In vitro propagation and homing of liver-derived dendritic cell progenitors to lymphoid tissues of allogeneic recipients. Implications for the establishment and maintenance of donor cell chimerism following liver transplantation

Abstract

Dendritic cell (DC) progenitors were propagated in liquid culture from nonparenchymal cells resident in normal mouse (B10.BR; H-2k, I-E+) liver in response to granulocyte-macrophage colony stimulating factor (GM-CSF). The liver-derived DC progenitors were MHC class II-/dim and did not express counter receptors for CTLA-4, a structural homologue of the T cell activation molecule CD28. Following subcutaneous or intravenous injection, these liver-derived cells migrated to T cell-dependent areas of lymph nodes and spleen of unmodified, allogeneic (B10; H-2b; I-E-) recipients, where they were identified 1-5 days, and 1 and 2 months after injection by their strong surface expression of donor MHC class II (I-Ek) and their dendritic morphology. Maximal numbers of liver-derived DC in the spleen were recorded 5 days after injection. Both clusters of strongly donor MHC class II+ cells--and (more rarely) dividing cells--could also be identified, suggesting cell replication in situ. Using the same techniques employed to generate DC progenitors from normal liver, GM-CSF-stimulated cells were propagated for 10 days from the bone marrow and spleen of nonimmunosuppressed mice sacrificed 14 days after orthotopic liver transplantation (B10;H-2b-->C3H;H-2k). Immunocytochemical staining for recipient and donor MHC class II phenotype revealed the growth both of host cells with DC characteristics, and of cells expressing donor alloantigens (I-Ab). These results are consistent with the growth, in response to GM-CSF, of donor-derived DC from progenitors seeded from the liver allograft to recipient lymphoid tissue. The functional activity of the progenitors of chimeric DC and the possible role of these cells in the establishment and maintenance of donor-specific tolerance following liver transplantation remain to be determined.

Figure 1

Cytocentrifuge preparation showing cells released from GM-CSF-stimulated liver cell aggregates (day 10), which exhibit irregularly shaped, eccentric nuclei, absence of prominent granules, and distinct cytoplasmic processes (giemsa; ×600).

Figure 2

Absence of allostimulatory activity of γ-irradiated, GM-CSF-stimulated, B10.BR mouse liver–derived DC progenitors [] using naive, B10 (I-E⁻) splenic T cells as responders. The nonadherent, low-buoyant-density cells were harvested from 10-day GM-CSF-stimulated cultures and set up at various concentrations, with 4×10⁵ responder T cells. Cultures were maintained for 72 hr; [³H]TdR was added 18 hr before harvesting. The MLR-stimulatory activity of freshly-isolated allogeneic (B10.BR) liver NPC [] and spleen cells [] and of syngeneic (B10; []) spleen cells is also shown. The results are expressed as mean counts per minute (cpm) ± 1 SD and are representative of 3 separate experiments.

Figure 3

(a–h). Homing ability of GM-CSF-stimulated B10.BR liver–derived cells DC progenitors released in culture from proliferating cell aggregates (harvested on day 10). The cells (2.5×10⁵) were injected s.c. (day 0) into one hind footpad of B10 (I-E⁻) recipients and detected by immunohistochemistry (I-E⁺) in cryostat sections of draining lymph nodes, spleen, or thymus 1 day to 2 months later. The sections were stained, using the ABC peroxidase procedure, with donor-specific mouse anti-I-E^k mAb, together with appropriate controls, (a) day 1, spleen; a strongly donor MHC class II–positive cell in the T-dependent area of recipient spleen, close to an arteriole (arrow); (b), day 1, lymph node; a single positive cell in the subcapsular space and some additional structures (arrows) that may represent lightly positive cells; (c) day 1, thymus; a positive cell in the medulla; (d) day 5, spleen; several strongly positive cells with dendritic morphology in T-dependent area near an arteriole; (e) day 5, spleen; isolated donor class II⁺ cells with dendritic morphology and an aggregate of similar cells, each of which expresses donor phenotype; (f) 2 weeks and (g) 2 months, spleen; showing positive cells and (h) day 4, spleen; a dividing donor class II⁺ cell in late metaphase following the injection of Ia-depleted, liver-derived DC progenitors (all × 1000).

Figure 4

Mean number ± 1 SD of donor MHC class II⁺ cells with dendritic morphology observed in spleen per 100 high-power fields (hpf) 1–60 days after s.c. footpad injection of 2.5×l0⁵ GM-CSF-stimulated, liver-derived DC progenitors.

Figure 5

(a), donor MHC class II⁺ (I-A^b+) and (b), recipient MHC class II⁺ (I-E^k+) cells with dendritic morphology in 10-day GM-CSF-supplemented cultures of bone marrow-derived cells isolated from a C3H (H-2^k) mouse recipient of a B10 (H-2^b) liver allograft, 14 days after transplantation (×1000). (c), mouse IgG isotype control (×400). No staining of GM-CSF-stimulated C3H or B10 spleen cells (derived from normal mice) with anti-I-A^b or anti-I-E^k, respectively, was observed (data not shown).