Tijana Martinov

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR targeting a tumor epitope, is a form of adoptive cell therapy (ACT) that has exhibited promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, making them attractive targets for TCR-T cell therapy. However, class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence an epitope encompassing such mutations can bind HLA-A2 and induce T cell responses. We report post-translational modifications (PTMs) on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS _G12V peptide, rather than the unmodified epitope, may be presented in HLA-A2 by tumor cells and impact TCR recognition. Using a novel computationally guided approach, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate/demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these new TCRs. Our findings highlight the role of PTMs in immune evasion and suggest identifying and targeting such modifications should make effective ACTs available for a substantially greater range of tumors than the current therapeutic landscape. One-sentence summary: Tumor cell methylation of KRAS _G12V epitope in HLA-A2 permits immune evasion, and new TCRs were generated to overcome this with engineered cell therapy.

Purpose of Review Programmed death-1 (PD-1) is an inhibitory receptor that controls T and B cell proliferation and function through interacting with its ligand PD-L1 or PD-L2. PD-1/PD-L1 blockade reboots anti-tumor immunity and is currently used to treat > 15 different types of cancer. However, the response rate is not at 100% and some patients relapse. Importantly, up to 37% of patients treated with PD-1/PD-L1 blocking antibodies develop immune-related adverse events, including overt autoimmunity, such as type 1 diabetes (T1D). Herein, we discuss the role of PD-1, PD-L1, and PD-L2 signaling in pre-clinical models of T1D, including recent work from our laboratory. Recent Findings We highlight ongoing efforts to harness PD-1/PD-L1 signaling and treat autoimmunity. We also evaluate studies aimed at defining biomarkers that could reliably predict the development of immune-related adverse events after clinical PD-1/PD-L1 blockade. Summary With increasing use of PD-1 blockade in the clinic, onset of autoimmunity is a growing health concern. In this review, we discuss what is known about the role of PD-1 pathway signaling in T1D and comment on ongoing efforts to identify patients at risk of T1D development after PD-1 pathway blockade.

Type 1 diabetes (T1D) results from destruction of pancreatic beta cells by T cells of the immune system. Despite improvements in insulin analogs and continuous blood glucose level monitoring, there is no cure for T1D, and some individuals develop life-threatening complications. Pancreas and islet transplantation have been attractive therapeutic approaches; however, transplants containing insulin-producing cells are vulnerable to both recurrent autoimmunity and conventional allograft rejection. Current immune suppression treatments subdue the immune system, but not without complications. Ideally a successful approach would target only the destructive immune cells and leave the remaining immune system intact to fight foreign pathogens. This review discusses the autoimmune diabetes disease process, diabetic complications that warrant a transplant, and alloimmunity. First, we describe the current understanding of autoimmune destruction of beta cells including the roles of CD4 and CD8 T cells and several possibilities for antigen-specific tolerance induction. Second, we outline diabetic complications necessitating beta cell replacement. Third, we discuss transplant recognition, potential sources for beta cell replacement, and tolerance-promoting therapies under development. We hypothesize that a better understanding of autoreactive T cell targets during disease pathogenesis and alloimmunity following transplant destruction could enhance attempts to re-establish tolerance to beta cells.

Type 1 diabetes (T1D) results from T cell–mediated destruction of insulin-producing β-cells. Insulin represents a key self-antigen in disease pathogenesis, as recent studies identified proinsulin-responding T cells from inflamed pancreatic islets of organ donors with recent-onset T1D. These cells respond to an insulin B-chain (InsB) epitope presented by the HLA-DQ8 molecule associated with high T1D risk. Understanding insulin-specific T-cell frequency and phenotype in peripheral blood is now critical. We constructed fluorescent InsB10–23:DQ8 tetramers, stained peripheral blood lymphocytes directly ex vivo, and show DQ8+ patients with T1D have increased tetramer+ CD4+ T cells compared with HLA-matched control subjects without diabetes. Patients with a shorter disease duration had higher frequencies of insulin-reactive CD4+ T cells, with most of these cells being antigen experienced. We also demonstrate that the number of insulin tetramer+ effector memory cells is directly correlated with insulin antibody titers, suggesting insulin-specific T- and B-cell interactions. Notably, one of four control subjects with tetramer+ cells was a first-degree relative who had insulin-specific cells with an effector memory phenotype, potentially representing an early marker of T-cell autoimmunity. Our results suggest that studying InsB10–23:DQ8 reactive T-cell frequency and phenotype may provide a biomarker of disease activity in patients with T1D and those at risk.

