Girtman, Megan A.

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 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 Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum resident protein that is widely expressed. Although it has been demonstrated to participate in the tail-anchored protein insertion pathway, its physiological role in the mature immune system is unknown. In this work, we show that mature, peripheral T cells require CAML for survival specifically following TCR-induced activation. In this study, we examined mature T cells from spleen and lymph nodes of tamoxifen-inducible CAML knockout mice (tCAML−/−). Whereas CAML-deficient T cells were able to express the early activation markers CD25 and CD69, and produce IL-2 normally upon stimulation, deficient cells proliferated less and died. Cells did not require CAML for entry into the S phase of the cell cycle, thus implicating its survival function at a relatively late step in the T cell activation sequence. In addition, CAML was required for homeostatic proliferation and for Ag-dependent cell killing in vivo. These results demonstrate that CAML critically supports T cell survival and cell division downstream of T cell activation.