A distinct role of CD4+ Th17- and Th17-stimulated CD8+ CTL in the pathogenesis of type 1 diabetes and experimental autoimmune encephalomyelitis

Abstract

Both CD4(+) Th17-cells and CD8(+) cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG(35-55) peptide-pulsed dendritic cells (DC(OVA) and DC(MOG)) with CD4(+) T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8(+) T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2K(b-/-) mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.

Fig. 1

Phenotypic characterization of OVA-specific CD4⁺ Th17 cells. a Naïve CD4⁺ T cells and DC_OVA-activated CD4⁺ Th17 cells derived from OT II mice were stained with a panel of biotin-conjugated Abs (solid lines) followed by staining with FITC-conjugated avidin and analyzed by flow cytometry. Irrelevant isotype-matched biotin-conjugated Abs were used as controls (light dotted lines). b In vitro DC_OVA-activated CD4⁺ Th1 and Th17 cells were double-stained with FITC-anti-IL-17 Ab and PE-anti-IFN-γ Ab, and analyzed by flow cytometry. c RNA extracted from DC_OVA-activated CD4⁺ Th17 and Con A-stimulated CD4⁺ T (control) cells were analyzed by RT-PCR for expression of Th17 cell specific transcription factor ROR-γt. d Purified active CD4⁺ Th17 cells were stained with PE-anti-CD4 and FITC-anti-CD11c Abs and analyzed by flow cytometry. e CD4⁺ Th17 and (K^b−/−)Th17 cells were stained with FITC-anti-pMHC I antibody (solid lines) and irrelevant isotype-matched antibody was used as control (dotted lines). One representative experiment of two experiments is shown

Fig. 2

CD4⁺ Th17 cells induced CTL leads to diabetes in transgenic RIP-mOVA mice. a In vitro CD8⁺ T cell proliferation assay. Irradiated DC_OVA, CD4⁺ Th17, CD4⁺ Th17 with anti-IL-2 Ab and (K^b−/−)Th17 cells, and their twofold dilutions were co-cultured with naïve OTI CD8⁺ T cells. After 2 days, the proliferative responses of CD8⁺ T cells were determined by overnight ³H-thymidine uptake assay. b In vitro cytotoxicity assay. Th17-activated OTI CD8⁺ Tc1 cells were used as effector (E) cells and in another experiment, Th17-activated CD8⁺ T cells with or without preincubation of concanamycin A (CMA, 1 μM) or emetin (5 μM) for 2 h were used as effector (E) cells, while ⁵¹Cr-labeled EG7 and EL4 cells were used as target (T) cells in a chromium release assay. c In tetramer staining assay, the tail blood samples and pancreatic lymph node cells of transgenic RIP-mOVA mice adoptively transferred with CD4⁺ Th17 cells, DC_OVA, (K^b−/−)CD4⁺ Th17 cells, and PBS (controls) were stained with PE-H-2K^b/OVAI (PE-tetramer) and FITC-CD8 Ab (FITC-CD8), and then analyzed by flow cytometry. The values in each panel represent the percentage of tetramer-positive CD8⁺ Tcells versus the total CD8⁺ Tcell population. The value in parenthesis represents the standard deviation. In in vivo cytotoxicity assay, 16 h after target cell delivery, the residual OVAI-pulsed CFSE^high and Mut1-pulsed CFSE^low target cells remaining in the spleens of the above cohorts of mice were sorted and analyzed by flow cytometry. The value in parenthesis represents the standard deviation; (n=6, average±SD), *p<0.05 versus cohorts of mice adoptively transferred with DC_OVA (Student’s t test). d In tetramer staining assay, the tail blood samples of wild-type C57BL/6 and perforin^−/− mice adoptively transferred with CD4⁺ Th17 cells were stained with PE-H-2K^b/OVAI (PE-tetramer) and FITC-CD8 Ab (FITC-CD8), and then analyzed by flow cytometry. In in vivo cytotoxicity assay, 16 h after target cell delivery, the residual OVAI-pulsed CFSE^high and Mut1-pulsed CFSE^low target cells remaining in the spleens of the above cohorts of mice were sorted and analyzed by flow cytometry. The value in parenthesis represents the standard deviation; (n=6, average±SD), *p<0.05 versus cohorts of perforin^−/− mice (Student’s t test). e Urine test for diabetes. Glucose levels in urine samples from transgenic RIP-mOVA mice adoptively transferred with irradiated CD4⁺ Th17 cells, DC_OVA, (K^b−/−)CD4⁺ Th17 cells, and PBS (controls). The cutoff line of urine glucose concentration for diabetes is shown. f Hematoxylin and eosin-stained sections from Th17- and PBS-injected mice at higher magnification showing extensive cellular infiltration in Th17-injected mice compared to control. Magnifications, ×10 and ×20. One representative experiment of two in the above different experiments is shown

