Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft-versus-host disease

Abstract

In acute graft-versus-host disease (GVHD), naive donor CD4(+) T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (Th1, Th2, and Th17 cells), but the role of Th subsets in GVHD pathogenesis is incompletely characterized. Here we report that, in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, WT donor CD4(+) T cells predominantly differentiated into Th1 cells and preferentially mediated GVHD tissue damage in gut and liver. However, absence of interferon-gamma (IFN-gamma) in CD4(+) T cells resulted in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; absence of both IL-4 and IFN-gamma resulted in augmented Th17 differentiation and preferential, although not exclusive, tissue damage in skin; and absence of both IFN-gamma and IL-17 led to further augmentation of Th2 differentiation and idiopathic pneumonia. The tissue-specific GVHD mediated by Th1, Th2, and Th17 cells was in part associated with their tissue-specific migration mediated by differential expression of chemokine receptors. Furthermore, lack of tissue expression of the IFN-gamma-inducible B7-H1 played a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate donor CD4(+) T cells can reciprocally differentiate into Th1, Th2, and Th17 cells that mediate organ-specific GVHD.

Figure 1

IFN-γ^−/− donor CD4⁺ T cells induced preferential tissue damage in lung and skin in association with augmented differentiation of Th17 and Th2 cells. (A-C) Percent survival, percentage of mice with diarrhea, and body-weight change after HCT. There were 12 mice in each group, combined from 3 replicate experiments. (D-E) Hematoxylin and eosin (H&E) staining of colon, liver, lung, and skin sections of recipients 20 to 25 days after HCT and mean ± SE of histopathology scores (n = 12). (F) Mean ± SE of T-bet, Gata-3, and RORγt expression in sorted splenic CD4⁺ T cells (purity > 90%) from recipients given IFN-γ^−/− donor cells 5 days after HCT (n = 4). Relative gene expression levels were normalized within each sample to GAPDH and were presented relative to the expression of WT control. Data are representative of 2 replicate experiments. (G) Intracellular cytokine profiles of splenic CD4⁺ T cells 7 days after HCT. Gated CD4⁺ T cells are shown in CD4 versus cytokines. A representative of 4 replicate experiments is shown. (H) Seven days after HCT, sorted H-2b⁺CD4⁺ T cells from the spleen of recipients given WT or IFN-γ^−/− donor cells were restimulated with plate-bound anti-CD3/CD28. Mean ± SE of supernatant cytokines 24 hours after culture. These were 4 samples in each group, combined from 2 replicate experiments.

Figure 2

IFN-γ^−/−IL-17^−/− donor CD4⁺ T cells induced little tissue damage in gut, liver, and skin, but severe damage in lung. (A-C) Percent survival, percentage of mice with diarrhea, and body-weight change after HCT. There were 12 mice in each group, combined from 3 replicate experiments. (D-E) H&E staining of colon, liver, lung, and skin tissue sections of recipients 20 to 25 days after HCT and mean ± SE of histopathology scores (n = 12).

Figure 3

IFN-γ^−/−IL-17^−/− donor CD4⁺ T cells predominantly differentiated into Th2 and induced lung damage. (A) Intracellular cytokine profiles of splenic CD4⁺ T cells 7 days after HCT. Gated CD4⁺ T cells are shown in CD4 versus cytokines. A representative of 3 replicated experiments is shown. (B) Mean ± SE of serum IgE (n = 8) combined from 2 replicate experiments. (C) Mean ± SE of yield of eosinophil (CCR3⁺Gr-1⁺) in lungs 10 days after HCT (n = 4). (D) Intracellular cytokine profiles of CD4⁺ T cells among lung mononuclear cells 10 days after HCT. Gated CD4⁺ T cells were shown as IL-4/IL-5/IL-13 versus TNF-α or IL-10. Percentage of TNF-α⁺IL-4/IL-5/IL-13⁺ or IL-10⁺IL-4/IL-5/IL-13⁺ cells were calculated among total CD4⁺ T cells. A representative of 3 replicate experiments is shown. (E-F) Clinical scores and percent survival of recipients treated with TNFR-IgG or control IgG are shown. There were 12 mice in each group, combined from 3 replicate experiments. (G-H) H&E staining of lung tissue sections of recipients 15 days after HCT and mean ± SE of histopathology scores (n = 6). (I-J) BALB/c recipients given IFN-γ^−/−IL-17^−/− donor cells were treated with anti–IL-4 or rat IgG. H&E staining of lung tissue sections of recipients 15 days after HCT and mean ± SE of histopathology scores (n = 6). (K-L) WT or IL-4Rα^−/− BALB/c recipients were transplanted with IFN-γ^−/−IL-17^−/− donor cells. H&E staining of lung tissue sections of recipients 15 days after HCT and mean ± SE of histopathology scores (n = 6).

