Peripheral proinsulin expression controls low-avidity proinsulin-reactive CD8 T Cells in type 1 diabetes

Low-avidity autoreactive CD8 T cells (CTLs) escape from thymic negative selection, and peripheral tolerance mechanisms are essential for their regulation. We report the role of proinsulin (PI) expression on the development and activation of insulin-specific CTLs in the NOD mouse model of type 1 diabetes. We studied insulin B-chain–specific CTL from different T-cell receptor transgenic mice (G9Cα−/−) expressing normal PI1 and PI2 or altered PI expression levels. In the absence of PI2 (Ins2−/−), CTL in pancreatic lymph nodes (PLNs) were more activated, and male G9Cα−/− mice developed T1D. Furthermore, when the insulin-specific CTLs developed in transgenic mice lacking their specific PI epitope, the CTLs demonstrated increased cytotoxicity and proliferation in vitro and in vivo in the PLNs after adoptive transfer into NOD recipients. Dendritic cell–stimulated proliferation of insulin-specific T cells was reduced in the presence of lymph node stromal cells (LNSCs) from NOD mice but not from mice lacking the PI epitope. Our study shows that LNSCs regulate CTL activation and suggests that exposure to PI in the periphery is very important in maintenance of tolerance of autoreactive T cells. This is relevant for human type 1 diabetes and has implications for the use of antigen-specific therapy in tolerance induction

A reproducible method for the expansion of mouse CD8+ T lymphocytes

Murine adoptive CD8 + T-cell immunotherapy studies require the generation of large numbers of high viability CD8 + cells. Here we report a tissue culture protocol for the reliable expansion of CD8 + T-cells derived from murine spleen to give a 20-fold expansion after 4 days in culture. The cells were transfected with an mRNA GFP construct and transferred into NOD mice. GFP positive cells could be detected 7 days after transfer thus confirming that the cells survive and are functional for up to 1 week

Measurement of pre- and post-synaptic proteins in cerebral cortex: Effects of post-mortem delay

Assessments of synaptic density in human brain are often based on measurements of synaptic proteins. Little information is available on their post-mortem stability. We have investigated this by ELISAs of the pre-synaptic proteins syntaxin and synaptophysin, and the post-synaptic protein PSD-95, in rat and human cortex. The rat brains were cooled in situ from 37 to 20 or 4°C over 3 h, and then kept at 20 or 4°C for a further 24-72 h, to simulate post-mortem storage at room temperature or in a mortuary refrigerator. Synaptophysin and PSD-95 levels in rat cerebral cortex were not significantly decreased after 72 h of incubation at 20°C. Syntaxin was stable for 24 h but decreased by 39-44% at 48-72 h. Storage at 4°C resulted in a similar reduction of syntaxin levels over 72 h. In human brain tissue from 160 people aged 24-102 years, post-mortem delay had little effect on synaptic protein levels in superior temporal cortex, but was associated with a decline in PSD-95 and syntaxin in mid-frontal cortex after 24 h. The more robust stability of synaptophysin may be related to its multi-transmembrane structure. © 2004 Elsevier B.V. All rights reserved

Inhibition of AMP-activated protein kinase protects pancreatic β-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity

OBJECTIVE—Apoptotic destruction of insulin-producing pancreatic β-cells is involved in the etiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic β-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of β-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8+ T-cells. RESEARCH DESIGN AND METHODS— Clonal MIN6 β-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/null), constitutively active AMPK (AMPK-CA), or dominant-negative AMPK (AMPK-DN) and exposed or not to tumor necrosis factor-α, interleukin-1β, and interferon-γ. Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8+ purified T-cells was examined against pancreatic islets from NOD mice infected with either null or the AMPK-DN–expressing adenoviruses. RESULTS— Exposure to cytokines, or expression of AMPK-CA, induced apoptosis in clonal MIN6 β-cells and CD1 mouse pancreatic islets. By contrast, overexpression of AMPK-DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8+ T-cells toward NOD mouse islets. CONCLUSIONS— Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T-cells. AMPK may therefore represent an interesting therapeutic target to suppress immune-mediated β-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes

Immunotargeting of insulin reactive CD8 T cells to prevent diabetes

Insulin is one of the earliest targeted autoantigens in the immune destruction of insulin-producing beta cells by autoreactive CD4 and CD8 T cells in type 1 diabetes. In this study, we used Non-obese diabetic (NOD) transgenic T cells engineered to express MHC class I-insulin peptide complexes linked to a T cell activation component (InsCD3-ζ), to target insulin-reactive CD8 T cells. We showed that activated, but not naïve, InsCD3-ζ CD8 T cells killed diabetogenic insulin-reactive CD8 target cells in vitro, inducing antigen-specific cell death mediated via both the release of perforin and the Fas–Fas ligand pathway. In vivo, InsCD3-ζ CD8 T cells migrated to the pancreatic lymph nodes of NOD mice after adoptive transfer. Concomitant with this, infiltration of CD8 T cells was also reduced in the pancreatic islets. Finally, in vivo, we showed that diabetes induced by adoptive transfer of insulin-reactive T cells was reduced following injection of activated InsCD3-ζ CD8 T cells. Furthermore, young NOD mice injected with InsCD3-ζ CD8 T cells developed a lower incidence and delayed onset of diabetes. Thus, using this novel system we have demonstrated that InsCD3-ζ CD8 T cells can directly kill insulin-reactive CD8 T cells in vitro and by targeting insulin-specific CD8 T cells early in the course of disease alter the progression of spontaneous diabetes in vivo in NOD mice

Pseudo-Anaphylactic Reactions to Pfizer BNT162b2 Vaccine: Report of 3 Cases of Anaphylaxis Post Pfizer BNT162b2 Vaccination

10.3390/vaccines9090974VACCINES9