GM-CSF Therapy Expands Regulatory T Cells and Protects Against Chronic Graft Versus Host Disease

Regulatory T cells (Tregs) possess the ability to suppress chronic graft-versus-host disease (cGVHD). Hence, the in vivo expansion of Tregs can be used as therapy against cGvHD. In addition to IL-2, Tregs require TCR and costimulatory signals from antigen presenting cells such as dendritic cells (DCs) for their optimal proliferation. Both fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony stimulation factor (GM-CSF) induce the development of DCs and promote the proliferation of Tregs in a DC-dependent manner. GM-CSF preferentially increases CD11c+CD8a- DCs, whereas FLT3L more equally supports the development of many DC subsets. However, it is unknown whether GM-CSF-mediated CD11c+CD8a- DC expansion leads to the proliferation of Tregs and contributes to the inhibition of alloimmune responses against host antigens. To test whether the injection of GM-CSF augments Tregs and ameliorates cGVHD, we used a MHC-matched mouse cGVHD model (B10.D2 → Balb/c). Balb/c mice were lethally irradiated (850 cGy) and transplanted with 8 × 106 T cell-depleted bone marrow cells and 3.5 × 106 CD4+ T cells from either syngeneic or B10.D2 mice. Host mice were treated with vehicle or GM-CSF (in the form of immune complexes; GM-CSF ICs) for 3 days (days 17-19) and monitored for skin GVHD score and mortality. We used GM-CSF ICs because the injection of GM-CSF ICs but not GM-CSF itself increased splenic CD11c+CD8a- DCs and Tregs. The administration of GM-CSF to allogeneic host mice significantly protected against GVHD-induced skin diseases (p<0.001) (Fig 1). Similar results were obtained when GM-CSF ICs were administrated at a later stage (days 27-29). Although CD11c+CD8- DCs were decreased in mice transplanted from the allogeneic donor compared with mice transplanted from the syngeneic donor, the administration of GM-CSF increased the CD11c+CD8a-/CD11c+CD8a+ DC ratio. However, the expansion of macrophages was not observed in mice administered GM-CSF. Expectedly, the administration of GM-CSF increased Tregs in the peripheral blood and the peripheral lymph nodes (PLNs) (P<0.05) (Fig 2). We investigated the production of proinflammatory cytokines (IFN-g, IL-17) of CD4+ T cells in the spleen, PLNs, and the skin by intracellular cytokine staining, as these cytokines are important for cGVHD pathogenesis in this model. The proportion of IFN-g+CD4+ T cells in the spleen and PLNs was slightly but not significantly decreased in GM-CSF-administered mice. The proportion of IL-17+CD4+ T cells in the skin was decreased in GM-CSF-administered mice compared to vehicle-administered mice. We next investigated the production of IL-2 and IL-10, as these cytokines are associated with survival and function of Tregs. Skin infiltrating Tregs were not increased in GM-CSF-administered mice compared to vehicle-administered mice; however, the proportion of IL-10+ Tregs was increased in GM-CSF-administered mice. The proportion of IL-2+CD4+ T cells was comparable in the allogeneic host with or without GM-CSF, indicating that GM-CSF-induced Treg expansion did not result from an increase in IL-2 production by CD4+ T cells. Together, these data suggest that GM-CSF induces the proliferation of Tregs by expanding CD11c+CD8a- DCs, and can regulate alloimmune responses in a cGVHD mouse model. Our findings indicate the potential of GM-CSF as a therapeutic strategy to ameliorate cGVHD. Disclosures No relevant conflicts of interest to declare.

Prevention of GVHD while sparing GVL effect by targeting Th1 and Th17 transcription factor T-bet and RORγt in mice

Allogeneic hematopoietic cell transplantation (HCT) is effective therapy for hematologic malignancies through T cell–mediated GVL effects. However, HCT benefits are frequently offset by the destructive GVHD, which is also induced by donor T cells. Naive Th can differentiate into Th1 and Th17 subsets and both can mediate GVHD after adoptive transfer into an allogeneic host. Here we tested the hypothesis that blockade of Th1 and Th17 differentiation is required to prevent GVHD in mice. T cells with combined targeted disruption of T-bet and RORγt have defective differentiation toward Th1 and Th17 and skewed differentiation toward Th2 and regulatory phenotypes, and caused ameliorated GVHD in a major MHC-mismatched model of HCT. GVL effects mediated by granzyme-positive CD8 T cells were largely preserved despite T-bet and RORγt deficiency. These data indicate that GVHD can be prevented by targeting Th1 and Th17 transcription factors without offsetting GVL activity.

CELL INTERACTIONS BETWEEN HISTOINCOMPATIBLE T AND B LYMPHOCYTES

The adoptive transfer of 2,4-dinitrophenyl(DNP)-keyhole limpet hemocyanin(KLH)-primed lymphocytes into a heavily irradiated allogeneic recipient permits the development of a secondary anti-DNP antibody response to DNP-bovine gamma globulin(BGG) whether or not the irradiated allogeneic host possesses BGG-specific helper T cells. This "allogeneic effect" has been demonstrated to result from the capacity of residual, apparently radioresistant, T cells in the irradiated host to exert an active effect on the transferred histoincompatible B lymphocytes. This conclusion derives from two corroborative experiments. In the first, an allogeneic effect was shown to occur on DNP-primed F1 spleen cells that had been adoptively transferred to irradiated parental recipients; the second experiment demonstrated the development of an allogeneic effect on anti-θ-treated, DNP-specific donor cells transferred to irradiated allogeneic hosts. These results emphasize the extreme caution required in designing and interpreting experiments that may involve adoptive cell transfers into histoincompatible hosts, and illustrate why such models are unsuitable for investigation of the question of physiologic cooperative interactions between T and B lymphocytes. Suitable approaches are described in the accompanying paper.