Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL

Abstract

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.

Figure 1

IL-21R is up-regulated on donor T cells early after BMT but is not required for efficient alloactivation. (A) WT B6 T cells were cultured in vitro for 24 hours in the presence or absence of PMA and ionomycin, then stained for FACS analysis of IL-21R levels. (B) WT B6 BM-TCD and T cells (1 × 10⁶) were transplanted into lethally irradiated BALB/c recipients, which were killed on day 4 or day 7 after BMT. Splenocytes were harvested and stained for FACS analysis of donor T-cell IL-21R levels. Data are combined from 2 independent transplantations per time point, with 8-10 total recipients per group. **P < .01 and ***P < .001.

Figure 2

IL-21R KO T cells cause decreased GVHD and lead to increased regulatory T cells. (A) Lethally irradiated BALB/c mice were reconstituted with either BM-TCD only (n = 9), 1 × 10⁶ IL-21R KO T cells + BM-TCD (n = 15), or 1 × 10⁶ WT T cells + BM-TCD (n = 15). Curves represent combined data from 2 independent experiments. (B) Lethally irradiated BALB/c mice received either BM-TCD only (n = 9), 5 × 10⁵ IL-21R KO CD4 T cells + BM-TCD (n = 15), or 5 × 10⁵ WT CD4 T cells + BM-TCD (n = 15). Curves represent combined data from 2 independent experiments. (C,D) Transplant recipients were scored on a weekly basis for 5 clinical GVHD parameters: weight loss, activity, kyphosis, fur ruffling, and skin flaking. Scores range from 0 to 2 for each parameter, and animals are killed once a total score of 5 is attained. Curves represent the average score of each group at each time point. Shown are combined data from 2 independent experiments. (E) Lethally irradiated BALB/c mice were reconstituted with B6 BM-TCD and 1 × 10⁶ B6 WT or 1 × 10⁶ B6 IL-21R KO T cells. Spleens were harvested on days 7, 14, and 21 after BMT and stained for FACS analysis of donor CD4⁺CD25⁺Foxp3⁺ T-regs or CD4⁺Foxp3⁻ nonregulatory T cells. Data represent 2 combined independent experiments on day 7 (n > 19), day 14 (n = 18), and day 21 (n = 15). (F) Lethally irradiated BALB/c mice were reconstituted with CD45.1 B6 BM-TCD and 5 × 10⁵ CD45.2 B6 MACS-purified T cells. Transplanted T-cell populations were either unfractionated or depleted of T-regs by sorting for CD25⁻ cells. Spleens were harvested on day 21 after BMT and analyzed by FACS for Foxp3 expression among donor CD4 T cells. Data represent 2 combined independent experiments with 8-11 total recipients per group. *P < .05, **P < .01, and ***P < .001 for WT versus KO T cells.

Figure 3

IL-21 signaling is not required for GVL against hematopoietic malignancy. (A) Lethally irradiated BALB/c mice were transplanted with either B6 BM-TCD alone or with A20 tumor cells (2.5-5 × 10⁵) with or without WT versus IL-21R KO T cells (0.5-1 × 10⁶). Recipients were followed for mortality, and curves represent combined data from 4 independent experiments (n = 19-20 for non–T-cell groups, n = 39 for T-cell groups). (B) Lethally irradiated B6 × DBA/2 F1 mice were transplanted with either B6 BM-TCD alone or with P815 tumor cells (1 × 10³) with or without WT versus IL-21R KO T cells (1 × 10⁶). Recipients were followed for mortality, and curves represent combined data from 2 independent experiments (n = 10 for non–T-cell groups, n = 20 for T-cell groups). ***P < .001 for WT versus KO T cells.

Figure 4

IL-21R KO T cells have decreased intestinal infiltration and LPAM expression after BMT. (A) Lethally irradiated BALB/c mice were transplanted with B6 BM-TCD and WT or IL-21R KO T cells (1 × 10⁶). Skin, liver, small intestine (S.I.) and large intestine (L.I.) were harvested 3 weeks after BMT. H&E-stained slides were scored for histopathologic damage and lymphocyte infiltration. Shown is the mean of each group from 2 combined independent experiments (n > 12). (B) Lethally irradiated BALB/c mice were transplanted with B6 BM-TCD and WT B6 T cells (1 × 10⁶). Lamina propria lymphocytes from small and large intestines were isolated 1 week after BMT and stained for IL-21R expression on donor T cells. Shown are combined data from 2 independent experiments with 7 total recipients per group. (C) Lethally irradiated LP mice were transplanted with B6 BM-TCD and WT or IL-21R KO B6 T cells (1 × 10⁶). Liver, small intestine, and large intestine were harvested 3 weeks after BMT, and H&E-stained slides were scored for histopathologic damage. Shown are combined data from 2 independent experiments with 17 total recipients per group. (D) Lethally irradiated BALB/c mice were transplanted with B6 BM-TCD and WT or IL-21R KO T cells (1 × 10⁶). Splenocytes were harvested 1 week after BMT and stained for FACS analysis of donor T-cell CD103, CCR9, and LPAM. Shown are combined data from 4 to 5 independent experiments with 20-25 total recipients per group. *P < .05 and ***P < .001 for WT versus KO T cells (A,C,D) or WT T cells in small versus large intestine (B).

Figure 5

IL-21R KO T cells are decreased in MLNs after BMT. (A) Lethally irradiated BALB/c mice were transplanted with B6 BM-TCD and WT or IL-21R KO T cells (1 × 10⁶). Spleens and MLNs were harvested 1 week after BMT and stained for FACS analysis of donor CD4 and CD8 T cells. Shown are combined data from 4 independent experiments with 20 total recipients per group. (B) CD45.1 B6 and IL-21R KO (CD45.2) T cells were cotransferred into lethally irradiated BALB/c recipients. Spleens and MLNs were harvested 4 hours after transfer and stained for FACS analysis of donor T cells. The ratio of spleen to MLN donor T cells was calculated, and connecting lines indicate WT (CD45.1) and KO (CD45.2) donor T cells isolated from individual recipients. Shown is 1 of 3 independent experiments. (C) Freshly isolated MLNs were harvested from untransplanted WT or CD45.1 B6 and IL-21R KO B6 mice and stained for FACS analysis of T-cell CCR7. Shown are combined results of 4 independent experiments with a total of 14 mice per group. *P < .05 and ***P < .001 for WT versus KO T cells.

Figure 6

IL-21R KO CD4 T-cell IFN-γ and TNF-α are maintained in the spleen but reduced in MLNs. Lethally irradiated BALB/c mice were transplanted with B6 BM-TCD and WT or IL-21R KO T cells (1 × 10⁶). (A) MLNs and (B) spleens were harvested 1 week after BMT and stained for intracellular cytokine FACS analysis of donor CD4 T cells. Shown are combined data from 4 independent experiments with 20 total recipients per group. **P < .01 and ***P < .001 for WT versus KO T cells.