Fig. 3. EBNA1 and CRYAB immunization cross-prime CD4⁺ T cells in vivo.

Draining lymph node lymphocytes from nonimmunized mice (n = 3) or mice immunized with recombinant CRYAB (n = 4), EBNA1_380–641 (n = 3), or Freund’s complete adjuvant (FCA; n = 3) were examined for antigen reactivity using recall stimulations and intracellular cytokine staining flow cytometry. (A) CD4⁺ T cells responding to restimulation after 10 days with bead-bound antigens. Data are presented as the fold change of %CD3⁺CD4⁺IFNγ⁺ over the naked bead (NB)–negative stimulation control. (B) Comparison of CD4⁺ responses within the EBNA1_380–641 and CRYAB immunization groups. Data are presented as in (A). (C) Responses of CD8⁺ T cells from draining lymph node lymphocytes responding to restimulation as in (A). Data are presented as the fold change of %CD3⁺CD8⁺IFNγ⁺ over the NB negative control. (D) Comparison of CD8⁺ responses within the EBNA1_380–641 and CRYAB immunization groups [as in (C)]. Each dot represents one biological replicate. P values were calculated using a two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, comparing each different restimulation within the immunization group and indicated where significant. Bars and staples denote means ± SEM. *P < 0.05; ***P < 0.001. RASGRP2, RAS guanyl-releasing protein 2 (29); SYNB, synuclein beta (28); GDPLFS, GDP-l-fucose synthase.