γδ T cells are essential effectors of type 1 diabetes in the nonobese diabetic mouse model

Abstract

γδ T cells, a lineage of innate-like lymphocytes, are distinguished from conventional αβ T cells in their Ag recognition, cell activation requirements, and effector functions. γδ T cells have been implicated in the pathology of several human autoimmune and inflammatory diseases and their corresponding mouse models, but their specific roles in these diseases have not been elucidated. We report that γδ TCR(+) cells, including both the CD27(-)CD44(hi) and CD27(+)CD44(lo) subsets, infiltrate islets of prediabetic NOD mice. Moreover, NOD CD27(-)CD44(hi) and CD27(+)CD44(lo) γδ T cells were preprogrammed to secrete IL-17, or IFN-γ upon activation. Adoptive transfer of type 1 diabetes (T1D) to T and B lymphocyte-deficient NOD recipients was greatly potentiated when γδ T cells, and specifically the CD27(-) γδ T cell subset, were included compared with transfer of αβ T cells alone. Ab-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection, supporting a nonredundant, pathogenic role of γδ T cells in this model. The potent contributions of CD27(-) γδ T cells and IL-17 to islet inflammation and diabetes reported in this study suggest that these mechanisms may also underlie human T1D.

Fig 1. Both αβT and γδT cells are required for efficient T cell-mediated T1D transfer to NOD.SCID recipients

Splenocytes were harvested from 12- to 14-week old NOD mice. B cells and APC, and in some cases γδTCR+ cells were depleted by magnetic bead purification. Purification of γδT only was performed by FACS sorting. Either 10⁷ splenic total T cells (containing αβT and γδT), 10⁷ splenic αβT cells alone, or 200,000 γδT alone were transferred to 4-5-week old female NOD.SCID recipients by tail vein i.v. injection. Recipients of NOD total T cells (αβT+γδT, black solid trace), all developed T1D within 100 days. In contrast, recipients of NOD αβT cells alone (αβT, dashed trace) were protected from T1D compared to recipients of both T cell subsets (p<0.0001). γδT cells alone were not able to transfer T1D in this setting (p=0.0141 compared to NOD αβT cells alone; no recipients diabetic). P values are Log-rank Mantel-Cox test comparisons of survival curves, n≥9 recipients per group).

Fig 2. CD27⁻ γδTcR⁺ cells infiltrate NOD islets during T1D pathogenesis

A) Flow cytometry-based enumeration of γδT cells in the pancreatic islets of 8- and 12-week old NOD mice. Pancreata were perfused with collagenase and the islets were prepared (Materials and Methods). Cells of interest were gated based on excluding propidium iodide (live cells), followed by forward and side scatter profile. Stains for both αβTcR and CD19 were included to exclude B cells and αβT cells from subsequent analysis. Within the CD19- αβTCR- population, an intra-islet γδTCR⁺ population was identified in both 8- and 12-week old NOD mice. Representative plots were chosen from n≥5 biological replicates. B) The frequency of γδTCR⁺ cells among non-B, non-αβT cells is shown for islet samples from 8- and 12-week old NOD mice. The frequency of intra-islet γδT cells is significantly higher among 12-week-old NOD mice, compared to 8-week old NOD mice (p<0.05, 2-tailed non-parametric t-test, n≥5 biological replicates). C) Within the γδTCR⁺ population, the expression of CD27 and CD44 was used to discriminate two subsets: the CD27⁺CD44^low (putatively IFN-γ secreting), and the CD27⁻ CD44^hi (putatively IL-17 secreting) γδT cells. FACS profiling of these subsets was performed in spleen, various lymph nodes, and islets from 8- and 12-week old NOD mice. Representative plots were chosen from n≥5 biological replicates. D) Statistical of analysis of multiple lymphoid compartments reveals that CD27⁻CD44^hi γδT cells were present at high frequency in the islets of 8-week old NOD mice, compared to all other lymphoid compartments in 8-week old NOD mice (p<0.01, 2-tailed non-parametric t-test, n≥5 biological replicates). E) In samples from 12-week old NOD mice, CD27⁻CD44^hi γδT cells were present at elevated frequencies in the spleen and islets, compared to all lymph nodes tested (p<0.01, 2-tailed non-parametric t-test, n=8 biological replicates).

Fig 3. CD27⁻ γδT cells mediate T1D transfer via an IL-17 dependent mechanism

A) Cells from NOD spleen, thymus, or pooled peripheral LN were cultured in the presence of 50ng/mL PMA and 1ug/mL Ionomycin for 5 hours. Cells were stained for surface markers (γδTCR, αβTCR, CD19, CD44, CD27, and a fixable viability dye), then permeabilized and stained with antibodies specific for IL-17 and IFN-γ. Contour plots in the upper row show production of IL-17 and IFN-γ by γδT cells. Histograms in the lower row show CD27 expression on either IL17+ γδT cells (blue trace) or IFN-γ+ γδT cells (red trace). Representative plots were chosen from n=4 biological replicates. B) 10⁷ purified splenic T cells, containing αβT and either total γδT cells (grey traces) or purified CD27⁻ γδT cells only (black traces), were transferred from 12- to 14-week old NOD donors into 4-5-week old NOD.SCID recipients. CD27⁻ γδT cells were present in the inoculum at their orthtopic frequency (see Materials and Methods). On the same day, and twice a week thereafter, NOD.SCID recipients were injected i.p. with either anti-IL-17 neutralizing antibody or a rat IgG2a isotype control antibody. Antibody injection was continued for 10 weeks post-T cell transfer and recipients were monitored for T1D. All recipients of the isotype control antibody progressed to T1D within the 100-day observation period. Recipients of the IL-17 neutralizing antibody were protected from T1D (total γδT cells p=0.0006; CD27⁻ γδT cells p<0.0001, n≥10 for each condition) compared to cohorts receiving isotype control antibody. P values represent pair-wise Log-rank Mantel-Cox tests of survival curves.

Fig 4. NOD.TcRδ-HET mice had half the frequency of γδT cells compared to wild-type NOD mice

Multicolour flow cytometry was performed to define frequencies of γδT cells in CD4⁻CD8⁻ thymocytes (thymic DN), pooled peripheral lymph nodes (LN), spleen, and small intestinal intraepithelial lymphocytes (IEL). NOD.TCRδ-HET mice displayed γδTCR⁺ cell frequencies intermediate between parental NOD and NOD.TCRδ-KO mice. Representative plots from n=3 per genotype.

Fig 5. Gene dosage at the TCRδ locus determines T1D susceptibility

A) Longitudinal assessment of T1D was performed in cohorts of NOD, NOD.TCRδ-HET and NOD.TCRδ-KO mice. In contrast to NOD females, NOD.TCRδ-KO mice were protected from diabetes (p<0.0001), NOD.TCRδ-HET mice displayed an intermediate phenotype different from both parental NOD (p=0.0296) and NOD.TCRδ-KO (p=0.0118) animals. P values represent pair-wise Log-rank Mantel-Cox tests of survival curves, n>22 per genotype. B) Insulitis severity was assessed in the pancreata of non-diabetic female NOD and NOD.TCRδ-KO mice, at the ages indicated (see Materials and Methods). NOD mice showed a progressive increase in insulitis severity from ages 80 to 240 days. In contrast, NOD.TCRδ-KO mice showed a low and constant level of insulitis throughout this time frame. Graph depicts mean and SEM (≥7 biological replicates per condition). p<0.05, 2-tailed t-test comparing NOD to NOD.TCRδ-KO within the indicated time point.