Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets

Abstract

STAT4 and STAT6 are transcription factors that play crucial roles in responding to IL-12 and IL-4, respectively. STAT4 gene knockout (STAT4(-/-)) mice have markedly reduced Th1 responses and enhanced Th2 responses. STAT6(-/-) mice show the inverse phenotype. We compared the ability of bone marrow transplantation (BMT) with the inclusion of spleen cells from STAT6(-/-), STAT4(-/-), and wild-type (WT) mice to produce graft-versus-host disease (GVHD) in lethally irradiated MHC-mismatched recipients. Acute GVHD mortality was more rapid when induced by cells from STAT6(-/-) mice than when induced by STAT4(-/-) cells. However, cells from STAT4(-/-) and STAT6(-/-) donors both induced delayed GVHD mortality compared with WT controls, or compared with combined STAT4(-/-) and STAT6(-/-) cells, indicating a contribution of both Th1 cells and Th2 cells to acute GVHD. Recipients of STAT6(-/-) BMT showed evidence of acute GVHD with severe diarrhea and marked weight loss. Recipients of STAT4(-/-) BMT showed signs of GVHD with only initial transient weight loss and later development of severe skin GVHD. Histopathology showed that Th2 responses were required for the induction of both hepatic and severe skin GVHD. In contrast, both Th1 cells and Th2 cells were capable of causing intestinal pathology of GVHD. Our studies demonstrate an additive role for Th1 and Th2 cells in producing acute GVHD, and suggest a cytokine-directed approach to treating end-organ manifestations of GVHD.

Figure 1

Both Th1 cells and Th2 cells contribute to GVHD mortality. Lethally irradiated B6 mice received 5 × 10⁶ T-cell–depleted (TCD) B6 BMC and either syngeneic spleen cells (SYN) (n = 18), BALB/c WT BMC and spleen cells (WT) (n = 38), STAT4^–/– (Th2) BMC and spleen cells (n = 31), or STAT6^–/– (Th1) BMC and spleen cells (n = 30). Each BALB/c (WT, STAT4^–/–, or STAT6^–/–) inoculum contained 13 × 10⁶ spleen cells plus 10 × 10⁶ BMC. Pooled data from 6 experiments, involving a total of 117 animals, are shown.

Figure 2

The addition of STAT4^–/– cells to WT BMC plus spleen cell inocula does not protect from GVHD. Lethally irradiated B6 mice received TCD B6 BMC and either syngeneic spleen cells (n = 3), BALB/c WT BMC and spleen cells (WT) (n = 6), STAT4^–/– (Th2) BMC and spleen cells (n = 8), or WT BMC and spleen cells plus STAT4^–/– BMC and spleen cells (STAT4^–/– + WT) (n = 3). Each inoculum contained 13 × 10⁶ BALB/c spleen cells plus 10 × 10⁶ BALB/c BMC (WT-induced control GVHD); 13 × 10⁶ BALB/c STAT4^–/– spleen cells plus 10 × 10⁶ BALB/c STAT4^–/– BMC (STAT4^–/–-induced GVHD); or 6.5 × 10⁶ BALB/c WT spleen cells plus 6.5 × 10⁶ BALB/c STAT4^–/– spleen cells plus 5 × 10⁶ BALB/c WT BMC plus 5 × 10⁶ STAT4^–/– BMC (STAT4^–/– + WT cells). Results of one of two similar experiments are shown.

Figure 3

Weight loss and mortality for WT-, Th1-, and Th2-mediated GVHD in a single representative experiment. GVHD mortality is shown in the upper panel, and GVHD-induced weight loss is shown in the lower panel. Lethally irradiated B6 mice received 5 × 10⁶ TCD B6 BMC and either syngeneic spleen cells (n = 3), BALB/c WT BMC and spleen cells (WT) (n = 6), STAT4^–/– (Th2) BMC and spleen cells (n = 8), or STAT6^–/– (Th1) BMC and spleen cells (n = 8). Each BALB/c (WT, STAT4^–/–, or STAT6^–/–) inoculum contained 13 × 10⁶ spleen cells plus 10 × 10⁶ BMC.

Figure 4

Histological evidence of GVHD in the liver, skin, and large intestine. Liver, skin, and large intestine sections were obtained from lethally irradiated B6 recipients reconstituted with syngeneic BMT (a, e, i), WT BMT (b, f, j), STAT6^–/– BMT (c, g, k), or STAT4^–/– BMT (d, h, l). Lethally irradiated B6 mice received 5 × 10⁶ TCD B6 BMC and either syngeneic spleen cells, BALB/c WT BMC and spleen cells (WT), STAT4^–/– (Th2) BMC and spleen cells, or STAT6^–/– (Th1) BMC and spleen cells. Each BALB/c (WT, STAT4^–/–, or STAT6^–/–) inoculum contained 13 × 10⁶ spleen cells plus 10 × 10⁶ BMC. These samples, with the exception of those from recipients of syngeneic BMT, were harvested from animals that died during the third week after BMT from two experiments in which the severity of GVHD was similar. Animals that died at later time points showed similar pathological changes. Syngeneic BMT recipients were sacrificed 200 days after BMT. Photographs were taken through a ×50 objective lens.

Figure 5

Serum IgE (a) and IgG₁ (b) levels in GVHD induced by WT, STAT6^–/–, and STAT4^–/– cells. C57BL/6 animals were transplanted with syngeneic, WT BALB/c, STAT4^–/–, or STAT6^–/– cells. Lethally irradiated B6 mice received 5 × 10⁶ TCD B6 BMC and either syngeneic spleen cells, BALB/c WT BMC and spleen cells (WT), STAT4^–/– (Th2) BMC and spleen cells, or STAT6^–/– (Th1) BMC and spleen cells. Each BALB/c (WT, STAT4^–/–, or STAT6^–/–) inoculum contained 13 × 10⁶ spleen cells plus 10 × 10⁶ BMC. For syngeneic BMT and for WT- and STAT6^–/–-induced GVHD, animals were sacrificed at 5 months after BMT. Recipients of STAT4^–/– cells were bled and sacrificed at 2 weeks, at 3 months, and at 5 months after BMT. Sera were collected and Ig levels were measured by ELISA. For each time point for each group, 2–4 animals were analyzed. MSTs in days for this experiment were 26 for recipients of WT cells, 54 for recipients of STAT4^–/– cells, and 35 for recipients of STAT6^–/– cells. Concentrations of IgE and IgG₁ in sera are expressed as mean ± SEM.

Figure 6

Cytokine profile of splenocytes from mice with WT- or STAT4^–/–-induced GVHD. Lethally irradiated C57BL/6 host mice transplanted with 5 × 10⁶ TCD B6 BMC and either 13 × 10⁶ WT spleen cells plus 10 × 10⁶ WT BMC (n = 2) or 13 × 10⁶ STAT4^–/– spleen cells plus 10 × 10⁶ STAT4^–/– BMC (n = 4) were sacrificed at 2 weeks after BMT, and splenocyte suspensions were prepared. Their donor-derived phenotype was demonstrated by FACS^®. The cells were cultured with irradiated splenocytes from C57BL/6 mice. Supernatants were harvested 72 hours after initiation of MLC, and cytokine levels were determined by ELISA. MSTs for this experiment were 26 days for recipients of WT cells and 54 days for recipients of STAT4^–/– cells. Concentrations of IFN-γ, IL-2, IL-4, and IL-10 in cell culture supernatants are expressed as mean ± SEM (pg/mL). ^AP < 0.01 vs. WT group.