Emergent autoimmunity in graft-versus-host disease

Abstract

Donor T-cell recognition of host alloantigens presented by host antigen-presenting cells (APCs) is necessary for the induction of graft-versus-host disease (GVHD), but whether direct alloreactivity is sufficient for the propagation of GVHD is unknown. In this study, we demonstrate that GVHD cannot be effectively propagated through the direct pathway of allorecognition. Rather, donor T-cell recognition of antigens through the indirect pathway is necessary for the perpetuation of GVHD. Furthermore, GVHD results in the breaking of self tolerance, resulting in the emergence of donor T cells that can cause autoimmune disease in syngeneic recipients. Notably, GVHD-induced autoreactivity is donor APC dependent, transferable into secondary hosts, and involves cells of the innate immune system. These results indicate that donor T-cell--mediated pathologic damage during GVHD becomes donor APC dependent and provide a mechanistic explanation for the long-standing observation that GVHD is associated with autoimmune clinical manifestations.

Figure 1.

GVHD is not effectively propagated through direct allorecognition. (A) Lethally irradiated (900 cGy) Balb mice received transplants of TCD B6 BM alone (□, n = 3) or together with 3 × 10⁵ B6 spleen cells (∼ 1 × 10⁵ T cells; ▪, n = 8). Mice were killed 18 to 24 days after bone marrow transplantation (BMT). (A) Serial weight curves are shown in panel A. Mean overall GVHD score and pathology score in the colon are shown in panel B. Results are representative of 5 independent experiments. Pooled spleen cell suspensions from mice that had received transplants as in panel A (containing either 0.4 or 1 × 10⁶ T cells in 2 independent experiments) were then transplanted along with B6 Rag-1 BM (10 × 10⁶ cells) into lethally irradiated Balb mice (n = 7). Data indicate percentage of body weight change (C) and mean overall and colon GVHD scores (D). Error bars depict SEM.

Figure 2.

Donor APCs are required for the propagation of GVHD. Lethally irradiated (900 cGy) Balb Rag mice received transplants of B6 Rag BM to create chimeric animals in which APCs were of donor origin. In reciprocal studies, lethally irradiated (1000 cGy) B6 Rag animals received transplants of Balb Rag BM. Chimeric mice were bled 60 days after transplantation, and peripheral blood cells were determined to be more than 95% donor origin. Spleen cells (containing 0.6 × 10⁶ T cells) from B6→Balb mice undergoing GVHD were then adoptively transferred into either B6 Rag BM→Balb Rag (▪, n = 10) or Balb Rag BM→B6 Rag (□, n = 8) chimeras 2 months after their initial transplantation. B6 Rag animals (n = 19) that received spleen cells containing 1 × 10⁶ T cells from normal B6 mice served as pathologic controls. Weight loss (A), incidence (B), and severity of colitis (C) are depicted. Results are cumulative data from 2 experiments. Error bars depict SEM.

Figure 3.

GVHD T cells cause colitis when transferred into syngeneic Rag mice. Lethally irradiated (900 cGy) Balb mice received transplants of TCD B6 BM plus 3 × 10⁵ B6 spleen cells. Mice were killed 20 to 27 days after transplantation. Spleen cell suspensions were then transferred into either B6 (▪, n = 18) or Balb (□, n = 17) Rag mice. In most experiments, spleen cells were pooled from multiple GVHD animals before transfer into Rag recipients. Mice in both cohorts received an equivalent number of T cells in the spleen cell suspension, ranging between 0.3 and 1 × 10⁶ in replicate experiments. Survival (A), serial weight curves (B), spleen cellularity (C), and total number of splenic T cells (D) are shown. Data in panels C and D are presented as the mean ± SEM. (E) Incidence of colitis in B6 and Balb Rag recipients of GVHD T cells. Data are derived from 6 independent experiments.

Figure 4.

Colitis is characterized by extensive infiltration of T cells and granulocytes. (A) Histologic analysis of colitis in B6 Rag recipients that received B6→Balb GVHD T cells, revealing marked infiltration of mononuclear cells into the lamina propria accompanied by depletion of goblet cells and sloughing of the mucosa into the colonic lumen along with (B) transmural inflammation extending out into the mesentery indicative of serosal inflammation (original magnification, × 200). (C) Neutrophils (dashed arrows) and eosinophils (solid arrows) in regions of histologically damaged colonic tissue. (D) Colonic mucosa in Balb-Rag recipients showing no evidence of colitis with preservation of goblet cells and the absence of inflammation in the lamina propria and muscularis mucosa. (E) Immunohistochemical staining showing extensive CD3⁺ T-cell infiltration (brown staining cells) throughout the lamina propria extending across the wall of the colon into the surrounding mesentery corresponding to sites of tissue damage in B6 Rag animals (ie, crypt abscesses, crypt cell apoptosis). (F) Absence of GVHD T-cell infiltration into the mucosa or lamina propria of Balb Rag mice. (G) Colonic tissue from B6 Rag recipients of B6→B10.BR spleen cells showing similar pathologic damage and (H) CD3⁺ T-cell infiltration as in panels A and E, respectively.

Figure 5.

Autoimmunity can be transferred in syngeneic recipients. Lethally irradiated (900 cGy) Balb mice received transplants of TCD B6 BM plus 3 × 10⁵ B6 spleen cells. Spleen cells were obtained from GVHD animals 22 days after BMT and transferred into B6 Rag recipients for 67 days. Mice were then killed, and pooled spleen cells were transferred into either B6 or Balb Rag animals (n = 4/group). Mice were killed 61 days after transfer and analyzed for overall spleen cellularity (A), absolute number of splenic T cells (B), and incidence of colitis (C). Data are presented as the mean ± SEM.

Figure 6.

Expansion of the innate immune system in mice with autoimmunity. Lethally irradiated Balb mice received transplants of TCD B6.SJL (CD45.1) BM plus 3 × 10⁵ B6.SJL spleen cells. Spleen cells were obtained from GVHD mice 3 to 4 weeks after BMT and transferred into either B6 (n = 6) or Balb (n = 6) Rag mice. Mice in each cohort were killed approximately 2 months after transfer and analyzed for spleen cellularity and absolute numbers of Gr-1⁺, Mac-1⁺, and CD11c⁺ cells (A) as well as the absolute numbers of Gr-1⁺, Mac-1⁺, and CD11c⁺ cells that were of donor (CD45.1) or host (CD45.2) origin (B). Data are cumulative results from 2 independent experiments. Histology of representative bone marrows from B6 (C) or Balb (D) Rag mice that received spleen cells from lethally irradiated Balb mice that had received transplants of TCD B6 BM plus B6 spleen cells to induce GVHD (original magnification, × 200). Marrow from B6 Rag mouse shows an almost homogenous population of mature neutrophils, while marrow from Balb Rag mouse demonstrates a heterogenous population of erythroid, myeloid, and megakaryocytic cells. In panels A and B, error bars indicate standard error measurement (SEM).