Fig 2. CD4⁺ T cells from Ech_0660 mutant vaccinated and wild-type E. chaffeensis infected animals proliferate in response to E. chaffeensis antigen.

PBMC from dogs vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis via needle inoculation (group 1, left panels), vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis via tick inoculation (group 2, middle panels), or unvaccinated and infected with wild-type E. chaffeensis or Ech_0480 via tick inoculation (group 3, right panels) were labeled with Cell Trace Violet, then cultured for 5 days at 4x10⁶ cells/mL in the presence or absence of 10 ug/mL E. chaffeensis host-cell free lysate grown in the tick ISE6 cell line. On day 5, CD4⁺ T cells were analyzed by flow cytometry for Cell Trace Violet dilution as a measure of proliferation. (A) Representative Cell Trace Violet dilution profiles, gated on total live cells and total CD3⁺CD4⁺ T cells. (B) The percentage of CD4⁺ T cells that have proliferated in response to E. chaffeensis antigens as measured over the course of the experiment. The background (mock stimulated) proliferation was subtracted, and results represent change over mock. Each line is representative of a single animal.