Pease, Larry R.

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Abstract Self-tolerant T cells can cross-react weakly on self, but are usually incapable of activation. However, when activated these T cells can cross-react effectively on cells expressing self antigens. We have observed that mutants of the MHC class I molecule H2-Kb have structural differences resulting in altered antigen presentation. We hypothesize that structural manipulation of MHC class I can increase stabile amino acid contacts with the TCR. Increasing stabile contacts may decrease the energy barrier required to overcome the threshold for activation, breaking tolerance for self antigens. Using computer modeling techniques, amino acid substitutions at specific sites of MHC class I were predicted to result in stabilization of the TCR:pMHC complex. These altered MHC molecules were made and expressed in cell lines. We have shown that altered MHC enhanced TCR binding. Altered MHC enhanced activation and proliferation of naïve T cells in response to a non-stimulatory, weak antigen. Altered MHC also induced CTL killing against self-antigens. In an in vivo transplant model, protective immunity was induced against a native tumor after an altered MHC expressing tumor challenge. By altering the structure of MHC class I we have activated normally tolerant T cells that are capable of cross-reacting on self.

Abstract Type 1 diabetes can be induced by virus expressing SIINFEKL in Rat Isulin Promoter (RIP)-OVA transgenic mice harboring adoptively transferred naïve antigen-specific OT1 T cells. By titrating the number of transferred OT-1 cells, disease outcome can be manipulated. Mice receiving high numbers of OT-1 cells develop autoimmunity and rapidly succumb to disease. Mice receiving limiting numbers develop hyperglycemia transiently and then over 10 days recover glucose homeostasis. Recovered mice exhibit restored normal resting blood sugar levels and are able to regulate glucose during a stress test. Histological examination of the pancreas showed extensive pancreatic destruction with no or very few remaining insulin-positive islets. Using RIP-OVA mice expressing luciferase under the mouse insulin promoter, pancreatic insulin promoter activity was extinguished, while liver expression increased in recovered animals, suggesting an ectopic source of insulin can support glucose homeostasis in mice with extensively damaged islets. Pancreatic infiltrates during peak inflammation contained host-derived CD8 T cells in addition to the infiltrating OT-1 cells. These host CD8 T cells were absent in CD8 deficient RIP-OVA mice, and diabetes occurred with fewer transferred OT-1 cells and was quickly fatal. Reconstitution of the CD8 compartment protected these animals. These findings indicate the importance of host CD8 T cells in limiting autoreactive T cell-mediated destruction of islets.

Abstract MHC multimers including tetramers and pentamers provide a powerful way to visualize antigen specific T cell responses in both experimental and clinical immune assays. The multimers can be acquired commercially, from the NIH, or assembled in the laboratory for measuring immune responses to commonly-studied or novel-hypothetical antigens. The multimer reagents can be expensive and time consuming to acquire and in many cases are uncontrolled reagents as robust positive control T cells used to assess the reagents functionally may not be available. This is particularly problematic when using MHC multimers to investigate less well-defined T cell responses, such as those characterized by low T cell frequencies or by specificity to hypothetical antigens relevant to cancer, infection, or autoimmune disease. These problems are magnified when studying irreplaceable clinical trial samples where having confidence that reagents are biochemically intact is critical. To address this problem, we have devised a versatile, flow cytometry-based, capture assay to probe the structural integrity of MHC multimer reagents. In principle, the idea is to immobilize on a bead antibody specific for conformational determinants expressed on a properly folded MHC molecule. The particle becomes labeled and can be visualized and quantified by flow cytometry when a fluorescently tagged tetramer is adsorbed to the particulate upon binding to the antibody.

Abstract The complexity of molecular networks regulating the immune response presents challenges to measuring immune response potential in human populations. We measured gene expression in CD4+ peripheral blood cells from healthy subjects using RNAseq. RNA expression was assessed twice, one month apart to identify the subset of genes expressed stably within individuals over time yet discriminating individuals in a cohort. This immune response signature gene set then was used to identify and successfully order the immune response phenotypes of independent, unrelated subjects. Following a brief 4 hr stimulation of freshly isolated cells with anti-CD3/C28 antibody-coated beads, changes in gene expression were measured using a similar approach, defining individual response profiles and clustering response patterns. Examination of the RNA expression signatures of unstimulated CD4 cells revealed a gene subset which when analyzed for gene expression levels correctly aligned independent test subjects into the defined response-phenotype clusters following stimulation. Therefore, there exists a subset of genes stably expressed over time in circulating CD4+ cells which discriminate among individuals in a manner associated with immune response potential. Thus a systems approach can be used to describe the complex immune phenotypes of individuals in ways that might be useful in determining how individuals will respond to complex stimuli or in disease and treatment settings.

Abstract We have engineered an attenuated picornavirus vaccine using Theiler’s murine encephalomyelitis virus (TMEV) and are exploring its usefulness in immunotherapy. We find that two different insertion sites within the leader protein of the TMEV genome allow for effective production of virus vaccine strains. One site is at the amino terminal end of the protein after the third amino acid position (p3); the other is at the XhoI restriction site further downstream. We find that vaccine antigen insertion at p3 disrupts the ability of the virus to inhibit type I interferon production by infected cells, whereas XhoI insertion does not. Further, the p3 modified virus can be cleared from animals lacking functional T and B-cells but replicates to high titer in IFNα/β receptor knockout mice, suggesting that innate immune mechanisms are sufficient for viral clearance. In spite of rapid clearance, p3 vaccines readily stimulate proliferation of transgenic T-cells and induce de novo responses to T-cell antigens in wild-type mice, although to a lesser extent than the XhoI modified vaccines strains. The use of either vaccine however effectively delays tumor growth and enhances survival in mice bearing melanoma and breast tumors. This vaccine provides a safe and effective vaccine strategy for the induction of CD8+ T-cell immunity directed towards cancer and provides support for the development of picornaviruses as vaccine vectors for treating human disease.

