Fig. 4: CLH001 and PBK001 vaccines generate antigen specific CD4⁺ T cells in the lung following intranasal immunization.

At day 21 post vaccination, lungs of C57BL/6 mice receiving PBS, CLH001, or PBK001 were collected and disrupted cells were co-cultured with BSA (mock) or BMDC pulsed with 1 μg of heat-killed Bpm K96243 WCL. The cultured cells were harvested after incubation at 37 °C and 5% CO₂ for 72 h, with addition of a Golgi protein transport inhibitor during the last 4 h of incubation. The markers were used to identify CD3 and CD4 T-cell populations and intracellular cytokines. a Representative gating strategy used to identify live cells with forward and side scatter properties of lymphocytes followed by selection of cells expressing the T lymphocyte markers CD3 and CD4. Intracellular cytokines including IFN-γ, TNF-α, IL-2, and IL-17 were further detected, along with the Ki-67 proliferation marker, following fixation and permeabilization of cells. Expression of intracellular cytokines IFN-γ (b), IL-17A (c), proliferation marker Ki-67 (d), TNF-α (e), and IL-2 (f) by CD3 + CD4⁺ T cells was determined in lungs of vaccinated and non-vaccinated mice. Data are represented as mean ± SEM from 10 mice/group. Significant values were analyzed using t-test or Mann–Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant).