Allogeneic hematopoietic cell transplantation (HCT) offers great promise for the treatment of hematologic malignancies. However, HCT benefits are frequently offset by graft-versus-host disease (GVHD). Donor T cells can differentiate into Th1 or Th17 subset that contribute to GVHD. The T-box transcription factor T-bet is important for promoting the differentiation of naïve CD4+T cells into Th1 phenotype, while simultaneously inhibiting Th2 and Th17 lineage commitment. Published data indicate that donor T cells deficient for IFN-γ induce exacerbated GVHD. In contrast, our recent study showed that T cells deficient for T-bet were impaired in the induction of GVHD. Given T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ, the underlining mechanisms accounted for the distinct outcomes caused by T-bet- versus IFN-γ-deficient donor T cells are not clear.
We evaluated the roles of T-bet and IFN-γ in acute GVHD induced by naïve CD4+ T cells or polarized Th17 cells using murine allogeneic bone marrow transplantation (allo-BMT) model. WT, T-bet knockout (KO) and IFN-γ KO mice on C57BL/6 (B6) background were used as donors, and lethally irradiated BALB/c mice were used as recipients. Pathologic analysis and serum cytokine detection were done 14 days after adoptive transfer of WT, T-bet–/–, and IFN-γ–/– CD4+ T cells. Using microarray technology, gene expression profile on donor T cells was analyzed 7 days after adoptive transfer by sorting donor-derived CD4+ T cells from the recipients of WT, T-bet–/– or IFN-γ–/– CD4+ T cells.
We compared the ability of WT, T-bet–/–, and IFN-γ–/– CD4 T cells in the induction of acute GVHD. In the comparison with WT cells, IFN-γ–/– CD4 T cells caused similar or even more severe GVHD as expected. In sharp contrast, T-bet–/– CD4 T cells induced much ameliorated GVHD, as significantly higher survival and less body weight loss were observed in the recipients of T-bet–/–T cells. Pathology study on GVHD target organs showed that recipients of T-bet–/– donor T cells had markedly reduced T cell infiltration and tissue damage in liver, gut, and skin, when compared with those of WT or IFN-γ–/– T cells. Reduced GVHD in the recipients of T-bet–/– T cells was consistent with significantly lower levels of pathogenic cytokines IFN-γ, TNF-α, and IL-2 but higher IL-10 (anti-inflammatory), IL-6 (Th17 related) and IL-4 (Th2 related) in serum as compared with those in the recipients of WT T cells. Mechanistic studies in vitro revealed that T-bet–/– CD4 T cells expressed significantly lower levels of IFN-γ, CXCR3 (Th1 specific chemokine receptor) and CD122 (T cell activation marker), but higher levels of IL-17 (Th17 cytokine) and CCR6 (Th17 specific chemokine receptor) compared with WT CD4 T cells, indicating that T-bet–/– T cells impaired in differentiating into Th1 cells and instead into Th17 cells. Given Th17 subset only is capable of causing GVHD and T-bet–/– T cells are prone to Th17-differentiation, we assessed the role of T-bet or IFN-γ in the development of GVHD by comparing the pathogenicity of in vitro polarized WT, T-bet–/– and IFN-γ–/– Th17 cells. While IFN-γ–/– Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, T-bet–/– Th17 cells had reduced pathogenicity, and caused ameliorated GVHD. Furthermore, microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet−/− not IFN-γ–/–T cells in the induction of GVHD.
We conclude that T-bet is required for Th1 differentiation and optimal function of Th17 cells, and it can also control T cell infiltration into GVHD target organs by regulating chemokines and their receptors. Thus, failure in Th1 generation, migration and reduced activity of polarized Th17 cells are likely accounted for impaired ability of T-bet−/− CD4 T cells in the induction of acute GVHD. The current study suggests that targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD after allogeneic HCT in clinic.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.