TRANSIENT ARREST OF DENDRITIC CELL MATURATION: A WINDOW OF OPPORTUNITY FOR PRIMATE TOLERANCE INDUCTION? (1999)

813 Introduction: According to current concepts, dendritic cells (DC) in peripheral blood (PB) and in organ transplants represent immature precursors (DCp) that process antigen but lack costimulatory molecules for presentation. DCp maturation is driven by proinflammatory cytokines that depend on activation and nuclear translocation of NF-κB transcription factors, particularly Rel-B. We have observed in nonhuman primates (NHP) that, following depletion of T cells in lymph nodes (LN) and blood after day-of-transplant induction with anti-CD3 immunotoxin (IT), stable tolerance to MHC mismatched renal allografts is favored when proinflammatory cytokine responses are inhibited by brief treatment with the NF-κB inhibitor, 15-deoxyspergualin (DSG). This finding led us to examine whether the synergistic effect of DSG in this model might be to block DCp maturation during the T cell recovery phase after IT induction. Methods: Normal 3 kg male rhesus monkeys received a renal allograft from an unrelated, MHC mismatched donor. Immunosuppression consisted of 3 injections of IT (150 μg/kg total dose over days 0 to +2, methylprednisolone (MP) tapered from 7 mg/kg/d to 0 over days 0 to +3, and DSG (2.5 mg/kg/d) on days 0 to 4 (n=5) or 0 to +14 (n=5). Additional recipients had IT with MP alone (n=3) or MP plus Neoral (120 mg/kg/d) on days 0-4(n=3). Inguinal and axillary LN biopsied at 5, 15, and 30 days were examined by immunohistochemistry for presence of mature DC expressing membrane CD83, DR, and CD86 and nuclear Rel-B. To directly examine DCp maturation, we cultured isolated rhesus PB DCp in medium containing monocyte conditioned medium (MCM) plus TNFα. DSG (0.6-10 mg/ml) was added to DCp cultures prior to adding MCM/TNFα. Expression of DC maturation markers was assessed by flow cytometry and by immunocytochemical staining of cytocentrifuge preparations. Results: Renal allografts survived without histologic or clinical evidence of acute or chronic rejection in 80% of IT-treated recipients given MP plus DSG ×15d, in 40% in those given MP plus DSG × 5d, and in 0% of those given only MP or MP plus Neoral. The 3 longest DSG survivors are>830 days (2.3 years) with normal renal function and immunologic indication of specific tolerance. Compared to LN from transplants without DSG, the day +5 LN tissue from DSG-treated recipients showed extreme reduction in mature DC, i.e., a mean 58-fold±9.9 reduction in membrane expression for CD83, 49-fold ± 9.0 for CD86, and 81-fold±13.5 for DR. Rel-B nuclear positive cells were reduced 46-fold±7.1. Partial recovery occurred by day 15, and by day 30, DR+, CD83+, and Rel-B+ cells were within control range. CD86+ cells were still reduced (by 7.5-fold ±1.8). The results are consistent with NF-κB inhibition and DCp maturation arrest by DSG. A dose-dependent DSG effect was confirmed by in vitro analysis of MCM/TNFα-driven DCp maturation. The results showed a shift from nuclear and membrane staining to cytoplasmic staining of Rel-B and CD83, respectively, reflecting a dominant immature DCp phenotype in the DSG treated cultures. Conclusions: These studies show the NF-κB inhibitor, DSG, blocks maturation of DCp in vivo and in vivo in NHP. We hypothesize that the unusual synergy of IT and DSG in promoting tolerance in this model is due in part to depletion of memory as well as naïve T cells by IT. Conceptually, this would create a transitional circumstance in which repopulating naïve T cells are committed to DC costimulatory requirements for activation, while DCp acquisition of costimulatory function is arrested by DSG. The net result is a fleeting lapse into a "non-dangerous" milieu, akin to the neonate's, coincidentally reduced both in T cell mass and DC maturity, thereby favoring tolerance.