Figure 9

CD4⁺CD62L^lowmemory/effector subset produces Th2-type cytokines and directly induces transfer-mediated AHR. (A and B)CD4⁺CD62L^low memory/effector subset proliferates and produces Th2-type cytokines with antigen stimulation. On day 18, CD4⁺ T cells (CD4), CD4⁺CD62L^high T cells (CD62Lhi), or CD4⁺CD62L^low T cells (CD62Llo) from OVA-sensitized mice were positively selected by magnetic cell sorting as described in Methods. Then, these cells were cultured with freshly isolated mitomycin C-treated splenocytes in the presence of OVA. After 48 hours, the proliferation was assessed by BrdU incorporation using ELISA (A). The maximum proliferation observed in response to OVA for CD4⁺CD62L^low T cells from OVA-sensitized mice was set as control (100%). After 72 hours, Th2-type cytokine levels in the supernatants were assayed using ELISA (B). *** p < 0.001 compared with the value of CD4⁺CD62L^high T cells. (C and D) CD4⁺CD62L^low memory/effector T cells induce AHR. Recipients received CD4⁺ (CD4; 1.25 × 10⁷), CD4⁺CD62L^high (CD62Lhi; 8.5 × 10⁶), or CD4⁺CD62L^low (CD62Llo; 4 × 10⁶) cells from OVA-sensitized mice. AR was measured 4 days after the transfer. (C) AR was measured by Penh methods (n = 5–6 per group). * p < 0.05 compared with PC₂₀₀Mch of mice that received CD4⁺CD62L^high T cells. (D) AR was assessed by measurement of Raw (n = 6–8 per group). * p < 0.05 compared with PC₂₀₀Mch Raw of mice that received CD4⁺CD62L^high T cells. (E)Fluorescence study. CD4⁺CD62L^low cells from OVA-sensitized mice (4 × 10⁶) were labeled with fluorescent dye, and then transferred into recipients. Five-micrometer sections of the lungs were observed by fluorescence microscopy 24 hours after the transfer (CD62Llo). Scale bar, 100 μm. (E) Double staining analysis by immunohistochemistry. T cells were detected by staining for CD3 (cytoplasm, blue). Proliferation was assessed by staining for PCNA (nucleus, brown). Lung sections from mice that received OVA-sensitized CD4⁺CD62L^low cells (CD62Llo; 4 × 10⁶) are shown. Proliferating T cells were clearly detected (black arrow). Scale bar, 40 μm.