Figure 4.

R21/LMQ and R21/SQ induce disparate CD4⁺ T cell responses

(A) Summary of the experimental protocol. C57BL/6 mice require three vaccine doses to develop full response against R21.⁴³

(B) Representative FACS plots of R21 peptide pool stimulated splenocytes from mice with indicated genotype and vaccine adjuvant used. Graphs show IFN-γ and TNF-α double-positive CD4⁺ T cells. Cells were pre-gated on live single cells, CD19⁻, CD3⁺, and CD8⁻.

(B and C) Representative data from two experiments; mean ± SEM; n = 3 for WT/R21, WT/LMQ, and WT/SQ; n = 4 for Nlrp3^−/−/R21; n = 5 for Nlrp3^−/−/LMQ and Nlrp3^−/−/SQ.

(C) Quantification and summary of data represented in (B).

(D) IFN-γ and IL-13 in supernatants of R21 peptide pool stimulated splenocytes from mice with indicated genotype and vaccine adjuvant used, as measured by ELISA. Representative data from two experiments.

(E and F) Pooled data from three experiments; median + replicates; n = 9, WT/R21; n = 13, Nlrp3^−/−/R21; n = 12, WT/LMQ; n = 22, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 20, Nlrp3^−/−/SQ. (E) Anti-NANP serum antibody titers of indicated isotypes. C57BL/6 WT or Nlrp3^−/− mice were vaccinated and sampled as indicated in (A). Titers were assessed by ELISA. (F) Detection of anti-NANP IgG subclasses by proportional ELISA. Each sample was diluted and normalized to 80 ng/mL of total IgG. Graphs show optical density against indicated IgG subclasses. Pooled data from three independent experiments; mean ± SEM; n = 12, WT/LMQ; n = 22, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 20, Nlrp3^−/−/SQ.

(G) T_H1/T_H2 index of adjuvant-induced subclass patterns. Index was calculated as ([IgG2c + IgG3]/2)/IgG1.⁴⁴ Increased T_H1/T_H2 index indicates T_H1-skewed immune response. Pooled data from three independent experiments; median + replicates; n = 12, WT/LMQ; n = 21, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 19, Nlrp3^−/−/SQ; all statistical analyses were done using two-way ANOVA of adjuvanted groups with Bonferroni’s multiple comparisons; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.