Nonobese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D), progression of which is similar to that in humans, and therefore are widely used as a model for understanding the immunological basis of this disease. The incidence of T1D in NOD mice is influenced by the degree of cleanliness of the mouse colony and the gut microflora. In this report, we show that the T1D incidence and rate of disease progression are profoundly influenced by the pH of drinking water, which also affects the composition and diversity of commensal bacteria in the gut. Female NOD mice that were maintained on acidic pH water (AW) developed insulitis and hyperglycemia rapidly compared with those on neutral pH water (NW). Interestingly, forced dysbiosis by segmented filamentous bacteria (SFB)-positive fecal transfer significantly suppressed the insulitis and T1D incidence in mice that were on AW but not in those on NW. The 16S rDNA–targeted pyrosequencing revealed a significant change in the composition and diversity of gut flora when the pH of drinking water was altered. Importantly, autoantigen-specific T-cell frequencies in the periphery and proinflammatory cytokine response in the intestinal mucosa are significantly higher in AW-recipient mice compared with their NW counterparts. These observations suggest that pH of drinking water affects the composition of gut microflora, leading to an altered autoimmune response and T1D incidence in NOD mice.

Recently, a new developmental pathway for CD4 T cells that is mediated by major histocompatibility complex class II–positive thymocytes was identified (Choi, E.Y., K.C. Jung, H.J. Park, D.H. Chung, J.S. Song, S.D. Yang, E. Simpson, and S.H. Park. 2005. Immunity. 23:387–396; Li, W., M.G. Kim, T.S. Gourley, B.P. McCarthy, D.B. Sant'angelo, and C.H. Chang. 2005. Immunity. 23:375–386). We demonstrate that thymocyte-selected CD4 (T-CD4) T cells can rapidly produce interferon γ and interleukin (IL) 4 upon in vivo and in vitro T cell receptor stimulation. These T-CD4 T cells appear to be effector cells producing both T helper type 1 (Th1) and Th2 cytokines, and they maintain a potential to produce Th2 cytokines under Th1-skewing conditions in a signal transducer and activator of transcription 6–independent manner. The IL-4 mRNA level is high in CD4 single-positive thymocytes if they are selected on thymocytes, which is at least partly caused by enhanced histone acetylation of the IL-4 locus. However, mice that can generate T-CD4 T cells showed attenuated immune responses in an allergen-induced airway inflammation model, suggesting a protective role for T-CD4 T cells during an airway challenge. Our results imply that this thymic selection pathway plays an important role in determining the effector function of the resulting CD4 cells and in regulating immune response.

Chronic graft-versus-host disease (cGVHD) is characterized as autoimmune-like fibrosis and antibody production mediated by pathogenic T cells and B cells. MicroRNA-17-92 (miR-17-92) influences the survival, differentiation, and function of lymphocytes in cancer, infections, and autoimmunity. To determine whether miR-17-92 regulates T- and B-cell responses in cGVHD, we generated mice conditionally deficient for miR-17-92 in T cells, B cells, or both. Using murine models of allogeneic bone marrow transplantation, we demonstrate that expression of miR-17-92 in donor T and B cells is essential for the induction of both scleroderma and bronchiolitis obliterans in cGVHD. Mechanistically, miR-17-92 expressed in T cells not only enhances the differentiation of pathogenic T helper 1 (Th1) and Th17 cells, but also promotes the generation of follicular Th cells, germinal center (GC) B cells, and plasma cells. In B cells, miR-17-92 expression is required for autoantibody production and immunoglobulin G deposition in the skin. Furthermore, we evaluated a translational approach using antagomirs specific for either miR-17 or miR-19, key members in miR-17-92 cluster. In a lupus-like cGVHD model, systemic administration of anti-miR-17, but not anti-miR-19, alleviates clinical manifestations and proteinuria incidence in recipients through inhibiting donor lymphocyte expansion, B-cell activation, and GC responses. Blockade of miR-17 also ameliorates skin damage by reducing Th17 differentiation in a scleroderma-cGVHD model. Taken together, our work reveals that miR-17-92 is required for T-cell and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the prevention of cGVHD.

MHC class II-expressing double-positive thymocytes induce progression of CD4(+) T cell development as efficiently as cortical thymic epithelial cells do. Because double-positive thymocytes expressing CD1d select natural killer T (NKT) cells, we investigated whether thymocyte-selected CD4(+) (T-CD4) T cells require the same signaling components as NKT cells. Using bone-marrow chimeras, we found that the signaling molecules SAP, Fyn, and PKCtheta were essential for T-CD4 T cell generation, whereas mutations in the Ly108 receptor, interleukin-15 receptor alpha, or the transcription factor T-bet had a marginal effect. Furthermore, SAP was critical for IL-4 production by T-CD4 T cells, but the PKCtheta deficiency did not alter the ability of T-CD4 T cells to produce cytokines. T-bet was necessary to produce the maximum amount of IFN-gamma for CD4(+) T cells regardless of the selection pathway. Thus, in contrast to epithelial cell-selected CD4(+) T cells, the two distinct lineages of T cells selected by thymocytes--i.e., T-CD4 and NKT cells--both utilize the SAP-Fyn-PKCtheta pathway for their development and function.

Adoptive natural regulatory T cell (nTreg) therapy has improved the outcome for patients suffering from graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (Allo-HCT). However, fear of broad immune suppression and subsequent dampening of beneficial graft-versus-leukemia (GVL) responses remains a challenge. To address this concern, we generated alloreactive induced Tregs (iTregs) from resting CD4(+) or CD8(+) T cells and tested their ability to suppress GVH and maintain GVL responses. We utilized major mismatched and haploidentical murine models of HCT with host-derived lymphoma or leukemia cell lines to evaluate GVH and GVL responses simultaneously. Alloreactive CD4(+) iTregs were effective in preventing GVHD, but abrogated the GVL effect against aggressive leukemia. Alloreactive CD8(+) iTregs moderately attenuated GVHD while sparing the GVL effect. Hence, we reasoned that using a combination of CD4(+) and CD8(+) iTregs could achieve the optimal goal of Allo-HCT. Indeed, the combinational therapy was superior to CD4(+) or CD8(+) iTreg singular therapy in GVHD control; importantly, the combinational therapy maintained GVL responses. Cellular analysis uncovered potent suppression of both CD4(+) and CD8(+) effector T cells by the combinational therapy that resulted in effective prevention of GVHD, which could not be achieved by either singular therapy. Gene expression profiles revealed alloreactive CD8(+) iTregs possess elevated expression of multiple cytolytic molecules compared to CD4(+) iTregs, which likely contributes to GVL preservation. Our study uncovers unique differences between alloreactive CD4(+) and CD8(+) iTregs that can be harnessed to create an optimal iTreg therapy for GVHD prevention with maintained GVL responses.