Fig. 6.

CD40 targeting is required for optimal induction of CD8⁺Tscm cells. (a) Proportion of CD8⁺ Tscm, CM, and EM cells among human CD8⁺ T cells in the spleens of HIS-mice one week after the last vaccination. The HIS-mice were vaccinated twice with the CD40.CoV2 vaccine alone, the CD40.CoV2 vaccine plus poly-ICLC, the Pfizer BNT162b2 mRNA vaccine, or IgG4.CoV2. Mock animals received two injections of PBS or poly-ICLC. The CD8⁺ Tscm and CD8⁺ EM cell induction was compared between the CD40.CoV2 group versus IgG4.CoV2 or BNT162b2 mRNA groups using the Mann–Whitney test with a Bonferroni correction. Median and individual values from two independent experiments are shown. ∗p < 0.05, ∗∗p < 0.01. (b) Proportion of Wuhan- and XBB.1.5-specific CD8⁺ Tscm cells (CD3⁺ CD8⁺ AIM⁺ CD62L⁺ CD44^- CCR7⁺ CD95⁺ Sca-1⁺) with a non-exhausted or exhausted (PD-1⁺ TIGIT⁺) phenotype in the spleen of hCD40 transgenic mice one week after the last vaccination (the gating strategies are shown in Figure S2b and d). The hCD40 Tg mice received an injection of CD40.CoV2 adjuvanted with the poly-ICLC (10 μg of vaccine with 50 μg of poly-ICLC) or Comirnaty XBB.1.5 mRNA vaccine (1 μg) at day 0 and day 21. Spleen cells collected at day 28 were re-stimulated with Wuhan or XBB.1.5 RBD OLPs for 20 h. The frequencies of memory CD8⁺ T cell subsets were examined by flow cytometry among the specific AIM⁺ (CD25⁺ CD69⁺) CD8⁺ T cells (see the gating strategy in Figure S2b). (c) Proportion of Wuhan- or XBB.1.5 RBD-specific CD8⁺ EM and CM T cells in the hCD40 Tg mice. The CD40.CoV2 group versus controls or Comirnaty mRNA group were analyzed using the Mann–Whitney test. Geometric means ± SEM and individual values from two independent experiments are shown. ∗p < 0.05.