P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells

Abstract

T lymphocytes have been found to harbor P-glycoprotein (Pgp) and to demonstrate modulation of its ion channel transporter function according to the state of activation of T lymphocytes. We hypothesized that cytotoxic chemicals that are extruded by Pgp could be used to specifically eliminate immunoreactive T-cell populations. In this study, we evaluated the capacity of 4,5-dibromorhodamine methyl ester (TH9402), a photosensitizer structurally similar to rhodamine, a dye transported by Pgp, and which becomes IntroductionGraft-versus-host disease (GVHD) is the principal cause of mortality and the primary limitation to the early and widespread use of allogeneic stem cell transplantation (SCT), a treatment that often represents the only curative option for numerous patients with malignant diseases and hereditary metabolic disorders. Depletion of T cells capable of recognizing and mounting an immune response toward host cells from stem cell grafts abrogates GVHD. [1][2][3][4] However, the elimination of T cells also results in delayed T-cell reconstitution and, thus, an increased rate of infection, particularly with viral agents such as cytomegalovirus, herpes zoster, and Epstein-Barr virus. [5][6][7] In addition, the eradication of mature T cells is associated with an increased risk of graft rejection and an increased incidence of relapse of malignant disease. 1,5,[8][9][10] Thus, T cells are required early after allogeneic transplantation and depleting the graft of its T-cell content is not an ideal approach to prevention of complications after transplantation. Although new immunosuppressive agents offer options to decrease the incidence and severity of GVHD, most of the time these strategies are only partially effective and may also increase the incidence of viral and fungal infections and other adverse effects of profound immunosuppression. To provide a solution to this conundrum, selective inactivation or elimination of alloreactive donor T lymphocytes could allow early immune recovery and response toward infectious agents, and potentially preserve graft-versus-leukemia (GVL) activity. [11][12][13] In addition, a strategy to selectively eliminate immunoreactive T cells could represent an important advance for the treatment of a large number of patients with autoimmune disorders.Recently, there have been significant efforts to try to identify and eliminate T-cell subsets capable of mounting an immune response toward host cells and mediating GVHD. Strategies targeting CD6 ϩ or CD8 ϩ T cells demonstrated convincing potential for the prevention of GVHD in HLA-matched transplants using related and even unrelated donors. [14][15][16] Interestingly, the elimination of donor T-cell subsets did not translate into a greater incidence of graft rejection. 17 Moreover, the partial loss of GVL activity that occurs with T-cell depletion was able to be restored through the administration of donor lymphocyte infusions at a time when patients are at lower risk for GVHD. 18 Delaying the infusion of donor T cells until the early po...

Functional assessment and specific depletion of alloreactive human T cells using flow cytometry

“…33 The degree of allodepletion we achieved (Ն 1-2 logs) was similar to that obtained using immunotoxin-based purging or through ex vivo approaches designed to induce anergy of alloreactive T cells. 26,27,30,32 Consequently, we believe that standard apheresis products from healthy donors should yield sufficient quantities of alloreactivity-depleted T cells for clinical use, even if we assume that the precursor frequencies of alloreactive T cells in the haploidentical or mismatched setting may be significantly lower than those observed here. Given the demonstrated breadth of TCR diversity in the residual T-cell population following depletion of alloreactivity, relatively modest numbers of these cells (eg, 10 4 -10 6 /kg) might be sufficient to transfer meaningful infectious immunity without the need for prolonged cell sorting of the entire lymphoid fraction of an apheresis product.…”

“…Other investigators have developed alternate approaches to deplete [23][24][25][26][27][28][29][30][31] or anergize 32 alloreactive T cells. In one general strategy, cells expressing the CD25 antigen associated with the Figure 5.…”

“…Further experiments demonstrated that the residual PBMCs lacked the ability to respond to the initial stimulus but retained the ability to respond to alloantigens or viral antigens in cells stimulated with CMV or alloantigens, respectively, and retained a diverse TCR repertoire. Finally, we applied these methods to clinically obtained apheresis products, resulting in the depletion of specific alloreactivity in a one-way MLR with the preservation of CMV-specific T-cell responses and T cells capable of responding to third-party alloantigens.Other investigators have developed alternate approaches to deplete [23][24][25][26][27][28][29][30][31] or anergize 32 alloreactive T cells. In one general strategy, cells expressing the CD25 antigen associated with the Figure 5.…”

Epidermal Growth Factor and Perlecan Fragments Produced by Apoptotic Endothelial Cells Co-Ordinately Activate ERK1/2-Dependent Antiapoptotic Pathways in Mesenchymal Stem Cells

“…These hMSC were grown in MSC growth medium (MSCGM) (Cambrex, Waskerville, MD, http://www.cambrex.com) and used at passages 2 through 9. The MSC phenotype was evaluated by direct immunofluorescence according to standard techniques [37,38], with monoclonal antibodies directed against the human antigens CD90, CD44, CD45, CD14, CD117, CD11b, CD34, CD38, CD33, CD32, CD10, CD64, CD71, CD13, and CD62L (Coulter Immunology, Beckman Coulter, Mississauga, ON, Canada, http:// www.beckmancoulter.com) (online supporting information Table 1). Immunofluorescence reactivity was determined by automated multiparameter flow cytometry analyzing a minimum of 10 4 cells (FACScan, Becton, Dickinson and Company, Mississauga, ON, Canada, http://www.bd.com).…”

Optimizing a photoallodepletion protocol for adoptive immunotherapy after haploidentical SCT

“…This method demonstrated that alloreactive T cells were selectively eliminated, as anti-third party, anti-CMV T-cell responses and T-regulatory cell content were preserved in vitro in matched and mismatched pair MLRs. 10,11 When mice in an allogeneic BMT model were infused with photoallodepleted donor T cells, the GVL effect was preserved without GVHD. 12 In order to apply photodynamic purging of alloreactive T cells in the human haploidentical transplant setting, we investigated and reported a range of experimental conditions for optimal T-cell allodepletion with preservation of pathogen-specific responses and T-regulatory cells.…”