Fig. 3

MOG peptide immunization stimulate MOG-specific CTL responses and induce EAE. a Wild-type C57BL/6 and CD4⁺ T cell- or CD8⁺ T cell-deficient Ia^b and H-2K^b mice were immunized with MOG_35-55 + CFA. C57BL/6 mice immunized with CFA only were used as control. Clinical EAE was scored according to 0–5 scale. The difference between C57BL/6 and CD4⁺ T cell-depleted mice (two asterisks) or CD8⁺ T cell-depleted C57BL/6 mice (single) is very significant (p<0.01) or significant (p<0.05; Mann–Whitney U test). b The tail blood samples of mice immunized with MOG peptide or OVAI peptide (control) were stained with PE-H-2D^b/MOGI pentamer (PE-pentamer) and FITC-anti-CD8 Ab (FITC-CD8), and then analyzed by flow cytometry. The value in each panel represents the percentage of pentamer-positive CD8⁺ T cells versus the total CD8⁺ T cell population. c In in vivo cytotoxicity assay, 16 h after target cell delivery, the residual MOGI-pulsed CFSE^high and Mut1-pulsed CFSE^low target cells remaining in the spleens of the above immunized mice were sorted and analyzed by flow cytometry. The value in parenthesis represents the standard deviation. d Photographs of sections of spinal cords derived from mice with EAE; tissue sections were stained with Luxol fast blue along with H&E counterstaining. Control mice (a and c) and MOG-immunized mice (b and d). Magnifications, ×5 (a and b) and ×20 (c and d). Inflammatory infiltration and demyelination are shown with arrows. e Mean scores of inflammation and demyelination±SD. *p<0.01 versus cohorts of the control groups (Student’s t test). One representative experiment of three in the above experiments is shown

Fig. 4

In vitro-activated MOG-specific CD4⁺ MOG-TCR-Th17 cells stimulate MOG-specific CD8⁺ CTL responses and induce EAE. a Phenotypic analysis of MOG-specific CD4⁺ MOG-TCR-Th17 cells. MOG-specific CD4⁺ MOG-TCR-Th17 cells derived from transgenic MOG-TCR mice were stained with a panel of biotin-conjugated Abs (solid lines) followed by staining with FITC-conjugated avidin and analyzed by flow cytometry. Irrelevant isotype-matched biotin-conjugated Abs were used as controls (light dotted lines). b RNA extracted from MOG-specific CD4⁺ MOG-TCR-Th17 and Con A-stimulated CD4⁺ T (control) cells were analyzed by RT-PCR for assessment of expression of RORγt. c Pentamer staining assay. The tail blood samples of mice adaptively transferred with CD4⁺ MOG-TCR-Th17 cells or Con A-stimulated CD4⁺ T (control) cells were stained with PE-H-2D^b/MOGI pentamer (PE-pentamer) and FITC-anti-CD8 Ab (FITC-CD8), and then analyzed by flow cytometry. The value in each panel represents the percentage of pentamer-positive CD8⁺ T cells versus the total CD8⁺ T cell population. The value in parenthesis represents the standard deviation. d In vivo cytotoxicity assay. Sixteen hours after target cell delivery, the residual MOGI-pulsed CFSE^high and Mut1-pulsed CFSE^low target cells remaining in the spleens of the above cohorts of mice were sorted and analyzed by flow cytometry. The value in parenthesis represents the standard deviation. e Wild-type C57BL/6 mice were adoptively transferred with MOG-specific MOG-TCR-Th17 cells or OVA-specific Th17 cells (control). The clinical EAE was scored according to 0–5 scale. f Photographs of sections of spinal cords derived from mice with EAE; tissue sections were stained with Luxol fast blue along with H&E counterstaining. Control mice (a and c) and MOG-immunized mice (b and d). Magnifications, ×5 (a and b) and ×20 (c and d). Inflammatory infiltration and demyelination are shown with arrows. g Mean scores of inflammation and demyelination±SD. *p<0.01 versus cohorts of the control groups (Student’s t test). One representative experiment of three in the above experiments is shown

Fig. 5

In vivo-generated MOG-specific CD4⁺ Th17 cells stimulate MOG-specific CD8⁺ CTL responses and induce EAE. a Phenotypic analysis of in vivo-generated MOG-specific CD4⁺ Th17 cells. MOG-specific CD4⁺ Th17 cells derived from MOG peptide-immunized mice with EAE and expanded in vitro by co-culturing with MOG peptide-pulsed splenocytes were stained with a panel of biotin-conjugated Abs (solid lines) followed by staining with FITC-conjugated avidin and analyzed by flow cytometry. Irrelevant isotype-matched biotin-conjugated Abs were used as controls (light dotted lines). b RNA extracted from MOG-specific CD4⁺ Th17 and Con A-stimulated CD4⁺ T (control) cells were analyzed by RT-PCR for assessment of expression of RORγt. c Pentamer staining assay. The tail blood samples of mice adoptively transferred with CD4⁺ Th17 cells or Con A-stimulated CD4⁺ T (control) cells were stained with PE-H-2D^b/MOGI pentamer (PE-pentamer) and FITC-anti-CD8 Ab (FITC-CD8), and then analyzed by flow cytometry. The value in each panel represents the percentage of pentamer-positive CD8⁺ T cells versus the total CD8⁺ T cell population. The value in parenthesis represents the standard deviation. d In vivo cytotoxicity assay. Sixteen hours after target cell delivery, the residual MOGI-pulsed CFSE^high and Mut1-pulsed CFSE^low target cells remaining in the spleens of the above cohorts of mice were sorted and analyzed by flow cytometry. The value in parenthesis represents the standard deviation. e Wild-type C57BL/6 mice were adoptively transferred with MOG-specific Th17 cells or OVA-specific Th17 cells (control). The clinical EAE was scored according to 0–5 scale. f Photographs of sections of spinal cords derived from mice with EAE; tissue sections were stained with Luxol fast blue along with H&E counterstaining. Control mice (a and c) and MOG-immunized mice (b and d). Magnifications, ×5 (a and b) and ×20 (c and d). Inflammatory infiltration and demyelination are shown with arrows. g Mean scores of inflammation and demyelination±SD. *p<0.01 versus cohorts of the control groups (Student’s t test). One representative experiment of three in the above experiments is shown

Fig. 6

Distinct role of CD4⁺ Th17- and Th17-stimulated CD8⁺ CTL in pathogenesis of T1D and EAE. Both CD4⁺ Th17 cells and Th17-stimulated CD8⁺ CTLs are involved in pathogenesis of T1D and EAE. However, T1D is directly mediated by Th17-stimulated CD8⁺ CTLs to destroy OVA-expressing pancreatic islets of RIP-mOVA mice via perforin-mediated cytotoxicity. On the contrary, CD4⁺ Th17 cells play a major role in pathogenesis of EAE by Th17 cytokine-mediated tissue inflammation leading to demyelination in the central nervous system