Fig 3. Extrinsic MyD88 regulates T cell contraction.

(A) 10⁶ CD4⁺ purified, CFSE-labeled, naïve 1807 Tg cells were transferred into wild type and Myd88 ^-/- mice that were then vaccinated s.c. with 10⁶ heat-killed vaccine yeast or not. At day 7 post-vaccination, the skin draining lymph nodes (sdLN) were harvested and the numbers of activated (CD44⁺) 1807 T cells and endogenous CD4⁺ T-cells enumerated by FACS. (B) T cell proliferation of 1807 T cells is indicated by CFSE loss. (C). BMDC from wild type and Myd88 ^-/- mice were co-cultured with vaccine yeast for 24 h and transcript analyzed by RT-PCR. * P < 0.05 vs. increase in transcript in wild type DC. (D) At day 11 post-vaccination, the frequency of cytokine producing 1807 T cells was determined at the site of vaccination. Dot plots show the sum of concatenated events from 6 mice/group and the numbers indicate the mean ± SEM of cytokine producing 1807 T cells. (E) Upon recall at day 4 post-infection, the frequency of IL-17 producing 1807 T cells was quantified in the lung. Data are representative of two independent experiments. (F) At day 35 post-vaccination, endogenous CD4⁺ T cells were purified from the skin draining lymph nodes (sdLN) and spleen of vaccinated wild type and Myd88 ^-/- mice and stimulated with CW/M antigen for 3 days. Cytokines were measured in the cell-culture supernatants by ELISA. *P < 0.05 vs. cytokine production by CD4⁺ T cells from vaccinated wild type controls. (G, H) 10⁶ purified naïve CD4⁺ 1807 T cells were transferred into wild type and Myd88 ^-/- mice that were vaccinated s.c. with 10⁶ heat-killed vaccine yeast or not. At serial time points post-vaccination, the sdLN and lung were harvested and the number of CD44⁺ 1807 T cells enumerated by FACS. * P < 0.05 vs. number of activated CD4⁺ T cells from vaccinated wild type controls. Data are representative of three independent experiments.