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. Author manuscript; available in PMC: 2009 Apr 15.

Published in final edited form as: J Immunol. 2008 Apr 15;180(8):5187–5200. doi: 10.4049/jimmunol.180.8.5187

OVA-pσ1-induced unresponsiveness is mediated by CD4⁺ T cells. (A) CD4⁺ T cells isolated from CLNs and spleens of mice adoptively transferred with CLN-derived CD4⁺ T cells from OVA-pσ1- or OVA-dosed mice were cultured in vitro without or with 1 mg of OVA for 5 days. ³H-TdR incorporation was measured and expressed as a stimulation index (SI). For CLN, ^§ p ≤ 0.001 versus i.m. OVA; for spleen** p = 0.003, ***p = 0.006 vs. i.m. OVA, calculated by Student’s t test. (B, C) BALB/c mice were adoptively transferred with Vybrant-labeled DO11.10 Tg CD4⁺ T (responder) cells and with the designated T cell population (donor CD4⁺ T cells) isolated from OVA-pσ1- or OVA-dosed mice. (B) In vivo suppression of proliferation of DO11.10 Tg CD4⁺ T cells following challenge with OVA was measured by FACS. * p ≤ 0.001 vs. mice given CD4⁺ T cells from OVA-dosed mice, calculated by Student’s t test. (C) Lymphocytes pooled from HNLNs, MLNs, and spleens from recipient mice were stained with anti-CD4, anti-CD25, and anti-FoxP3 mAbs and analyzed by FACS. The mean percentage (± SD) of FoxP3⁺ CD25⁻CD4⁺ T cells and FoxP3⁺ CD25⁺CD4⁺ T cells as a (left panel) proportion of total CD4⁺ T cells, or (right panel) DO11.10 CD4⁺ T cells is depicted. * p ≤ 0.001 vs. CD25⁺CD4⁺ T cells from mice given CD4⁺ T cells from OVA-dosed mice; ^§ p ≤ 0.001, ** p ≤ 0.05 vs. CD25⁻CD4⁺ T cells from mice given CD4⁺ T cells from OVA-dosed mice, calculated by Student’s t test. (D, E) FACS analysis was performed on lymphocytes isolated from C57BL/6 mice (D) naïve or (E) nasally dosed with OVA-pσ1 three times at weekly intervals. Depicted are percentages of unstimulated T_reg cells isolated from HNLNs. Percentage of FoxP3⁺ T_reg cells (upper filled histogram) and FoxP3⁺ CD25⁻CD4⁺ T cells (lower filled histograms) as proportion of CD4⁺ T cells are plotted versus isotype control for FoxP3 (empty histogram). Results are presented as an average ± SD of 5 animals per tissue.

OVA-pσ1-induced unresponsiveness is mediated by CD4⁺ T cells. (A) CD4⁺ T cells isolated from CLNs and spleens of mice adoptively transferred with CLN-derived CD4⁺ T cells from OVA-pσ1- or OVA-dosed mice were cultured in vitro without or with 1 mg of OVA for 5 days. ³H-TdR incorporation was measured and expressed as a stimulation index (SI). For CLN, ^§ p ≤ 0.001 versus i.m. OVA; for spleen** p = 0.003, ***p = 0.006 vs. i.m. OVA, calculated by Student’s t test. (B, C) BALB/c mice were adoptively transferred with Vybrant-labeled DO11.10 Tg CD4⁺ T (responder) cells and with the designated T cell population (donor CD4⁺ T cells) isolated from OVA-pσ1- or OVA-dosed mice. (B) In vivo suppression of proliferation of DO11.10 Tg CD4⁺ T cells following challenge with OVA was measured by FACS. * p ≤ 0.001 vs. mice given CD4⁺ T cells from OVA-dosed mice, calculated by Student’s t test. (C) Lymphocytes pooled from HNLNs, MLNs, and spleens from recipient mice were stained with anti-CD4, anti-CD25, and anti-FoxP3 mAbs and analyzed by FACS. The mean percentage (± SD) of FoxP3⁺ CD25⁻CD4⁺ T cells and FoxP3⁺ CD25⁺CD4⁺ T cells as a (left panel) proportion of total CD4⁺ T cells, or (right panel) DO11.10 CD4⁺ T cells is depicted. * p ≤ 0.001 vs. CD25⁺CD4⁺ T cells from mice given CD4⁺ T cells from OVA-dosed mice; ^§ p ≤ 0.001, ** p ≤ 0.05 vs. CD25⁻CD4⁺ T cells from mice given CD4⁺ T cells from OVA-dosed mice, calculated by Student’s t test. (D, E) FACS analysis was performed on lymphocytes isolated from C57BL/6 mice (D) naïve or (E) nasally dosed with OVA-pσ1 three times at weekly intervals. Depicted are percentages of unstimulated T_reg cells isolated from HNLNs. Percentage of FoxP3⁺ T_reg cells (upper filled histogram) and FoxP3⁺ CD25⁻CD4⁺ T cells (lower filled histograms) as proportion of CD4⁺ T cells are plotted versus isotype control for FoxP3 (empty histogram). Results are presented as an average ± SD of 5 animals per tissue.