Preterminal host dendritic cells in irradiated mice prime CD8+ T cell-mediated acute graft-versus-host disease

Abstract

To understand the relationship between host antigen-presenting cells (APCs) and donor T cells in initiating graft-versus-host disease (GVHD), we followed the fate of host dendritic cells (DCs) in irradiated C57BL/6 (B6) recipient mice and the interaction of these cells with minor histocompatibility antigen- (miHA-) mismatched CD8+ T cells from C3H.SW donors. Host CD11c+ DCs were rapidly activated and aggregated in the T cell areas of the spleen within 6 hours of lethal irradiation. By 5 days after irradiation, <1% of host DCs were detectable, but the activated donor CD8+ T cells had already undergone as many as seven divisions. Thus, proliferation of donor CD8+ T cells preceded the disappearance of host DCs. When C3H.SW donor CD8+ T cells were primed in vivo in irradiated B6 mice or ex vivo by host CD11c+ DCs for 24-36 hours, they were able to proliferate and differentiate into IFN-gamma-producing cells in beta(2)-microglobulin-deficient (beta(2)m(-/-)) B6 recipients and to mediate acute GVHD in beta(2)m(-/-) --> B6 chimeric mice. These results indicate that, although host DCs disappear rapidly after allogeneic bone marrow transplantation, they prime donor T cells before their disappearance and play a critical role in triggering donor CD8+ T cell-mediated GVHD.

Figure 1

Development of acute GVHD in B6 recipients transplanted with C3H.SW T^–BM cells and CD8⁺ T cells. (a) Recipient weight loss and (b) actuarial survival rate. Recipients were conditioned with 9.5 Gy TBI and then administered 7 × 10⁶ C3H.SW T^–BM cells alone (n = 12; diamonds) or along with 3 × 10⁶ C3H.SW CD8⁺ T cells (n = 17; squares). Data are derived from three separate experiments. (c) Infiltration of lymphocytes and CD8⁺ T cells in liver and skin (arrows) during GVHD. Livers and skin were harvested from mice and sectioned for histologic staining with hematoxylin and eosin (HE) and immunohistochemical staining with anti-CD8 Ab as described in Methods. Magnification: left, ×100; right, ×200. (d) Intracellular IFN-γ expression in hepatic CD8⁺ T lymphocytes. Hepatic lymphocytes were isolated from mice and restimulated in vitro in the presence or absence of anti-CD3 Ab plus MC57SV cells (H-2D^b) irradiated with 30 Gy for 16 hours. Lymphocytes were then labeled with anti-CD8 and anti-CD11a Ab’s, fixed, permeabilized, and labeled with anti–IFN-γ Ab. The expression of intracellular IFN-γ in CD8⁺ T cells was quantitated by flow cytometry. Dot plots shown are from IFN-γ and CD11a labeling of gated CD8⁺ T cells.

Figure 2

Elimination of CD11c⁺ DCs in the spleens of irradiated mice. Spleen cells were isolated from lethally irradiated B6 mice 6 hours, 24 hours, and 5 days after TBI with or without transplantation of C3H.SW CD8⁺ T cells, and CD11c⁺ DCs were enumerated by flow cytometry. (a) Percentage and (b) absolute number of CD11c⁺ DCs in the spleens of B6 mice injected with PBS, irradiated B6 mice injected with PBS, or irradiated B6 mice infused with C3H.SW CD8⁺ T cells. Data are representative of three experiments.

Figure 3

Irradiation upregulates the expression of antigen-presenting and costimulatory molecules on DCs. Splenic DCs were isolated from (a) irradiated mice at 6 hours and 24 hours after irradiation or (b) irradiated mice infused with donor CD8⁺ T cells or donor CD8⁺ T cells + T^–BM cells. These purified DCs were double stained with anti-CD11c Ab coupled with anti-Ia Ab or anti-CD86 Ab as described in Methods. The expression of Ia and CD86 antigens on the CD11c⁺ cell population was analyzed by flow cytometry. Isotype-matched IgG was used as control. (c) IL-12 in serum from nonirradiated and irradiated B6 mice at 6 hours, 24 hours, and 5 days following TBI or in the supernatants of cultured host DCs was examined by ELISA. Host splenic DCs were separately purified from the spleen of B6 mice at 6 and 24 hours following TBI or from nonirradiated mice as described in Methods. The supernatants were collected from the cultures of DCs at 48 hours. *P < 0.05 compared with samples from nonirradiated mice or irradiated mice at 24 hours and 5 days after TBI. (d) Host DCs were isolated from nonirradiated B6 mice (diamonds) and from B6 mice at 6 hours (triangles) and 24 hours (circles) after irradiation. These cells were cocultured with BALB/c mouse–derived CD4⁺ T cells to examine their capacity to stimulate an allogeneic mixed lymphocyte reaction. Activated DCs that were induced by culturing naive B6 DCs in the presence of GM-CSF + TNF-α for 48 hours were used as positive control (squares). One representative experiment of three is shown.

Figure 4

Donor CD8⁺ T cells are rapidly recruited to the T cell areas of recipient spleens, in direct proximity to host CD11c⁺ DCs. CFSE-labeled CD8⁺ T cells (2 × 10⁶) derived from C3H.SW mice (CD45.2⁺) were intravenously injected into lethally irradiated B6/SJL mice (CD45.1⁺). (a) Aggregation of CD11c⁺ DCs in the T cell areas of the spleens of irradiated mice. Cryosections of spleen harvested from lethally irradiated B6 mice 6 hours after TBI were immunostained with anti-CD11c Ab (brown) and anti-CD4 Ab (gray-black). Magnification: top row, ×100; bottom row, ×400. Data are representative of three experiments. (b) Spleens were taken from B6 mice 6 hours after PBS injection and from irradiated B6 mice 6 hours after infusion of CFSE-labeled C3H.SW CD8⁺ T cells (green) and cryosectioned for immunofluorescent staining with anti-CD11c Ab (red). Magnification, ×100. (c) Splenocytes were prepared and counted at the indicated timepoints. Cells were sequentially labeled with Cychrome-conjugated anti-CD8 Ab coupled with biotinylated anti-CD45.2 and streptavidin-conjugated PE. The percentage of CD8⁺CD45.2⁺ cells was obtained by flow cytometry analysis. The recruitment rate (percentage) of donor CD8⁺ T cells in spleen was calculated by dividing the absolute number of donor CD8⁺ T cells recovered from the spleens of irradiated recipient mice by the total number of intravenously injected donor CD8⁺ T cells.

Figure 5

Activation of donor naive CD8⁺ T cells upon intravenous injection into lethally irradiated B6 recipients. (a) Naive CD8⁺CD45.2⁺ C3H.SW T cells (2 × 10⁶) were intravenously injected into lethally irradiated B6/SJL recipients (CD45.1⁺). At the indicated timepoints, splenocytes were prepared from these mice and tricolor-stained with anti-CD8, anti-CD45.2, and anti-CD25 Ab’s, which were separately revealed with FITC, Cychrome, and PE. After gating on CD45.2⁺ donor–derived T cells, the expression of CD25 on CD8⁺ T cells was analyzed by flow cytometry. (b) CFSE-labeled CD8⁺ T cells (2 × 10⁶) from C3H.SW donor mice (CD45.2) were transferred with or without mixed donor T^–BM cells into lethally irradiated B6/SJL recipient mice (CD45.1). At the indicated timepoints after transplantation, splenocytes were prepared from these mice, stained with anti-CD8 Ab conjugated with Cychrome, and then sequentially labeled with biotinylated anti-CD45.2 and streptavidin-conjugated PE. The division of donor CD8⁺ T cells in vivo was analyzed by flow cytometry by gating on CD8⁺CD45.2⁺ T cells. One representative experiment of three is shown.

Figure 6

The activation and proliferation of allogeneic CD8⁺ T cells depend on the presence of host DCs and require MHC class I expression on host cells. (a) CFSE-labeled CD8⁺ T cells (2 × 10⁶) from C3H.SW mice (CD45.2⁺) were transplanted into irradiated B6 recipients (CD45.1⁺) at the indicated timepoints after TBI. Five days later, splenocytes were prepared from these mice for flow cytometry analysis as described in Figure 5b legend. (b) C3H.SW CFSE-CD8⁺CD45.2⁺ T cells (2 × 10⁶) were intravenously transferred into lethally irradiated β₂m^–/– mice or congenic B6/SJL (CD45.1⁺) recipients. Splenocytes were prepared from these mice, and the division of donor CD8⁺ T cells was analyzed by flow cytometry. The absolute number (c) of C3H.SW CD8⁺ T cells in the spleens of recipients 5 days after transplantation was calculated based on the flow cytometry analysis. Data shown derive from five independent experiments. One representative experiment of three is shown.

Figure 7

Brief priming by host DCs triggers the committed differentiation of donor naive CD8⁺ T cells into effectors. (a) Naive C3H.SW CFSE-CD8⁺ T cells were intravenously injected into lethally irradiated B6/SJL (CD45.1⁺) mice. After 24 to 36 hours, these primed C3H.SW CFSE-CD8⁺ T cells were then purified and adoptively transferred into secondary irradiated β₂m^–/– recipient mice. Six days later, cells from the spleens and lymph nodes of secondary recipients were cultured in the presence or absence of CD3 Ab plus MC57SV cells irradiated with 30 Gy for measuring IFN-γ production as described in Methods. Dot plots shown represent IFN-γ and CFSE intensity measured in gated CD8⁺ T cells. Naive unprimed C3H.SW CFSE-CD8⁺ T cells were injected into lethally irradiated B6/SJL mice (CD45.1⁺) and β₂m^–/– mice as control. (b) C3H.SW CFSE-CD8⁺ T cells were stimulated ex vivo by host CD11c⁺ DCs for 24 hours, recovered, sorted, and adoptively transferred into irradiated β₂m^–/– mice. Six days later, cells from spleens and lymph nodes were separated from the secondary recipients and stained with anti-CD8 Ab for measuring donor CD8⁺ T cell divisions. These cells were also cultured for 16 hours as described in a, to measure IFN-γ secretion. (c) Survival rate of β₂m^–/– B6 → B6 BM chimeric mice that received either C3H.SW T^–BM alone, C3H.SW T^–BM + C3H.SW CD8⁺ T cells, or C3H.SW T^–BM + C3H.SW CD8⁺ T cells that were primed ex vivo by B6 CD11c⁺ DCs. Representative results from two independent experiments are shown.