Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes

Abstract

The functional maturation of dendritic cells (DC) and other antigen-presenting cells is believed to reflect the upregulation of cell surface major histocompatibility complex (MHC) class II and other T cell co-stimulatory molecules, especially the CD28 ligands B7-1 (CD80) and B7-2 (CD86). In this study, we propagated cells exhibiting characteristics of DC precursors from the bone marrow (BM) of B10 mice (H-2b; I-A+) in response to granulocyte-macrophage colony stimulating factor (GM-CSF). The methods used were similar to those employed previously to propagate DC progenitors from normal mouse liver. Cells expressing DC lineage markers (NLDC 145+, 33D1+, N418+) harvested from 8-10-day GM-CSF stimulated BM cell cultures were CD45+, heat-stable antigen+, CD54+, CD44+, MHC class II+, B7-1dim but B7-2- (costimulatory molecule-deficient). Supplementation of cultures with interleukin-4 (IL-4) in addition to GM-CSF however, resulted in marked upregulation of MHC class II and B7-2 expression. These latter cells exhibited potent allostimulatory activity in primary mixed leukocyte cultures. In contrast, the cells stimulated with GM-CSF alone were relatively weak stimulators and induced alloantigen-specific hyporesponsiveness in allogeneic T cells (C3H; H-2k; I-E+) detected upon restimulation in secondary MLR. This was associated with blockade of IL-2 production. Reactivity to third-party stimulators was intact. The hyporesponsiveness induced by the GM-CSF stimulated, costimulatory molecule-deficient cells was prevented by incorporation of anti-CD28 monoclonal antibody in the primary MLR and was reversed by addition of IL-2 to restimulated T cells. The findings show that MHC class II+ B7-2- cells with a DC precursor phenotype can induce alloantigen-specific hyporesponsiveness in vitro. Under the appropriate conditions, such costimulatory molecule-deficient cells could contribute to the induction of donor-specific unresponsiveness in vivo.

Figure 1

FACScan immunophenotypic profiles of GM-CSF-stimulated B10 (H-2^b) mouse BM-derived cells released from cell aggregates in liquid culture (day 10) and examined using rat, hamster, or mouse mAbs. Further details are provided in Materials and Methods or have been described elsewhere (7). Unshaded profiles denote appropriate Ig isotype subclass controls. Note the absence of expression of lymphoid cell markers and the staining for three murine DC-restricted antigens NLDC 145, N418 (CD11c) and 33D1. The result is representative of 4 separate experiments.

Figure 2

Expression of MHC class II (I-A^b), B7–1 and B7–2 on (above) 8-day GM-CSF and (below) 8-day GM-CSF + IL-4–stimulated B10 BM-derived bone marrow cells. The result is representative of 3 separate experiments.

Figure 3

The MLR stimulatory capacity of B10 (H-2^b) mouse BM-derived cells (day 8) propagated in response to (0) GM-CSF (B7–2⁻) or (●) GM-CSF + IL-4 (B7–2⁺). Graded concentrations of washed, γ-irradiated stimulator cells were cultured with 2 × 10⁵ purified allogeneic (C3H; H-2^k) T cells. The stimulatory activity of freshly-isolated (△) B10 and (■) C3H (syngeneic) spleen cells and of (□) GM-CSF + IL-4 stimulated (C3H) syngeneic cells is also shown. Cells were pulsed with [³H]TdR for the final 18 hr of 3-day cultures. The results are expressed as mean counts per minute (cpm)±1 SD and are representative of three separate experiments.

Figure 4

Stimulation with B7–2⁻ BM-derived cells during a primary MLR induces hyporesponsiveness upon restimulation in secondary MLR. C3H splenic T cells (2×10⁶/ml at 1:2 [S:R]) ratio were stimulated during a 2-day primary MLR with γ-irradiated (○) GM-CSF-stimulated B10 BM-derived cells (B7–2⁻) (●) GM-CSF + IL-4–stimulated B10 BM-derived cells (B7–2⁺), or (△) fresh B10 spleen cells. The T cells were repurified and rested for 2 days, then restimulated for 3 days with graded concentrations of freshly isolated B10 spleen cells. The results are expressed as mean cpm ± 1 SD and are representative of four separate experiments.

Figure 5

T cells primed by B7–2⁻ BM-derived cells are specifically hyporesponsive to donor alloantigens. C3H splenic T cells were stimulated during a 2-day primary MLR with either (△) freshly-isolated B10 bulk spleen cells or (○) B7–2⁻ B10 cells. They were repurified, rested for 2 days, and restimulated for 3 days with C3H, B10 or BALB/c (third party) spleen cells. Results are expressed as mean counts per minute (cpm) ± 1 SD and are representative of 3 separate experiments.

Figure 6

Addition of anti-CD28 mAb (25 μg/ml) to primary MLR can prevent T cell hyporesponsiveness induced by B7–2⁻ BM-derived cells. C3H splenic T cells were stimulated during a 2-day primary MLR with (□), fresh B10 spleen cells; (■), B7–2⁻ B10 cells; (□) B7–2⁻ B10 cells + hamster IgG or (□), or B7–2⁻ B10 cells + anti-CD28. The T cells were repurified, rested for 2 days, and restimulated in 3-day secondary MLR with freshly isolated B10 spleen cells. The results are expressed as mean counts per minute (cpm) ± 1 SD and are representative of three separate experiments.