IL-17 contributes to CD4-mediated graft-versus-host disease

Abstract

CD4(+) interleukin-17 (IL-17)(+) T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4(+) donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17(-/-) T-cell recipients. However, upon transfer of murine IL-17(-/-) CD4(+) T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4(+) T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17(-/-) CD4(+) T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-gamma-secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-gamma, IL-4, and IL-6) in recipients of IL-17(-/-) CD4(+) T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.

Figure 1

Th17 cells are found in lymphoid organs after BMT. Lethally irradiated (850 cGy) BALB/c mice were reconstituted with 5 × 10⁶ WT TCD-BM and 0.5 × 10⁶ CD4⁺ T cells. One representative staining for intracellular IFN-γ and IL-17 on CD4⁺ T cells from (A) untreated B6 mice (n = 5) and donor-derived CD4⁺ T cells from the MLN from mice with GVHD (B) on day 14 (n = 8) and (C) day 21 after BMT (n = 9). The absolute number of donor-derived CD4⁺ T cells expressing IL-17, IL-17F, or IL-22 in the (D-F) spleen, (G-I) MLN, and (J-L) PLN on days 7 (syngeneic n = 10; allogeneic n = 16), 14 (syngeneic n = 10; allogeneic n = 14), and 21 (syngeneic n = 5; allogeneic n = 14) after BMT. The mean and SD of each group is shown. Data represent 2 combined experiments. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

Figure 2

Th17 cells play a role in the early stage of CD4-mediated GVHD. (A) Lethally irradiated (850 cGy) BALB/c mice were reconstituted with 5 × 10⁶ WT B6 TCD-BM alone, or with TCD-BM and 10⁶ nylon wool–passed (NWP) WT B6 T cells or IL-17^−/− T cells. Data represent 3 combined experiments: mice either received TCD-BM only (n = 15; dotted line, black square), IL-17^−/− T cells + TCD-BM (n = 30; gray line, gray triangles) or WT T cells ⁺ TCD-BM (n = 30; solid line, black squares). (B) Lethally irradiated BALB/c mice received 5 × 10⁶ WT B6 TCD-BM and 0.5 × 10⁶ CD4⁺ WT B6 T cells or IL-17^−/− T cells. Data represent 4 combined experiments: mice received either TCD-BM only (n = 30) (dotted line, black square), IL-17^−/− CD4⁺ T cells + TCD-BM (n = 35) or WT B6 CD4⁺ T cells + TCD-BM (n = 45), P < .04. (C) Lethally irradiated (1300 cGy) C3FeB6F1 mice were reconstituted with 5 × 10⁶ WT B6 TCD-BM and 2 × 10⁶ NWP T cells WT B6 T cells or IL-17^−/− T cells. Data represent 2 combined experiments: mice received TCD-BM only (n = 10; dotted line, black square), IL-17^−/− T cells ⁺ TCD-BM (n = 20; gray line, gray triangles) or WT T cells + TCD-BM (n = 20; solid line, black squares). (D) Lethally irradiated BALB/c mice were reconstituted with 5 × 10⁶ WT B6 TCD-BM (n = 20; dotted line, black square), or 5 × 10⁶ WT B6 TCD-BM + 0.5 × 10⁶ A20 cells (n = 20; gray line, gray circles), and 0.5 × 10⁶ NWP WT B6 T cells + 0.5 × 10⁶ A20 cells (n = 40; solid line, black squares) or IL-17^−/− T cells + 0.5 × 10⁶ A20 cells (n = 40; gray line, gray triangles). Data represent 4 combined experiments. Survival curves are shown.

Figure 3

Recipients of IL-17^−/− CD4⁺ T cells have a decrease in splenic Th1 cells and produce decreased levels of proinflammatory cytokines. Lethally irradiated BALB/c mice were reconstituted with 5 × 10⁶ WT TCD-BM and 0.5 × 10⁶ WT or IL-17^−/− CD4⁺ T cells. Spleens from recipient mice were harvested on days 7, 14, and 21 after BMT. (A) The absolute number of splenoctyes in recipients of CD4⁺ WT (gray) versus CD4⁺IL-17^−/− (black). The mean of each group is shown; data represent 3 combined experiments on days 7 (n = 48) and 14 (n = 28) and one experiment on day 21 (n = 9). (B) The percentage of donor-derived CD4⁺CD25⁺Foxp3⁺ cells (black) and donor-derived CD4⁺Tbet⁺ cells (gray) in the spleen was determined using flow cytometry. The mean of each group is shown; data represent one of 3 experiments (days 7 and 14), day 7 (n = 6), day 14 (n = 9), or day 21 (n = 9; 1 experiment on day 21). (C) The number of IFN-γ⁺ cells on day 7 was determined by intracellular cytokine staining in recipients of CD4⁺ WT (gray) and CD4⁺ IL-17^−/− (black) T cells. Two combined experiments (n = 12). (D) Splenocytes from recipients of WT B6 CD4⁺ T cells (gray) and IL-17^−/− CD4⁺ T cells (black) on day 7 after BMT were stimulated in vitro for 4 hours, the supernatant was collected, and a CBA or an ELISA was performed to determine cytokine levels. Graphs represent the amount of cytokine secreted per 10⁴ cells stimulated. The mean of each group is shown (n = 8); data represent one experiment of 2. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

Figure 4

Recipients of IL-17^−/− CD4⁺ T cells have decreased frequency of CD4⁺ Tbet⁺ cells in the MLN and decreased IFN-γ levels in the serum on day 7 after BMT. Lethally irradiated BALB/c mice were reconstituted with 5 × 10⁶ WT TCD-BM and 0.5 × 10⁶ WT or IL-17^−/− CD4⁺ T cells. MLN from recipient mice were harvested on days 7, 14, and 21 after BMT. (A) The absolute number of MLN from recipients of CD4⁺ WT (gray) and CD4⁺IL-17^−/− (black) T cells. The mean of each group is shown; data represent 3 combined experiments on days 7 (n = 48) and 14 (n = 28), and 1 experiment on day 21 (n = 9). (B) The percentage of donor-derived CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells (black) and donor-derived CD4⁺Tbet⁺ cells (gray) in the MLN was determined using flow cytometry. The mean of each group is shown, and data represent one of 3 experiments (days 7 and 14), day 7 (n = 6), day 14 (n = 9) or a single experiment (day 21, n = 9). (C) MLN from recipients of WT B6 CD4⁺ T cells (gray) and IL-17^−/− CD4⁺ T cells (black) on day 7 after BMT were stimulated in vitro for 4 hours, the supernatant was collected, and a CBA or an ELISA was performed to determine cytokine levels. Graphs represent the amount of cytokine secreted per 10⁴ cells stimulated. The mean of each group is shown (n = 8), and data represent one experiment of 2. (D) Cytokine levels in the serum on day 7 after BMT were measured by CBA and ELISA. Shown is the mean for each group (n = 7), one representative experiment of 3. **P ≤ .01.