Superior depletion of alloreactive T cells from peripheral blood stem cell and umbilical cord blood grafts by the combined use of trimetrexate and interleukin-2 immunotoxin  Paul Szabolcs, Kyung-Duk Park, Luciana Marti, Divinomar DeOliveria, Young-Ah Lee, Michael O. Colvin, Joanne Kurzberg  Biology of Blood and Marrow Transplantation  Volume 10, Issue 11, Pages 772-783 (November 2004) DOI: 10.1016/j.bbmt.2004.07.005 Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Trimetrexate (TMT) depletes alloreactivity in a dose-dependent manner. A-E, FACS profile of proliferation (cells expressing IC KI-67) or apoptosis (cells expressing IC active caspase-3) among alloreactive peripheral blood T cells at the end of re-stimulation with the original APCs (EBV/LCLs) on day 5 of the secondary MLR. A, Unmanipulated alloreactive T cells were mock-treated while receiving maximal stimulation by EBV/LCLs. B-D, Proliferating alloreactive T cells expressing Ki-67+ (cells in green) were progressively depleted in a dose-dependent manner with an increased molar TMT concentration added to an MLR for the first 3 days. Simultaneously, the percentage of apoptotic T cells expressing active caspase-3 IC (cells in red) was increased similarly in a dose-dependent way. All depicted cells are gated on CD3+ T cells. E, IL-2 IT added on day 3 at 0.5 μg/mL for 30 minutes to a primary MLR induced antiproliferative and proapoptotic effects similar to TMT. F, Proliferation of alloreactive T cells was quantitated by 3H-thymidine incorporation and expressed in counts per minute (CPM). The responder cells were from the same MLRs as cells depicted in (A-E). Results are representative of 6 experiments. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Trimetrexate depletes alloreactivity in a time-dependent manner. The efficiency of alloreactive T-cell depletion was tested for its dependence on continued exposure to TMT at 10−6 mol, demonstrating increasing efficacy with each additional day passing (3 days’ exposure versus 4 or 5 days). Proliferation of alloreactive T cells was quantitated by 3H-thymidine incorporation and expressed in counts per minute (CPM). Results are representative of 6 experiments. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 TMT and IL-2 IT act synergistically in depleting PBSC or UCB alloreactive T cells. A, The effect of allodepletion methods on alloreactive T-cell proliferation was evaluated at the end of the primary MLR on the fifth day by measuring 3H-thymidine incorporation (counts per minute). Responder lymphocytes were generated from PBSC grafts and were stimulated by HLA-mismatched EBV/LCLs. TMT exposure was 3, 4, or 5 days during the MLR, as depicted, and IL-2 IT was administered on the fourth day of primary MLR for 30 minutes and was then washed out. Results are representative of 6 experiments. B, Cord blood alloreactive T cells stimulated by allogeneic MO/DCs were depleted by TMT plus IL-2 IT in a synergistic way. TMT exposure was for 5 days; IL-2 IT was administered on the fourth day of primary MLR. Results are representative of 5 experiments. C, Effect of allodepletion methods on alloreactive T-cell proliferation as evaluated at the end of re-stimulation (third day of secondary MLR; eighth day from obtaining the graft sample). PBSC responder lymphocytes from the primary MLR presented in Figure 3A were washed and then re-stimulated with original EBV/LCLs in a secondary MLR. Results are representative of 6 experiments. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 TMT and IL-2 IT deplete TNF-α-secreting alloreactive T cells. A, Percentage of T cells expressing IC TNF-α at the end of the primary MLR. Responder lymphocytes isolated from PBSCs were stimulated for 5 days by MO/DC stimulators in the primary MLR for 5 days and were manipulated as indicated except the unstimulated control. (“Unmanipulated” indicates full alloreactive activation without depletion efforts.) B, Percentage of T cells that expressed IC TNF-α in each group after re-stimulation with fresh MO/DCs in the secondary MLR. Results are representative of 4 experiments. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 TMT and IL-2 IT deplete alloreactive CD8+ T cells that express granzyme A. Responder lymphocytes isolated from PBSCs were stimulated for 5 days by MO/DCs in a primary MLR, after which each group was re-stimulated with the same APCs in the secondary MLR (except the unstimulated control that had no APC present). The percentage of CD8−APC+ T cells that expressed intracellular granzyme A/FITC after re-stimulation is presented. Results are representative of 4 experiments. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 TMT and IL-2 IT deplete alloreactive T cells but preserve overall T-cell responsiveness and third-party reactivity. Peripheral blood T cells were stimulated by irradiated EBV/LCLs and were treated with either TMT or IL-2 IT. Unmanipulated cells were rested without any alloactivation or treatment with depleting agents. On day 5, cells were washed and were re-stimulated with autologous monocytes and loaded with 5 μg/mL of PHA (A) for 3 days or 10 μg/mL of candida antigen (C) for 5 days before pulsing with 3H-thymidine. Proliferation is expressed in counts per minute (CPM). Third-party responsiveness (B) was tested in an allogeneic MLR. On day 5 at the end of the primary MLR stimulated by MO/DC responder cord blood, cells were washed off the deletion agents indicated and were re-stimulated with irradiated third-party EBV/LCLs for another 5 days before the cultures were pulsed with 3H-thymidine. Proliferation is expressed in CPM. Biology of Blood and Marrow Transplantation 2004 10, 772-783DOI: (10.1016/j.bbmt.2004.07.005) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions