Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients

Abstract

We have shown previously that granulocyte-macrophage colony-stimulating factor-stimulated mouse bone marrow-derived MHC class II+ dendritic cell (DC) progenitors that are deficient in cell surface expression of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) can induce alloantigen-specific T-cell anergy in vitro. To test the in vivo relevance of these findings, 2 x 10(6) B10 (H2b) mouse bone marrow-derived DC progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-/dim) that induced T-cell hyporesponsiveness in vitro were injected systemically into normal C3H (H2k) recipients. Seven days later, the mice received heterotopic heart transplants from B10 donors. No immunosuppressive treatment was given. Median graft survival time was prolonged significantly from 9.5 to 22 days. Median graft survival time was also increased, although to a lesser extent (16.5 days), in mice that received third-party (BALB/c; H2d) DC progenitors. Ex vivo analysis of host T-cell responses to donor and third-party alloantigens 7 days after the injection of DC progenitors (the time of heart transplant) revealed minimal anti-donor mixed leukocyte reaction and cytotoxic T lymphocyte reactivity. These responses were reduced substantially compared with those of spleen cells from animals pretreated with "mature" granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DC (MHC class IIbright, B7-1+, B7-2bright), many of which rejected their heart grafts in an accelerated fashion. Among the injected donor MHC class II+ DC progenitors that migrated to recipient secondary lymphoid tissue were cells that appeared to have up-regulated cell surface B7-1 and B7-2 molecule expression. This observation may explain, at least in part, the temporary or unstable nature of the hyporesponsiveness induced by the DC progenitors in nonimmunosuppressed recipients.

Figure 1

FACScan analysis of NLDC-145, MHC class II, and B7-2 expression on 6-day GM-CSF and 6-day GM-CSF+IL-4-stimulated B10, C3H, or BALB/c BM-derived DC. Further details are provided in Materials and Methods or have been described elsewhere (15). The appropriate Ig isotype subclass control is always to the left of the specific antibody profile. The results are representative of three separate experiments.

Figure 2

Mixed leukocyte responses of splenic T cells from normal C3H mice injected intravenously 7 days previously with either 2×106 (*) B10 BM-derived B7-2-DC progenitors or ([black small square]) B7-2+ DC. The C3H T cells were stimulated for 3 days with either B10 (allogeneic), C3H(syngeneic), or BALB/c (third party) spleen cells at various S:R ratios. Results are mean counts per minute +/- 1SD and are representative of three separate experiments.

Figure 3

CTL responses of splenic T cells from normal C3H (H2k) mice injected intravenously 7 days previously with either 2×106(*) B10 (H2b) BM-derived B7-2- DC progenitors or ([black small square]) B7-2+ DC. Freshly isolated C3H spleen cells were cultured for 6 days with [gamma]-irradiated B10 spleen cells, as described in Materials and Methods, then used as effectors against 51Cr-labeled allogeneic EL-4(H2b), syngeneic R1.1 (H2k), or third-party P815 (H2d) target cells. The results are expressed as mean% specific lysis in triplicate cultures.

Figure 4

Two color immunofluorescence labeling of B7-2 (CD86) on donor-derived DC (I-Ab+) within the T-dependent area of allogeneic spleen 7 days after the injection of GM-CSF-stimulated DC progenitors(B7-2-). (A) B7-2+ cells are stained green (FITC-conjugated secondary mAb) and, in addition, (B) donor MHC class II+ cells(I-Ab+) are stained red (steptavidin-conjugated Cy 3.18). (B) Both double-positive cells and less brightly staining (B7-2-) donor cells(I-Ab+; arrows) are visible (magnification ×250).