Combined Cytoreduction and Infusion of Anti-CD3/Anti-CD28 Bead-Activated T Cells Prevent Diabetes in NOD Mice. (2005)

Abstract Autoreactive T cells are found in many autoimmune diseases (AID). Most current therapies rely on chronic immunosuppression, which increases the risks of infection and cancer. More intensive therapies, such as high-dose chemotherapy and stem cell transplantation, have been used to treat some of these diseases, but this treatment can cause serious toxicities. As a safer alternative, we are developing a therapeutic strategy, which would use less toxic therapies to eliminate T cells from AID patients followed by infusing healthy T cells to restore their immune systems. In the Xcellerate Technology, T cells are activated and expanded ex vivo utilizing beads coated with Abs specific for CD3 and CD28 (3/28 beads). We and our colleagues have previously demonstrated that the Xcellerate Technology can be used to generate large numbers of T cells, known as Xcellerated T Cells, from patients with cancer and HIV. Several clinical trials have demonstrated that administration of Xcellerated T Cells is well-tolerated and associated with rapid and sustained immune reconstitution. By increasing the number of beads mixed with T cells, we have previously demonstrated that a stronger signal is delivered that is capable of specifically deleting memory T cells, which are sensitive to activation-induced cell death, while allowing the rapid expansion of other T cells (J. Immunother.27(5):405. 2004). Since memory T cells reactive against host tissues, so-called autoreactive T cells, are associated with AID, we hypothesized that activation and expansion of T cells with increased beads would generate a pool of healthy T cells with few, if any, pathogenic T cells. In a murine model of autoimmune disease, female NOD mice develop insulitis and diabetes at 10–12 weeks and 30 weeks, respectively; these time periods can be significantly shortened by treatment with a cytoreductive regimen using cyclophosphamide (Cy). We treated 7–8 week old Thy1.2+ female NOD mice with 200mg/kg Cy i.p. and 8 days later injected 2x107 Thy1.1+ congenic splenic T cells, which were either freshly isolated or undergone activation and expansion using 3/28 beads. At 30-days post-treatment, we evaluated glucose levels and then sacrificed animals to assess tissue distribution of infused T cells by flow cytometry, and islet pathology by immunohistochemistry. 3/28 bead-activated T cells engrafted better than non-activated T cells. Moreover, tetramer-based flow cytometric analysis detected large numbers of autoreactive T cells specific for islet antigens in the lymph node, spleen and blood of non-treated animals and animals treated with non-activated T cells. In contrast, fewer autoreactive T cells were found in animals, who received 3/28 bead-activated T cells. Finally, insulitis was decreased in mice that were treated with 3/28 bead-activated T cells. These data provide in vivo evidence of the potential of using the Xcellerate Technology to generate heathy T cells from AID patients, which may prove useful in treating these illnesses, when combined with lymphodepleting therapy. Adoptive Transfer of 3/28 Bead-Activated T Cells Diminishes Islet Pathology in NOD Mice Treatment Ave. Insulitis Score (day 30) Score based on insulin, glucagon, and H&E stains; 0=normal islet, 1=MNC in periphery of <25% of islet, 2=25–50% islets with MNC, 3=>50% islets with MNC infiltrate, 4=small, retracted islet with few MNC Cy (n=5) 3.2±0.45 Cy+non-activated T cells (n=5) 1.2±0.45 Cy+3/28 bead-activated T cells (n=6) 0.17±0.41