4-5-dibromorhodamine-123 and Graft-vs-Host-Disease

Graft versus Host Disease (GVHD) is the principal cause of morbidity and mortality in patients undergoing allogeneic stem cell transplant. T cell depletion has been recognized as a method of reducing the incidence of GVHD in allogeneic transplants. Until recently, most T cell depletion methods were non-selective in reducing lymphocytes. Rhodamine purging is one method, which selectively reduces alloreactive T cells preventing GVHD. We review here the methods of non-selective and selective T cell depletion, particularly the newer method of photodynamic purging utilizing rhodamine.

Topics: Animals; Graft vs Host Disease; Graft vs Leukemia Effect; Humans; Lymphocyte Depletion; Photochemotherapy; Photosensitizing Agents; Rhodamines; Stem Cell Transplantation; T-Lymphocytes; Transplantation Immunology; Transplantation, Homologous

A major goal in transplantation immunology is to develop strategies that can specifically promote tolerance to foreign cells and tissues without compromising other immune functions. Experimental induction of transplantation tolerance by dendritic cells (DCs) and/or T regulatory (Treg) cells can efficiently prevent graft-versus-host disease and organ graft rejection in animal models, and there is much hope that similar strategies in transplanted patients will provide an alternative to immunosuppression. Photodynamic therapy (PDT) is a therapeutic treatment for graft versus host disease and organ rejection that may operate via induction of tolerance. We investigated whether a new PDT therapy, based on exposure of cells to 4,5-dibromorhodamine methyl ester (TH9402), may operate via induction of tolerogenic DC. We developed an in vitro model to mimic the in vivo effect of re-infusing peripheral blood mononuclear cells (PBMCs) treated with PDT based on TH9402 (TH-PDT). TH-PDT-treated PBMCs were co-cultured with allogeneic immature monocyte-derived DCs. After 24 h, the phenotype and T-cell stimulatory capacity of the DCs was assessed. Following phagocytosis of TH-PDT PBMCs, DCs maintained an immature phenotype, produced significantly increased amounts of interleukin-10, and had a reduced allostimulatory capacity in comparison to mature DCs. In the context of transplantation, these data suggest that repeated exposure of circulating DCs to TH-PDT PBMCs may result in presentation of alloantigens under anti-inflammatory conditions and induction of antigen-specific tolerance.

Topics: Dendritic Cells; Graft vs Host Disease; Humans; In Vitro Techniques; Photochemotherapy; Rhodamines; Transplantation Tolerance

Even the most potent immunosuppressive drugs often fail to control graft-versus-host disease (GVHD), the most frequent and deleterious posttransplantation complication. We previously reported that photodepletion using dibromorhodamine (TH9402) eliminates T cells from healthy donors activated against major histocompatibility complex-incompatible cells and spares resting T cells. In the present study, we identified photodepletion conditions selectively eradicating endogenous proliferating T cells from chronic GVHD patients, with the concomittant sparing and expansion of CD4(+)CD25(+) forkhead box protein 3-positive T cells. The regulatory T-cell (Treg) nature and function of these photodepletion-resistant cells was demonstrated in coculture and depletion/repletion experiments. The mechanism by which Tregs escape photodepletion involves active P-glycoprotein-mediated drug efflux. This Treg-inhibitory activity is attributable to interleukin-10 secretion, requires cell-cell contact, and implies binding with cytotoxic T-lymphocyte antigen 4 (CTLA-4). Preventing CTLA-4 ligation abrogated the in vitro generation of Tregs, thus identifying CTLA-4-mediated cell-cell contact as a crucial priming event for Treg function. Moreover, the frequency of circulating Tregs increased in chronic GVHD patients treated with TH9402 photodepleted cells. In conclusion, these results identify a novel approach to both preserve and expand Tregs while selectively eliminating CD4(+) effector T cells. They also uncover effector pathways that could be used advantageously for the treatment of patients with refractory GVHD.

Topics: Cell Separation; Chronic Disease; Clinical Trials as Topic; Flow Cytometry; Graft vs Host Disease; Humans; Photosensitizing Agents; Rhodamines; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

In this study, we investigated the possibility of selective depletion of donor alloantigen-specific T cells from C57BL/6 (H-2(b)) mice to prevent graft-versus-host disease (GVHD). These cells were first activated with irradiated BALB/c (H-2(d)) host spleen cells in a 5-day mixed lymphocyte culture. Following this activation, a photoactive rhodamine derivative called 4,5-dibromorhodamine 123 (TH9402), was added. This compound is selectively retained in the mitochondria of activated host-reactive cells but not tumor- or third-party-specific resting cells. The treated cells were subsequently exposed to visible light (514 nm) to deplete the TH9402-enriched activated host-reactive cells. Treatment with photodynamic cell purging process (PDP) inhibited antihost responses measured by cytotoxic T lymphocytes (CTL) by 93%, and interferon-gamma production by 66%. By contrast, anti-BCL1 (BALB/c-origin leukemia/lymphoma) and anti-third-party C3H/HeJ (H-2(k)) responses were preserved. PDP-treated primed C57BL/6 cells were further tested in vivo. All lethally irradiated BALB/c mice inoculated with BCL1 cells and T-cell-depleted bone marrow cells developed leukemia by day +30, with 50% mortality by 100 days. All mice died of GVHD after addition of 5 x 10(6) untreated primed C57BL/6 cells. However, addition of same numbers of PDP-treated cells allowed 90% of the recipients to survive more than 100 days without detectable BCL1 tumor cells and free of GVHD. Moreover, PDP-treated primed C57BL/6 cells retained the ability to induce GVHD in the third-party C3H/HeJ mice. These data suggest that PDP can selectively deplete host alloantigen-specific T cells for GVHD prevention and immune and antileukemia function preserve.

Topics: Animals; Bone Marrow Transplantation; Female; Graft vs Host Disease; Graft vs Leukemia Effect; Histocompatibility; Light; Lymphocyte Depletion; Mice; Mice, Inbred Strains; Photosensitizing Agents; Rhodamines; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transplantation Immunology; Tumor Cells, Cultured