Figure 2

OVA-specific T helper cells of cMy-mOVA-OT-II mice are not anergic and proliferate in vitro in response to OVA. Splenocytes derived from 8 to 12 weeks old C57BL/6, cMy-mOVA, OT-II, and cMy-mOVA-OT-II mice (n = 8 for each strain) were stained with CFSE and cultured in absence (w/o OVA) or presence of 250 µM OVA (plus OVA) for 5 days. Afterwards, the cells were stained with anti-CD3 and anti-CD4 antibodies and the CFSE fluorescence of the CD3⁺CD4⁺ T cells was determined by flow cytometry. (a) Examples of gating of CD3⁺CD4⁺ T cells and the CFSE fluorescence on CD3⁺CD4⁺ T cells of a cMy-mOVA (upper panels) and a cMy-mOVA-OT-II mouse (lower panels) after culture in presence of OVA are shown. The fractions of proliferating cells with reduced CFSE intensity (CFSE^dim) were identified in the marked range. (b) Means plus standard errors of the mean (SEM) of the proportions of CD3⁺CD4⁺ T cells and (c) proliferating T helper cells (CD3⁺CD4⁺CFSE^dim) are shown. (d) The MFI of CFSE of CD3⁺CD4⁺ T cells was determined and means plus SEM are displayed. (e) In addition, the division index (DI) of the CD4⁺ T cells has been calculated and is shown as means plus SEM. Effects of the stimulation with antigen (w/o OVA versus plus OVA) and differences between the four mouse strains were analyzed by 2-way-ANOVA and the P-values are indicated above the diagrams. In addition, differences between the four mouse strains were analyzed separately for cultures without (strain (w/o OVA)) and with antigen (strain (plus OVA)) by 1-way-ANOVA. If significant differences were revealed, Bonferroni post hoc tests always indicated significant differences (P < 0.01) between the TCR-transgenic strains (OT-II and cMy-mOVA-OT-II) and the non-TCR-transgenic strains (C57BL/6 and cMy-mOVA) but none within these groups. Moreover, significant differences between control (w/o OVA) and antigen stimulated (plus OVA) cells within each of the four strains were evaluated by t-tests and are indicated in the panels (^# P < 0.001).