Roles of Host Nonhematopoietic Cells in Autoimmunity and Donor Cell Engraftment in Graft-versus-host Disease

Abstract

Background: Graft-versus-host disease (GVHD) is initiated when alloreactive donor T cells are primed by host APCs to undergo clonal expansion and maturation. Since there is a controversy regarding the role of nonhematopoietic cells in GVHD, we wanted to investigate the influence of MHC disparity on nonhematopoietic cells on the pathogenesis of GVHD in the MHC-haplomismatched C57BL/6 (H-2(b)) or DBA/2 (H-2(d))-->unirradiated (C57BL/6xDBA/2) F(1)(BDF(1); H-2(b/d)) murine model of acute GVHD (aGVHD) or chronic GVHD (cGVHD).

Methods: We generated (BDF(1)-->C57BL/6), (BDF(1)-->DBA/2), and (BDF(1)-->BDF(1)) chimeras and examined GVHD-related parameters and donor cell engraftment in those chimeras.

Results: Using this experimental system, we found that 1) severe aGVHD across MHC Ag barrier depends on the expression of nonhematopoietically rather than hematopoietically derived alloAgs for maximal GVHD manifestations; 2) host APCs were sufficient to break B cell tolerance to self molecules in cGVHD, whereas host APCs were insufficient to induce autoimmunity in aGVHD; 3) donor cell engraftment was greatly enhanced in the host with MHC-matched nonhematopoietic cells.

Conclusion: Taken together, our results provide an insight into how MHC disparity on GVHD target organs contribute to the pathogenesis of GVHD.

Keywords: Autoimmunity; Chimera; Graft-versus-host disease; Immune tolerance.

Figure 1

Confirmation of BM reconstitution. B6, DBA/2 and BDF₁ mice received 12 Gy irradiation and were reconstituted with 5×10⁶ T cell-depleted BDF₁ BM cells. PBMCs were analyzed by flow cytometry at days 45 and 55 after cell transfer. Percent of donor cells were counted by staining PBMCs with anti-H-2K^b or H-2K^d mAbs (n=10~20 per group).

Figure 2

MHC disparity on nonhematopoietic cells exacerbates aGVHD. Eighty-four days after BM reconstitution, aGVHD was induced by transferring 8×10⁷ B6 spleen/lymph node cells into three sets of mice (n=20 per group). (A) Loss of body weight. (B) Percent of survival. (C) Pathological scores for livers and colons. Organs were harvested at day 54 after disease induction (n=5~8 per group). ^†p<0.001, ^**p<0.01 and ^*p<0.05 between the indicated groups.

Figure 3

MHC match on nonhematopoietic cells increases donor cell engraftment in aGVHD. Eighty-four days after BM reconstitution, aGVHD was induced by transferring 8×10⁷ B6 spleen/lymph node cells into three sets of mice (n=20 per group). Splenocytes were harvested at day 54 after disease induction and stained with anti-H-2K^d plus anti-CD4, anti-CD8 or B220. (A) Percent of total donor cells. (B) Percent of donor B cells. (C) Percent of donor CD4⁺ T cells. (D) Percent of donor CD8⁺ T cells (n=5~8 per group). ^†p<0.001, ^**p<0.01 and ^*p<0.05 between the indicated groups.

Figure 4

MHC match on nonhematopoietic cells decreases activation of donor T cells in aGVHD. Eighty-four days after BM reconstitution, aGVHD was induced by transferring 8×10⁷ B6 spleen/lymph node cells into three sets of mice (n=20 per group). Splenocytes were harvested at day 54 after disease induction and stained with anti-H-2K^d plus anti-CD62L and anti-CD4 or anti-CD8. (A) Percent of donor CD4⁺CD62L^low T cells. (B) Percent of donor CD8⁺CD62L^low T cells (n=8 per group). ^†p<0.001, ^**p<0.01 and ^*p<0.05 between the indication groups.

Figure 5

MHC disparity on nonhematopoietic cells does not affect the development of cGVHD. B6, DBA/2 and BDF₁ mice received 12 Gy irradiation and were reconstituted with 5×10⁶ T cell-depleted BDF₁ BM cells. Eighty-four days after BM reconstitution, cGVHD was induced by transferring 8×10⁷ DBA/2 spleen/lymph node cells into three sets of mice (n=10 per group). (A) Serum samples were collected every 2 wk and assayed in duplicate by ELISA for IgG1 anti-DNA autoAb. The OD of duplicate samples for each mouse was measured at 450 nm, using serially diluted serum samples. (B) Percent of survival (n=10 per group). (C) Histology of kidneys harvested at day 54 after disease induction. Representative kidney sections are shown for H&E staining. ^*p<0.05 between the indicated groups.

Figure 6

MHC match on nonhematopoietic cells increases donor lymphocyte engraftment in cGVHD. Eighty-four days after BM reconstitution, cGVHD was induced by transferring 5×10⁶ DBA/2 spleen/lymph node cells into three sets of mice (n=20 per group). Splenocytes were harvested at day 54 after disease induction and stained with anti-H-2K^b plus anti-CD4, anti-CD8 or B220. (A) Percent of total donor cells. (B) Percent of donor B cells. (C) Percent of donor CD4⁺ T cells. (D) Percent of donor CD8⁺ T cells (n=5~8 per group). ^**p<0.01 and ^*p<0.05 between the indicated groups.

Figure 7

Donor lymphocyte engraftment correlates with donor CD8⁺ T cell activation in cGVHD. Eighty-four days after BM reconstitution, aGVHD was induced by transferring 5×10⁶ DBA/2 spleen/lymph node cells into three sets of mice (n=10 per group). Splenocytes were harvested at day 54 after disease induction and stained with anti-H-2K^b plus anti-CD62L and anti-CD4 or anti-CD8. (A) Percent of donor CD4⁺ CD62L^low T cells. (B) Percent of donor CD8⁺CD62L^low T cells (n=8 per group). ^*p<0.05 between the indicated groups.