Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells

Abstract

It is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4(+) and CD8(+) T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

Figure 1

Single H-Y Ag-specific T cells induce CD8⁺ and CD4⁺ T cell–mediated GVHD. (A) Survival in CD8⁺-mediated GVHD after infusion of 1 × 10⁶ CD8⁺ T cells from MataHari donors. Data are from 1 of 4 similar experiments. (B-E) GVHD analysis on day 5. Data are from 1 of 3 similar experiments. (B) Histopathologic analysis of liver (i-ii) and GI tract (iii-iv) by H&E stain (left) and scores (right). (C) Serum levels of TNF-α, IFN-γ, and IL-17A (ND indicates not detected). (D) Allo-H-Y Ag-specific donor CD45.2⁺CD8⁺ T-cell expansion in spleen. (E) Donor CD45.2⁺CD8⁺IFN-γ⁺ in spleen with representative histogram of IFN-γ expression gated by CD45.2⁺CD8⁺ T cells (left) and the absolute number of CD45.2⁺CD8⁺IFN-γ⁺ T cells in spleen (right). (F-G) Survival CD4⁺ GVHD. Data shown are from 1 of 3 similar experiments.

Figure 2

Donor T-cell precursor frequency is critical for induction of GVHD. Rachel T cells were transplanted along with TCD BM from B6♀ donors into lethally irradiated syngeneic B6♀ or allogeneic B♂ animals. The recipients were monitored for (A) survival and GVHD score. (B) Histopathology scores for liver and GI tract. (C) Skin H&E (left) and score (right). Data are from 1 of 3 similar experiments.

Figure 3

Polyclonal H-Y Ag-specific T cells induce GVHD. (A-B) Experimental schema: polyclonal T cells from WT B6♀ mice that are specific for H-Y Ags were generated after in vivo and subsequent ex vivo priming. These T cells were then transplanted along with TCD BM from B6♀ donors into lethally irradiated syngeneic B6♀ or allogeneic B6♂ animals. The animals were monitored for (C) survival and GVHD score. Data are from 2 combined experiments. **P < .01 compared with female recipients. (D) GVHD histopathology, scores of liver and GI tract and (E) lymphocyte infiltration in the spleen, intestine, and liver. Data are from 1 of 2 similar experiments. (F) Unprimed B6♀ T cells induce GVHD in allogeneic B6♂ recipients. CD90⁺ T cells (2 × 10⁶ or 15 × 10⁶) donor T cells were harvested from WT B6 female donors and transplanted without priming along with TCD BM into irradiated (11 Gy) syngeneic B6♀ and allogeneic B6♂ animals. The animals were monitored for clinical GVHD severity. (G) Histopathologic analysis, GVHD damage scores of liver and GI tract 70 days after allo-hematopoietic cell transplantation

Figure 4

Alloantigen expression and GVHD. (A) Experimental schema: the F→F, F→M, and M→F chimeras were generated such that male Ags are either not expressed or expressed only on the target tissues or only on the hematopoietic-derived cells. These chimeras were then irradiated and transplanted with Marilyn T cells along with TCD WT B6♀ BM cells and were monitored for (B) survival and (C) Serum levels of TNF-α, IFN-γ, and IL-17A on day 5. Data are from 1 of 2 similar experiments.

Figure 5

Presentation by donor and host nonhematopoietic APCs. (A) Experimental schema: B6♂ animals underwent thymectomy and were used for generation of (class II^−/−♀→B6♂) chimeras. Three months later, these chimeras were irradiated and transplanted with either B6 or Marilyn T cells and monitored for (B) survival. Data are from 2 combined experiments. (C) Donor APCs alone stimulate GVH responses. Experimental schema: TCD BM from either WT or class II^−/− B6♀ were transplanted along with WT or Marilyn T cells into lethally irradiated BALB/c males. (D) Total donor H2Kd⁺CD4⁺ T-cell expansion, H2kd⁺CD4⁺IFN-γ⁺ T-cell number, and serum IFN-γ concentration from day 7. Data are from 1 of 2 similar experiments. (E-I) Induction of GVHD in the absence of donor and radiosensitive host hematopoietic-derived APCs. (E) Experimental schema: B6Ly5.2 female and male animals were lethally irradiated, treated with anti–natural killer (NK) Ab and transplanted with either WT B6 or β2m^−/− BM. Three months later, these chimeras were transplanted with TCD BM from either WT or β2m^−/− B6♀ animals along with MataHari T cells. The animals were monitored for (F) survival and (G) clinical score. Data from 1 of 3 similar experiments are shown. (H) Donor CD45.2⁺CD8⁺ T cells expansion in spleen and (I) histopathology scores of liver and GI tract. Data from 1 of 2 similar experiments are shown.

Figure 6

Alloantigen presentation and GVHD in the absence of radiosensitive host hematopoietic-derived APCs. (A) Experimental schema: β2m^−/−♂→B6♂ chimeras were generated as described in “Methods.” They were then irradiated with 9 Gy and transplanted with TCD BM from β2m^−/− animals along with T cells from B6♂ or BALB/c♀ donors and analyzed for donor T-cell expansion. (B) Total donor CD45.1⁺CD8⁺ or H2kd⁺CD8⁺ T cells expansion. (C) Donor CD8⁺IFN-γ⁺ T cell number and (D) serum levels of IFN-γ. Data from 1 of 2 similar experiments are shown. (E) GVH responses induced by host nonhematopoietic APCs depend on T-cell precursor frequency. Survival and GVHD score. Data from 1 of 2 similar experiments are shown. (F-H) Male nonhematopoietic cells can stimulate both H-Y Ag-specific T cells and allogenic T cells. (F-G) Marilyn CD4 T cells against male liver endothelial cells (H) and epithelial cells of small intestine (G). (H) B6 male–derived liver endothelial cells can stimulate allogeneic BALB/c T cells. (I-J) Class II expression on liver endothelial cells gated by CD146⁺ cells, (right) naive and (left) IFN-γ stimulation for 48 hours (I). (J) Bar graph of class II expression on liver endothelial cells. Data are from 1 of 3 similar experiments.