Abstract We have induced acute diabetes following immune attack on the pancreas with islet specific CD8+ T cells, but surprisingly many animals subsequently recovered blood glucose homeostasis. The recovered animals exhibited nearly normal glucose tolerance and produced normal levels of blood insulin following glucose challenge. Remarkably very few islets were present in these animals and those remaining were virtually insulin free. In this model B6 RIP-OVAhi mice receive 100 to 600 naïve OT-1 CD8+ T cells prior to activation by an infection with Theilers murine encephalomyelitis virus encoding the SIINFEKL ovalbumin antigen. When greater numbers of T cells are transferred, this protocol causes acute and fatal diabetes. However, when limiting numbers of T cells are used, animals can recover from the acute phase of the disease. Under these limiting conditions, we find that the mice develop acute pancreatitis by day 5 following infection and stop producing insulin by day 8. By day 11 to 14 some animals begin to recover. Early in the recovery process, insulin positive beta cells are evident in islets. However, late after recovery (days >56) animals that regulate blood sugar normally are found devoid of insulin producing beta cells, though insulin levels are near normal. This raises important questions about how these animals recover glucose homeostasis following cellular immune attack on their islets. Our hypothesis is that insulin is produced outside of the pancreas in these animals.

Abstract We have developed a mouse model of diabetes by adoptively transferring small numbers of OT1 cells into animals expressing OVA under the rat insulin promoter (RIP-OVA). These cells are activated in vivo with a Picornavirus (TMEV) expressing OVA antigen. Titrating the dose of adoptively transferred cells, changes disease outcome. Mice receiving low amounts of cells develop hyperglycemia, and recover homeostasis. Investigation of recovered animals revealed normal blood glucose regulation. Pancreatic histology of recovered mice showed extensive pancreatic destruction with no or very few remaining insulin-positive islets. While adoptively transferred OT1 cells were present, pancreas infiltrating T cells were largely endogenous. To evaluate the functional importance of the host T cells, we tested disease progression using RAG KO, CD4 KO, and CD8 KO RIP-OVA hosts. Without RAG, CD4, or CD8 we observed increased disease severity. This change is not attributed to homeostatic proliferation in the CD4 and CD8 KO animals, as adoptively transferred OT1 cells behaved similarly in the KO hosts in comparison to RIP-OVA mice. However there is significant homeostatic proliferation occurring in the RIP-OVA RAG KO animals. Our studies reveal the importance of CD4 and CD8 T cells in limiting autoimmune attack in the pancreas. Although populations of CD4 T regulatory cells are well known, the importance of CD8 T cells in limiting inflammation are less well characterized.

Abstract IgM antibodies, as multivalent ligands, have the capacity to activate cells differentially by cross-linking targeted cell surface signaling molecules. For example, hIgM12 (B7DC Xab) activates dendritic cells (DCs), leading to potent immunomodulation, while hIgM22 binds glial cells and causes remyelination in mouse models of multiple sclerosis. Both antibodies activate common signaling intermediates in their respective cellular targets suggesting a common mechanism of action. Elucidation of these shared mechanisms became possible by targeting both antibodies to a common cell type. Our previous studies demonstrate that B7-DC expression is required to activate downstream signaling in DCs by hIgM12. We utilized a phage display system to identify 7mer peptides bound by each IgM and expressed each peptide attached to a surrogate cell surface carrier on retrovirally tranduced bone marrow-derived DCs from B7-DC -/- mice. Transduced cells were treated with hIgM12, hIgM22 or anti-carrier IgM, and subsequent activity was compared against B7-DC +/+ DCs or B7-DC-reconstituted B7-DC -/- DCs. DC activation by each IgM was specific to expression of their conjugate peptide construct. Activation by hIgM22 was indistinguishable from activation by hIgM12, while anti-carrier IgM caused no activation. This indicates that while their target specificity differs, once bound these IgMs activate cells via common mechanisms.

Abstract One limitation to the development of targeted tumor immunity is the availability of safe vectors that can drive strong T-cell responses capable of eradicating tumors. We have designed viral vectors using Theiler’s murine encephalomyelitis virus (TMEV) that effectively inhibit melanoma outgrowth. By cloning the TMEV genome into a eukaryotic expression vector that can easily be manipulated we were able to generate several novel tumor specific virus vaccines. We find that incorporation of defined MHC class I epitopes into sites within the leader protein of the virus can drive strong CD8+ T-cell responses against these target peptides. Further, we find that the site of incorporation can change the elicited cytokine profile and the virulence of the virus. Incorporation of the ovalbumen peptide after the third amino acid of the L protein (p3) generates a strong IFNβ response, whereas insertion within the downstream XhoI restriction site elicits a strong IL6 response similar to wild-type TMEV. Further, the p3 virus is not lethal to RAG-deficient mice, where the XhoI and wild-type TMEV virus are. However, p3 virus persists and kills IFNα/β receptor-deficient mice, suggesting that innate immune responses are responsible for viral clearance. In spite of these differences the p3 and XhoI viruses can stimulate antigen specific T-cell responses and inhibit tumor outgrowth equivalently, demonstrating the efficacy of TMEV and its potential as a therapeutic intervention.