Figure 5.

Polyclonal repertoire derived CD4⁺Foxp3⁺ Tregs inhibit diabetogenic T-cells in NSG recipients.

A) Diabetes incidence in NSG mice injected i.v. with 2.5×10⁶ monoclonal NOD-AI4 splenocytes either alone or admixed with total or CD4 T-cell depleted NOD splenocytes. Mice receiving CD4 T-cell deficient splenocytes were injected i.p. with 200μg of the depleting CD4 specific GK1.5 antibody once a week. The number of CD4 depleted NOD splenocytes was adjusted to inject 2×10⁵ CD8 T-cells an equivalent amount to that present in 5×10⁶ total NOD splenocytes. B) Diabetes incidence study comparing NOD.Foxp3-eGFP reporter mice with NOD/ShiLtDvs. Log-rank (Mantel-Cox) Test, ns p>0.05. C) Spleen cells from NOD.Foxp3-eGFP reporter mice were intra-cellularly stained with Allophycocyanine (AP) labelled anti-Foxp3 (FJK-16s) in order to test if Foxp3 and the fluorescent eGFP reporter were co-expressed in the same cells. Representative dot-plot graph gated on total CD4⁺ T-cells show that only Foxp3⁺ T-cells co-express eGFP (mean±SEM, n=5). D) Newly differentiated CD4⁺Foxp3⁺ Treg cells arise in NSG and NOD-scid recipients originally engrafted with reporter-cell depleted NOD.Foxp3-eGFP splenocytes. NOD.Foxp3-eGFP splenocytes depleted of reporter positive cells by flow cytometric sorting were injected i.v. into NSG and NOD.scid mice. The number of reporter cell depleted NOD.Foxp3-eGFP splenocytes was adjusted to inject 2×10⁵ CD8 T-cells. A group of NSG and NOD-scid mice receiving reporter-cell deficient NOD.Foxp3-eGFP splenocytes were also injected i.p. with 250 μg of the depleting CD25 specific PC61.5 antibody once a week to maintain Treg ablation (solid lines) and another group of each recipient type was kept without antibody treatment (dashed lines). At the indicated time point, PBL were analyzed by flow cytometry for proportions of eGFP reporter expressing Tregs. Data represent the mean ± SEM (n=5, each group). By 2 weeks post-transfer a significant increase in the proportion of CD4⁺Foxp3⁺ Tregs was observed in both types of recipients not receiving anti-CD25 antibody treatments. There were no significant differences between recipient strains in the anti-CD25 treated or untreated groups. ns: p>0.05, *** p<0.0001, two-way ANOVA and Bonferroni post-test. E) NSG mice were injected i.v. with 2.5×10⁶ monoclonal NOD-AI4 splenocytes either alone or admixed with total or reporter cell depleted NOD.Foxp3-eGFP splenocytes. The number of intact or reporter cell depleted NOD.Foxp3-eGFP splenocytes was adjusted to inject 2×10⁵ CD8 T-cells. Mice receiving reporter cell deficient NOD.Foxp3-eGFP splenocytes were also injected i.p. with 250μg of the depleting CD25 specific PC61.5 antibody once a week to maintain Treg ablation. F) NSG mice were injected i.v. with total or reporter-cell depleted NOD.Foxp3-eGFP splenocytes. Numbers of the two NOD.Foxp3-eGFP splenocyte inoculums were adjusted to inject 2×10⁵ CD8 T-cells. Mice receiving reporter-cell deficient NOD.Foxp3-eGFP splenocytes were also injected i.p. with 250μg of the depleting CD25 specific PC61.5 antibody once a week to maintain Treg ablation. The incidence of T1D in various groups was compared by Log-rank (Mantel-Cox) Test, *** p<0.0001.