P-Glycoprotein Modulation Facilitates the Selective Inhibition of Oxidative Phosphorylation in Alloreactive T Cells to Prevent Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplant

T lymphocytes play a central role in many human immunologic disorders including both autoimmune and alloimmune diseases. In hematopoietic stem cell transplant (HSCT), acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Ex vivo application of phototherapy to selectively deplete GVHD-causing cells prior to transplant may prevent GVHD. However, none of the photosensitive agents in use today have demonstrated selectivity without significant toxicity occurring in resting cells. We have designed the first-in-class, novel photosensitizer 2-Se-Cl with the ability to accumulate in pathogenic T cells in proportion to degree of oxidative phosphorylation (OXPHOS). Unique to 2-Se-Cl is the ability to potently stimulate P-glycoprotein (P-pg). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria, and protects resting lymphocytes essential for antipathogen and antitumor responses. To confirm that 2-Se-Cl selectively depletes antigen-specific immune responses, human lymphocytes were cultured with staphylococcal enterotoxin B (SEB) for 72 hours. Cells were then pulsed with 2-Se-Cl and exposed to 5 J/cm2 of light, which resulted in the impedance of OXPHOS and induction of apoptosis in stimulated T cells. After photodepletion (PD), resting cells remained intact with the ability to respond to stimulation by Toxic Shock Syndrome Toxin-1 (TSST-1) but not SEB. To evaluate the selective depletion of alloimmune responses, a well-established complete mHC antigen-mismatched murine model of HSCT was employed. To prepare the PD - treated primed splenocytes, donor C57BL/6 splenocytes were cocultured for 5 days with irradiated Balb/c splenocytes, and then photodepleted. In vitro analysis of PD - treated primed splenocytes demonstrated selective depletion of antihost immune responses by CFSE dilution, with complete retention of third-party responses (C3H/HeJ mice). On the day of HSCT, recipient Balb/c mice were irradiated with 900 cGy, and received 5x10⁶ T cell-depleted bone marrow cells accompanied by 5x10⁶ PD - treated (treatment group) or untreated (control group) primed splenocytes, and then monitored for signs of GVHD according to an established mouse GVHD grading system. Five animals underwent HSCT in each group in 3 independent experiments. All mice died of acute GVHD after the addition of 5x10⁶ untreated primed C57BL/6 cells at a median of 25 days after HSCT. In contrast, > 90% of mice that received the same number of PD - treated cells survived > 60 days without evidence of GVHD (see figure). Additionally, third-party C3H/Hej mice that received the same number of PD-treated cells died of GVHD, confirming the selective depletion of the alloimmune response for BalB/c by 2-Se-Cl. The high selectivity of this novel photosensitizer may have broad applications, including targeting the bioenergetics in alloimmune, autoimmune, and malignant T cells to provide alternative treatment options and improve clinical outcomes for patients with diseases such as GVHD, systemic lupus erythematosus, or peripheral T cell lymphoma.

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