Despite accounting for 10–30% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an autoimmune hazard by allowing self-reactive TCRs to escape thymic selection. We revisited this hypothesis using the NOD murine model of type 1 diabetes. We bred NOD mice hemizygous at both TCRα and β (TCRα^+/− β^+/−) loci, rendering them incapable of producing dual TCR T cells. We found that the lack of dual TCRα expression skewed the insulin-specific thymocyte population toward greater regulatory T (T_reg) cell commitment, resulting in a more tolerogenic T_reg to conventional T cell ratio and protection from diabetes. These data support a novel hypothesis by which dual TCR expression can promote autoimmunity by limiting agonist selection of self-reactive thymocytes into the T_reg cell lineage.

Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. Epidemiologic studies associate the risk of vulvodynia with a history of atopic disease. We used an established model of hapten-driven contact hypersensitivity to investigate the underlying mechanisms of allergy-provoked prolonged sensitivity to pressure. We sensitized female ND4 Swiss mice to the hapten oxazolone on their flanks, and subsequently challenged them four days later with oxazolone or vehicle for ten consecutive days on the labia. We evaluated labiar sensitivity to touch, local mast cell accumulation, and hyperinnervation after ten challenges. Oxazolone-challenged mice developed significant tactile sensitivity that persisted for over three weeks after labiar allergen exposures ceased. Allergic sites were characterized by mast cell accumulation, sensory hyper-innervation and infiltration of regulatory CD4⁺CD25⁺FoxP3⁺ T cells as well as localized early increases in transcripts encoding Nerve Growth Factor and nerve-mast cell synapse marker Cell Adhesion Molecule 1. Local depletion of mast cells by intra-labiar administration of secretagogue compound 48/80 led to a reduction in both nerve density and tactile sensitivity. Mast cells regulate allergy-provoked persistent sensitivity to touch. Mast cell-targeted therapeutic strategies may provide novel means to manage and limit chronic pain conditions associated with atopic disease.

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide–MHCII complexes.

Type 1 diabetes (T1D) is a CD4⁺ T cell-driven autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. Clinical evidence and studies in non-obese diabetic (NOD) mice suggest that insulin is a major autoantigen. With this in mind, we developed insulin B_10-23:IAg7 tetramer reagents to track insulin-specific CD4⁺ T cells in mice and interrogate the role of Programmed death-1 (PD-1) for peripheral tolerance. PD-1 is a T cell inhibitory receptor necessary to maintain tolerance and prevent T1D in NOD mice. PD-1 pathway inhibitors are increasingly used in the clinic for treating malignancies, and while some patients benefit, others develop adverse autoimmune events, including T1D. We therefore sought to understand the role of PD-1 in maintaining islet-specific tolerance in diabetes-resistant strains. B6.g7 mice express the same MHC Class II allele as NOD mice, have predominantly naïve insulin-specific CD4⁺ T cells in the periphery, and remain diabetes-free even after PD-1 pathway blockade. Here, we examined the trafficking potential of insulin-specific CD4⁺ T cells in NOD and B6.g7 mice with or without anti-PD-L1 treatment, and found that PD-L1 blockade preferentially increased the number of CD44^highCXCR3⁺ insulin-specific cells in NOD but not B6.g7 mice. Additionally, we investigated whether pancreatic islets in NOD and B6.g7 mice expressed CXCL10, a lymphocyte homing chemokine and ligand for CXCR3. Anti-PD-L1 treated and control NOD mice had detectable CXCL10 expression in the islets, while B6.g7 islets did not. These data suggest that islet tolerance may be in part attributed to the pancreatic environment and in the absence of pancreas inflammation, chemotactic cytokines may be missing. This, together with our previous data showing that PD-1 pathway blockade preferentially affects effector but not anergic self-specific T cells has implications for the use of checkpoint blockade in treating tumor patients. Our work suggests that determining tumor- and self-specific CD4⁺ T cell activation status (naïve, effector or anergic) prior to initiation of immunotherapy would likely help to stratify individuals who would benefit from this therapy versus those who might have adverse effects or incomplete tumor control.