Figure 4
Figure 4. Anti–IL-2Rα mAb blockade synergizes with TMZ-induced lymphodepletion to enhance antigen-specific immunity. (A) Forty-eight hours after TMZ treatment, lymphodepleted or untreated C57BL/6 mice received OVA vaccination with or without simultaneous anti–IL-2Rα mAb treatment (n = 5/group). Immune responses were monitored for 1 month by FACS analysis of peripheral blood OT-I T-cell levels. This experiment was repeated twice with similar results, and unpaired t tests were generated against the untreated + vaccine cohort; TMZ + vaccine and TMZ + vaccine + αIL-2Rα mAb cohorts were significantly different from untreated + vaccine for weeks 1 to 4. Baseline values were determined in untreated and TMZ-treated mice that did not receive vaccination. At week 4, the percentage of CD8+ T cells observed with TMZ significantly increased with the addition of anti–IL-2-Rα mAb (P = .036). (B) Representative FACS plots shown at 1 and 4 weeks after vaccination. (C) To determine the ratio of effector cells to TRegs, the absolute number of CD8+OVA+ effectors in peripheral blood 1 week after vaccination was assessed by FACS analysis with OVA+ tetramers and divided by the absolute number of CD4+CD25+Foxp3+ TRegs. (D) Peripheral blood was collected from untreated and TMZ-lymphodepleted (60 mg/kg/5 days) C57BL/6 mice treated with or without αIL-2Rα mAb 72 hours after the termination of TMZ administration. Cytokine levels in plasma were measured by Luminex according to manufacturer's instructions. For cytokine measurements, analysis of variance with interaction was conducted with TMZ and αIL-2Rα mAb as the main effects and their statistical interaction. TMZ + αIL-2Rα had a significant effect on IL-2 production (P = .0005), and TMZ alone had a significant effect on IL-7 production (P = .0001). Asterisks denote significance (P < .05) versus untreated. (E) Memory recall responses were evaluated 5 weeks after vaccination in treated mice by measurement of IFNγ secretion in peripheral blood lymphocytes using CBA. *P = .0082 and **P = .0198 by unpaired t test.

Anti–IL-2Rα mAb blockade synergizes with TMZ-induced lymphodepletion to enhance antigen-specific immunity. (A) Forty-eight hours after TMZ treatment, lymphodepleted or untreated C57BL/6 mice received OVA vaccination with or without simultaneous anti–IL-2Rα mAb treatment (n = 5/group). Immune responses were monitored for 1 month by FACS analysis of peripheral blood OT-I T-cell levels. This experiment was repeated twice with similar results, and unpaired t tests were generated against the untreated + vaccine cohort; TMZ + vaccine and TMZ + vaccine + αIL-2Rα mAb cohorts were significantly different from untreated + vaccine for weeks 1 to 4. Baseline values were determined in untreated and TMZ-treated mice that did not receive vaccination. At week 4, the percentage of CD8+ T cells observed with TMZ significantly increased with the addition of anti–IL-2-Rα mAb (P = .036). (B) Representative FACS plots shown at 1 and 4 weeks after vaccination. (C) To determine the ratio of effector cells to TRegs, the absolute number of CD8+OVA+ effectors in peripheral blood 1 week after vaccination was assessed by FACS analysis with OVA+ tetramers and divided by the absolute number of CD4+CD25+Foxp3+ TRegs. (D) Peripheral blood was collected from untreated and TMZ-lymphodepleted (60 mg/kg/5 days) C57BL/6 mice treated with or without αIL-2Rα mAb 72 hours after the termination of TMZ administration. Cytokine levels in plasma were measured by Luminex according to manufacturer's instructions. For cytokine measurements, analysis of variance with interaction was conducted with TMZ and αIL-2Rα mAb as the main effects and their statistical interaction. TMZ + αIL-2Rα had a significant effect on IL-2 production (P = .0005), and TMZ alone had a significant effect on IL-7 production (P = .0001). Asterisks denote significance (P < .05) versus untreated. (E) Memory recall responses were evaluated 5 weeks after vaccination in treated mice by measurement of IFNγ secretion in peripheral blood lymphocytes using CBA. *P = .0082 and **P = .0198 by unpaired t test.

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