Potent Strategy for Treatmet of Gvhd and Autoimmune-Diseased by Infusion of Donor-Typed or Even Third-Party Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment

Abstract 2777 We have reported that bone marrow derived dendritic cells with psoralen and UVA (PUVA-DCs) treatment acquired tolerogenicity in mice. With the purpose of potential application of PUVA-DCs in a clinical hematopoietic stem cell transplantations (HSCT) for graft-versus-host disease (GVHD), we showed that mixed lymphocyte reaction (MLR) was strongly inhibited when PUVA-DCs from the stimulator strain were added to the coculture (Stimulator (S): conventional DCs obtained from C57BL/6, Responder (R): splenocytes obtained from Balb/c, PUVA-DCs: C57BL/6). This suggests that infusion of host-typed PUVA-DCs would become a novel therapeutic approach for GVHD. However utilizing host-typed DCs has problems because of leukemic cell contaminations or low efficiency of cell culture from the patients receiving repetitive chemotherapy. Therefore next concern is whether PUVA-DCs generated from BM donor or even strangers would have same tolerogenicity as host-typed PUVA-DCs do. To test this, we performed MLR by adding PUVA-DC generated from the same strain of responder or third party strain (S: conventional DCs obtained from C57BL/6, R: splenocytes obtained from Balb/c, PUVA-DC: C57BL/6 or C3H). Proliferation was significantly inhibited when PUVA-DC generated from the stimulator strain were added to the coculture (p<0.05). Also significant inhibition was observed (p<0.05) when adding PUVA-DCs generated from third party, suggesting that PUVA-DCs have tolerogenicity in a MHC-independent manner. To clarify the mechanisms of how PUVA-DCs induce tolerogenicity, we performed MLR as mentioned above with the addition of neutralizing antibodies against IL-10 or TGF-beta1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner. This is the first report that PUVA-DCs prepared not only from host-typed but from donor-typed or even third-party could induce strong inhibition of alloreaction. Tolerogenic DCs prepared previously by several ways could not induce inhibition of alloreaction in vitro when these cells were prepared from donor-typed or third-party strains in mice. To apply tolerogenic dendritic cells for GVHD in clinical settings, it is necessary to obtain sufficient doses of PUVA-DCs with ease and safety guaranteed. Therefore in the future PUVA-DCs generated even from HLA mismatched iPS cells would be a promising approach. In conclusion, infusion of PUVA-DCs from donor-typed or even third party strain could have a potent strategy for treatment of lethal GVHD and autoimmune diseases. Disclosure: No relevant conflicts of interest to declare.

Strong Inhibition of Alloreaction by Newly Generated Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment.

Abstract 3686 Poster Board III-622 Dendritic cells (DCs) are a heterogenous population of antigen-presenting cells (APCs) that contribute to innate immunity and initiate the adaptive immune response. In addition, recent studies demonstrated the existence of tolerogenic DCs (TDCs) that suppress the immunoreaction. Although the tolerogenic mechanisms are not fully understood, there are some methods reported to generate TDCs from conventional DCs by using cytokines such as IL-10/TGF-beta, vasoactive intestinal peptide (VIP), and IL-21. With the purpose of potential application of TDCs in a clinical bone marrow transplantation for preventing graft-versus-host disease (GVHD), which is caused by strong immunoreaction between host-typed conventional DCs and donor-typed naïve T cells, the establishment of much safer and easier, and more efficient TDCs culture system would be needed. To this end, we investigated whether treatment of conventional DCs with psoralen plus UVA (PUVA), which is widely and safely available in the treatment of some human immune disease and organ transplantation for preventing graft rejection, induced a subset of highly potent TDCs. Bone marrow cells obtained from Balb/c (H-2d) or C57BL/6 (H-2b) were incubated in complete RPMI containing GM-CSF for 10 days to generate bone marrow derived DCs (BM-DCs). BM-DCs were cultured with Psoralen (200 ng/mL) for 30 miniutes and then exposed to UVA light (2J/cm2). After 24 hours UVA irradiation, PUVA-treated DCs were collected and used in all experiments. First, PUVA-treated or untreated DCs with irradiatin were used as stimulator for allogenic splenocytes in mixed leukocyte reactions (MLR). The immunostimulatory capacity of PUVA-treated DCs was significantly diminished compared to those of untreated DCs (p<0.01). The expression levels of CD80 and CD86 by FACS, both of which are costimulatory molecules for T cell activation, was significantly reduced after PUVA treatment (p<0.05). This might explain for the induction of hyporesponsiveness in part. Next we further evaluated whether PUVA-treated DCs directly suppress T cell alloreaciton by cell-to-cell contact. Proliferation was inhibited when PUVA-treated DCs from the stimulator strain were added to the coculture, with a maximum reduction in proliferation of 95% at a 1:1 or higher ratio of untreated DCs to PUVA-treated DCs (Attached file). In conclusion, PUVA-treated DCs directly inhibit T cell alloreaction. Infusion of host-typed PUVA-treated DCs would be potent strategy for preventing lethal acute GVHD. Disclosures: No relevant conflicts of interest to declare.