Induktion von Preproinsulin-spezifischen Foxp3+ CD4+ Treg Zellen in Mausmodellen des Typ 1 Diabetes
Zusammenfassung der Projektergebnisse
In this project, we continued to characterize expression/processing of autoantigens, priming of autoreactive effector CD8+ T cells or regulatory Foxp3+CD4+Treg cells by DNA immunization and their role for induction or prevention of autoimmune diabetes in ppins-inducible and spontaneous diabetes mouse models. Our new and exciting results are: We identified a novel beta cell-specific target molecule (free fatty acid receptor GPR40) for potential autoreactive CD8 T cells in immune-mediated diabetes mellitus. Interestingly, two newly identified MHC-I epitopes located in hydrophobic transmembrane helices. Priming/expansion of Kb/GPR40187-195-specific CD8 T cells required help from beta cell-specific, B7.1-mediated costimulatory signaling in RIP-B7-1 tg or from co-induced Kb/A12-21-specific CD8 T cells that apparently trigger initial hits in beta cells and thereby induce suitable conditions in the islets favouring the concomitant activation of autoreactive T cells. I-Ab/ppins76-90-specific CD4+ Tregs play a crucial role in the control of ppins-primed CD8 T cellmediated autoimmune diabetes in PD-1/PD-L1-deficient mice. Most interestingly, this epitope apparently cross-reacted with I-Ag7-specific CD4 T cells and also suppressed spontaneous diabetes development in female I-Ag7+ NOD/ShiLtJ (NOD) mice, when delivered by a chimeric HBVcore/ppins76-90 vector. The I-Ab/ppins76-90 peptide specifically stimulated the conversion of conventional Foxp3-/eGFP-CD4+Tconv cells (from Foxp3eGFP mice) into Foxp3+eGFP+CD4+Treg cells, upregulated expression of immune-suppressive mTGF-β and CTLA4 and secretion of TGF-β but not IL-10. In contrast, the pool of natural and induced Foxp3+eGFP+CD4+Tregs in these mice barely responded to the I-Ab/ppins76-90 peptide. Immune-suppressive Tregs were thus primarily recruited from mature CD4 T cells. Using a novel I-Ab/ppins76-90 and control I-Ab/CLIP tetramers from the NIH tetramer core facility, we have a novel tool to specifically detect Foxp3+CD4+Treg cells in vaccinated PD-1-/- mice. Injection of ppins/pins designer antigens (excluded from ER expression) into 10-12 weeks old female NOD mice efficiently suppressed spontaneous diabetes, though beta cells already showed signs of an initial disease with a prominent insulitis. In particular, we could detect an influx of Kd/ppins15-23 tetramer+ CD8 T cells into the pancreata of non-vaccinated early diabetic NOD mice, but had difficulties to specifically identify CD4 T cells directed against the overlapping I-Ag7/ppins9-23 epitope with I-Ag7/InsBp8E and I-Ag7/CLIP tetramers from NIH tetramer core facility). Using mono-specific cationic peptide vaccines, we could show that priming of Kd/B15-23-specific CD8 T cells and autoimmune diabetes apparently depends on an overlapping ppins9-23 CD4 T cell help. This argued for a prominent role of CD8 T cells in disease development. We established novel diabetes models based on B6.NOD-(D17Mit21-D17Mit10)/LtJ (B6.g7) mice, expressing the diabetes susceptible haplotype (Kd;Db;I-Ag7). In contrast to B6 mice, a single injection of ppins DNA into B6.g7 mice induced Kd/B15-23-specific CD8 T cells and autoimmune diabetes that was suppressed by both, anti CD8 and anti CD4 mAbs. Therefore, the CD4 T-cell help circumvents the PD-L1-mediated negative regulation of autoreactive CD8 T cells seen in ppins-immune H-2b B6 vs PD-L1/PD-1-/- mice. Interestingly, cytosolic/nuclear ppins-designer antigens also suppressed pCI/ppins-inducible diabetes in B6.g7 mice, but the analyses whether ppins76-90 reactive CD4 Tregs are involved in diabetes suppression are still in progress. In our hands, homozygous but not heterozygous B6.g7xRIP-B7.1 tg mice spontaneously developed severe autoimmune diabetes. Most interestingly, we could also detect tetramer+/IFN-γ+ Kd/B15-23-CD8 T cells in the pancreata of diabetic mice. These findings strongly argued for a ´cross-reactive´ B6-mediated immune suppressive factor that prevented full-blown diabetes in the heterozygous mice. Comparable to ppins, expression/processing in the ER was a prerequisite to prime Cripto-1 (Kb/cr16- 24)-specific CD8 T cells in B6 mice in a TAP- and ERAP-independent manner by DNA vaccination. Notably, we could also prime Kb/Cr16-24-specific CD8 T cells in B6 mice by an ER-associated chimeric ppinsΔA12-21/Cr12-24 antigen, confirming that no central or peripheral tolerance mechanisms operate against the ppinsΔA12-21 ´self´ carrier.
Projektbezogene Publikationen (Auswahl)
- (2016) Exploring the induction of preproinsulin-specific Foxp3(+) CD4(+) Treg cells that inhibit CD8(+) T cellmediated autoimmune diabetes by DNA vaccination. Sci Rep 6, 29419
Stifter, K., Schuster, C., Schlosser, M., Boehm, B. O., and Schirmbeck, R.
(Siehe online unter https://doi.org/10.1038/srep29419) - (2018) LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus. Nat Commun 9, 1488
E., Lopez-Noriega, L., Mellado-Gil, J. M., Romero-Zerbo, S. Y., Baquie, M., Lachaud, C. C., Stifter, K., Perdomo, G., Bugliani, M., De Tata, V., Bosco, D., Parnaud, G., Pozo, D., Hmadcha, A., Florido, J. P., Toscano, M. G., de Haan, P., Schoonjans, K., Sanchez Palazon, L., Marchetti, P., Schirmbeck, R., Martin-Montalvo, A., Meda, P., Soria, B., Bermudez-Silva, F. J., St-Onge, L., and Gauthier, B. R.
(Siehe online unter https://doi.org/10.1038/s41467-018-03943-0) - (2019) Preproinsulin Designer Antigens Excluded from Endoplasmic Reticulum Suppressed Diabetes Development in NOD Mice by DNA Vaccination. Mol Ther Methods Clin Dev 12, 123-133
Stifter, K., Schuster, C., Krieger, J., Spyrantis, A., Boehm, B. O., and Schirmbeck, R.
(Siehe online unter https://doi.org/10.1016/j.omtm.2018.12.002) - (2020) A dominant insulin-specific and islet-destructive T-cell response is sufficient to activate CD8 T cells directed against the fatty-acid receptor GPR40. Cell Mol Immunol 17, 659-661
Spyrantis, A., Krieger, J., Stifter, K., Boehm, B. O., and Schirmbeck, R.
(Siehe online unter https://doi.org/10.1038/s41423-019-0309-y) - (2020) An IFN-γ treatment protocol for MHC-Ilo, PD-L1+ tumor cells selectively restores their TAP-mediated presentation-competence to prime effector CD8 T cells. Journal for Immunotherapy of cancer (JITC)
Stifter, K., Krieger, J., Ruths, L., Gout, J., Mulaw, M., Lechel, A., Kleger, A., Seufferlein, T., Wagner,M., and Schirmbeck, R.
(Siehe online unter https://dx.doi.org/10.1136%2Fjitc-2020-000692)
