HF synergistically interacts with RAPA in the suppression of TCR stimulated T cell proliferation.

<p>T cell proliferation in response to the stimulation of anti-CD3 antibody binding to TCR complex was measured by using MTT assay or trypan blue exclusion. (A) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 1 nM of RAPA for 48 hrs. (B) Anti-CD3 antibody-stimulated splenocytes were treated with RAPA alone or in combination with 2.5 nM of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments, and were statistically analyzed using two-way ANOVA. P < 0.0001 (HF <i>vs</i>. HF + RAPA); P < 0.0001 (RAPA <i>vs</i>. RAPA + HF). γ = 0.1905 based on IC₇₀ values in these two experiments. (C) Anti-CD3 antibody-stimulated splenocytes were treated with HF (1 or 2 nM) or RAPA (0.5 nM) alone or in combination with both drugs for 48 hrs, followed by counting viable cells using trypan blue exclusion assay. Data are presented as mean ± SD of three experiments. The Bliss independence: τ < α. (D) The splenocyte cultures were treated with HF alone, RAPA alone or both HF and RAPA for 18 hrs, and the cell growth was determined by using MTT assay. The difference of inhibition between groups was analyzed using two-tailed <i>t</i>-test. Data are presented as mean ± SD of five experiments. The Bliss independence: τ < α. (E) The splenocyte cultures were treated with HF alone or in combination with 1 nM RAPA for 36 hrs. (F) The splenocyte cultures were treated with RAPA alone or in combination with 2.5 nM HF for 36 hrs. The cell growth was determined using MTT assay. Data are presented as mean ± SD of five experiments. The difference of drug inhibitions between groups was analyzed using two-way ANOVA. P < 0.0001 (HF <i>vs</i>. HF + RAPA); P < 0.0001 (RAPA <i>vs</i>. RAPA + HF). γ = 0.201 based on IC₇₀ values in these two experiments.