and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the ‘stereotypical’ TCR-MHC docking paradigm evolved from productive signaling geometries, and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

The T cell receptor - peptide-MHC interface is comprised of conserved and diverse regions, yet the relative contributions of each in shaping T cell recognition remain unclear. We isolated cross-reactive peptides with limited homology, allowing us to compare the structural properties of nine peptides for a single TCR-MHC pair. The TCR’s cross-reactivity is rooted in highly similar recognition of an apical ‘hotspot’ position in the peptide, while tolerating significant sequence variation at ancillary positions. Furthermore, we find a striking structural convergence onto a germline-mediated interaction between TCR CDR1α and the MHC α2 helix of twelve TCR-pMHC complexes. Our studies suggest that TCR-MHC germline-mediated constraints, together with a focus on a small peptide hotspot, may place limits on peptide antigen cross-reactivity.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1), a NAD+ dependent deacetylase, has been associated with induction of autophagy, reprogramming cellular metabolism, and regulating immune mediators. In this study, we investigated the role of SIRT1 in bone marrow dendritic cell (BMDC) function during RSV infection. SIRT1 deficient (SIRT1-/-) BMDC showed a defect in mitochondrial membrane potential (Δ⍦m) that worsens during RSV infection. This defect in Δ⍦m caused the generation of elevated levels of reactive oxygen species (ROS). Furthermore, the oxygen consumption rate (OCR) was reduced as assessed in Seahorse assays, coupled with lower levels of ATP in SIRT1-/-DC. These altered responses corresponded to altered innate cytokine responses in the SIRT1-/-DC in response to RSV infection. Reverse Phase Protein Array (RPPA) functional proteomics analyses of SIRT1-/-and WT BMDC during RSV infection identified a range of differentially regulated proteins involved in pathways that play a critical role in mitochondrial metabolism, autophagy, oxidative and ER stress, and DNA damage. We identified an essential enzyme, acetyl CoA carboxylase (ACC1), which plays a central role in fatty acid synthesis and had significantly increased expression in SIRT1-/-DC. Blockade of ACC1 resulted in metabolic reprogramming of BMDC that ameliorated mitochondrial dysfunction and reduced pathologic innate immune cytokines in DC. The altered DC responses attenuated Th2 and Th17 immunity allowing the appropriate generation of anti-viral Th1 responses both in vitro and in vivo during RSV infection thus reducing the enhanced pathogenic responses. Together, these studies identify pathways critical for appropriate DC function and innate immunity that depend on SIRT1mediated regulation of metabolic processes.

Microvascular complications account for the significant morbidity associated with diabetes. Despite tight glycemic control, disease risk remains especially in type 2 diabetes (T2D) patients and no therapy fully prevents nerve, retinal, or renal damage in type 1 diabetes (T1D) or T2D. Therefore, new antidiabetic drug classes are being evaluated for the treatment of microvascular complications. We investigated the effect of empagliflozin (EMPA), an inhibitor of the sodium/glucose cotransporter 2 (SGLT2), on diabetic neuropathy (DPN), retinopathy (DR), and kidney disease (DKD) in streptozotocin-induced T1D and db/db T2D mouse models. EMPA lowered blood glycemia in T1D and T2D models. However, it did not ameliorate any microvascular complications in the T2D model, which was unexpected, given the protective effect of SGLT2 inhibitors on DKD progression in T2D subjects. Although EMPA did not improve DKD in the T1D model, it had a potential modest effect on DR measures and favorably impacted DPN as well as systemic oxidative stress. These results support the concept that glucose-centric treatments are more effective for DPN in T1D versus T2D. This is the first study that provides an evaluation of EMPA treatment on all microvascular complications in a side-by-side comparison in T1D and T2D models.

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Non-stimulatory or endogenous pepMHC presented on the surface of APCs, either alone or alongside agonist pepMHC, play various roles in T cell selection and activation. To examine these properties in more detail, here we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and non-stimulatory ligands, and were restricted by class I MHC alleles with different properties. We observed CD8-independent antagonism from TCR:pepMHC interactions with very low affinities (e.g. KD = 300μM). In addition, endogenous pep/Ld complexes on APCs antagonized activation of co-receptor (CD8)-negative 2C T cells even by the strong agonist QL9/Ld. In contrast, TCRs m33 and 3D-PYY, restricted by Kb and Db, respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as co-agonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g. in cases of low co-receptor levels) or enhanced activity (e.g. in presence of co-receptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC, but also perhaps by the inherent properties of the presenting MHC allele.

Elimination of peripheral tumors by adoptively transferred tumor-specific T cells may require killing of cancer cells and tumor stromal cells. Tumor Ags are cross-presented on stromal cells, resulting in direct cytotoxic T cell (CTL) killing of both Ag-expressing cancer cells and stromal cells. Indirect killing of Ag loss variant cells also occurs. We show here that similar processes occur in a brain tumor stromal environment. We used murine cancer cell lines that express high or low levels of a peptide Ag, SIYRYYGL (SIY), recognized by transgenic 2C CD8+ T cells. The two cell lines are killed with equivalent efficiency by 2C T cells in vitro. Following adoptive transfer of 2C T cells into mice with established SIY-Hi or SIY-Lo brain tumors, tumors of both types regressed, but low-Ag-expressing tumors recurred. High-Ag-expressing tumors contained CD11b+ cells cross-presenting SIY peptide and were completely eliminated by 2C T cells. To further test the role of cross-presentation, RAG1−/− H-2b mice were infused with H-2k tumor cells expressing high levels of SIY peptide. Adoptively transferred 2C T cells are able to kill cross-presenting H-2b stromal cells but not H-2k tumor cells. In peripheral models, this paradigm led to a small static tumor. In the brain, activated 2C T cells were able to kill cross-presenting CD11b+ cells and completely eliminate the H-2k tumors in most mice. Targeting brain tumor stroma or increasing Ag shedding from tumor cells to enhance cross-presentation may improve the clinical success of T cell adoptive therapies.

Diabetes is a global healthcare problem associated with enormous healthcare and personal costs. Despite glucose lowering agents that control glycaemia, both type 1 (T1D) and type (T2D) diabetes patients often develop microvascular complications that increase morbidity and mortality. Current interventions rely on careful glycemic control and healthy lifestyle choices, but these are ineffective at reversing or completely preventing the major microvascular complications, diabetic peripheral neuropathy (DPN), diabetic retinopathy (DR), and diabetic kidney disease (DKD). Minocycline, a tetracycline antibiotic with anti-inflammatory and anti-apoptotic properties, has been proposed as a protective agent in diabetes. However, there are no reported studies evaluating the therapeutic efficacy of minocycline in T1D and T2D models for all microvascular complications (DPN, DR, and DKD). Therefore, we performed metabolic profiling in streptozotocin-induced T1D and db/db T2D models and compared the efficacy of minocycline in preventing complications to that of insulin and pioglitazone in both models. Minocycline partially ameliorated DR and DKD in T1D and T2D animals, but was less effective than insulin or pioglitazone, and failed to improve DPN in either model. These results suggest that minocycline is unlikely to improve outcomes beyond that achieved with current available therapies in patients with T1D or T2D associated microvascular complications.