Figure 4

Absence of IL-4 and IFN-γ led to augmented Th17 differentiation and exacerbated damage in skin but not in lung. BALB/c recipients given IFN-γ^−/− donor cells were treated with rat-IgG or anti–IL-4. (A) Intracellular cytokine profiles of splenic CD4⁺ T cells 13 days after HCT. Gated CD4⁺ T cells are shown in CD4 versus cytokines. Representative of 4 replicate experiments is shown. (B-C) H&E staining of lung- and skin-tissue sections of recipients 15 days after HCT and mean ± SE of histopathology scores (n = 8).

Figure 5

Tissue-specific GVHD caused by Th1, Th2, and Th17 cells was associated with tissue-specific T-cell migration. (A) Yield of H-2b⁺CD4⁺ T cells in spleen or tissues of recipients given WT or IFN-γ^−/− donor cells 13 days after HCT. Mean ± SE is shown (n = 8), combined from 3 replicated experiments. (B-C) Five days after HCT, splenocytes from recipients were stained for H-2^b, CD4, and chemokine receptors. Gated H-2b⁺CD4⁺ T cells are shown in CD4 versus chemokine receptors. Representative of 4 replicate experiments is shown. (D-E) Mean ± SE of chemokine expression levels by GVHD target tissues 5 days after HCT (n = 4). Relative gene expression levels were normalized within each sample to housekeeping gene GAPDH. Data are a representative of 2 replicated experiments.

Figure 6

Lack of host lung tissue expression of IFN-γ–inducible B7-H1 contributed to Th2-mediated idiopathic pneumonia. (A) Intracellular cytokine profiles of splenic CD4⁺ T cells 10 days after HCT. Gated CD4⁺ T cells are shown in CD4 versus cytokines. Representative of 4 replicate experiments is shown. (B-C) Percent survival and body-weight change of recipients after HCT. There were 12 mice in each group, combined from 3 replicate experiments. (D) Mean ± SE of histopathology scores (n = 6). (E) Ten days after HCT, cells from lungs of recipients were stained for CD45, CD11c, and B7-H1 or isotype control. Gated CD11c⁺ or CD45⁻ cells were shown in histogram for B7-H1 or isotype control. Representative of 3 replicate experiments is shown. (F) Immunohistochemical staining of B7-H1 in lung tissues harvested 6 days after HCT. One representative picture of 3 replicate experiments is shown. (G-H) IFN-γ^−/− or IFN-γR^−/− donor cells were transplanted into B7-H1^−/−, or B7-H1^−/− chimera recipients. Percent survival curves are shown. There were 6 to 8 mice in each group, combined from 2 replicate experiments. (I) IFN-γR^−/− donor cells were transplanted into WT, B7-H1^−/−, or B7-H1^−/− chimera recipients. Mean ± SE of histopathology scores is shown (n = 6). (J) IFN-γ^−/− donor cells were transplanted into WT or B7-H1^−/− recipients with or without injection of recombinant IFN-γ. Mean ± SE of histopathology scores is shown (n = 6).