Abstract
Background: Graft-versus-host disease (GVHD) remains one of the largest obstacles in allogeneic stem cell transplantation. Current therapies for GVHD generally involve systemic immunosuppression resulting in increased risk of infection and relapse. Extracorporeal Photopheresis (ECP) is an emerging therapy for chronic GVHD that offers the potential of selective immunomodulation. In ECP, patient leukocytes are treated with 8-methoxypsoralen (8-MOP) and UVA light, causing them to undergo apoptosis, and are re-infused into the patient. Because ECP has been studied mostly in the clinical setting, however, the mechanism by which this infusion of apoptotic cells impacts GVHD remains unclear. We hypothesized that ECP-treated cells interact with recipient dendritic cells (DCs) to induce tolerance. We applied ECP to a murine GVHD model and explored the effects of ECP-treated cells on DCs.
Methods: ECP: Splenocytes were incubated with 8-MOP at 200 ng/ml and exposed to UVA light (5 J/cm2).
Bone Marrow Transplant (BMT): C3H.SW mice were irradiated (1000 cGy) and given 10 × 106bone marrow and 5 ×106 lymph node C57BL/6 (B6) cells on day 0. 10 ×106 ECP-treated splenocytes were administered on day 1.
Co-culture: Female B6 BM-derived DCs were cultured for 7 days with GM-SCF and IL-4 and incubated overnight with untreated, irradiated (3000 cGy) or ECP-treated splenocytes. Anti-CD40 was used to activate DCs. DC phenotype and cytokine production were examined using flow cytometry.
Vaccine: Male B6 DCs were co-incubated with untreated, irradiated or ECP-treated female B6 splenocytes and injected into naive female B6 recipients on days 14 and 28. On day 42, responses against the male histocompatibility antigen complex (H-Y) were assayed using IFN-gamma ELISPOT.
Results: 85–94% of ECP-treated cells were Annexin+/7AAD+ 24 hours after ECP treatment. Infusion of ECP-treated cells diminished GVHD-associated weight loss regardless of the strain of the ECP donor (Fig. 1) and restored responsiveness to H-Y vaccination. In co-culture experiments, the presence of ECP-treated cells did not alter phenotypic maturation of DCs. However, co-culture of DCs with ECP-treated cells did alter DC cytokine production resulting in less IL-12 production when compared to anti-CD40 alone (1.0% vs. 4.2% of CD11b+/CD11c+ cells). Finally, immunization of naive female mice with male DCs co-cultured with ECP-treated cells resulted in a modest but statistically significant (p=.0499) decrease in subdominant, class I H-Y responses compared to DCs incubated with untreated splenocytes.
Conclusion: The administration of ECP-treated cells in vivo appears to lessen the severity of GVHD, allowing mice to maintain immunocompetence. DCs cultured in the presence of ECP-treated cells can mature phenotypically but exhibit altered cytokine production. Activated, co-incubated DCs are less potent when used as a vaccine. This data suggests that perhaps the alteration of recipient DCs may contribute to the beneficial effect of ECP in GVHD. Ongoing experiments continue to explore the role of recipient DCs in ECP treatment of GVHD.
Figure 1. Female C3.HSW mice were lethally irradiated and received 10 × 106 bone marrow and 5 × 106 lymph node C57BL/6 (B6) cells on Day 0. On Day 1, 10 × 106 ECP-treated cells from either recipient (C3.HSW), donor (B6) or 3rd Party (BALB.B) mice were administered. Weight loss was monitored twice weekly. Figure 1. Female C3.HSW mice were lethally irradiated and received 10 × 106 bone marrow and 5 × 106 lymph node C57BL/6 (B6) cells on Day 0. On Day 1, 10 × 106 ECP-treated cells from either recipient (C3.HSW), donor (B6) or 3rd Party (BALB.B) mice were administered. Weight loss was monitored twice weekly.
Extracorporeal Photopheresis Attenuates Murine Graft-versus-Host Disease via Bone Marrow–Derived Interleukin-10 and Preserves Responses to Dendritic Cell Vaccination