Figure 2.

Antagonistic CD4⁺ CTL and Tfh Development

(A) Cd4 and Gzmb expression, assessed by single-cell RNA sequencing, in env-reactive donor EF4.1 CD4⁺ T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Each symbol shows the log₂-transformed normalized reads from an individual cell from one of two experiments. Numbers within the plots denote the number of cells positive for expression of the indicated gene.

(B) Tcf7 and Bcl6 expression in the same cells as in (A).

(C) Expression of Tcf7 and Bcl6, relative to Hprt, assessed by qRT-PCR in bulk env-reactive donor EF4.1 CD4⁺ T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Plotted are the mean values (±SEM) of four technical replicates from two experiments with four mice per group per experiment.

(D) Heatmap of gene expression, assessed by single-cell RNA sequencing, comparing Gzmb⁺ and Gzmb⁻ subsets in env-reactive donor EF4.1 CD4⁺ T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and priming. CD4⁺ T cells from both FV infection and Ad5.pIX-gp70 immunization are included. A select set of genes from the complete list in Table S1 is shown.

(E) Flow-cytometric correlation of intracellular GzmB and surface markers CXCR6, CD39, CD150, and CD226 (left) and frequency of surface marker⁺ cells separately in GzmB⁻ and GzmB⁺ cells within env-reactive (CD44⁺) donor EF4.1 CD4⁺ T cells (right) in the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization. In the scatterplots, each symbol represents an individual recipient.

(F) Expression of Prdm1, Bcl6, Lef1, Tcf7, Zbtb7b, and Runx3 assessed by single-cell RNA sequencing, separately in Gzmb⁺ and Gzmb⁻ env-reactive donor EF4.1 CD4⁺ T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization.

See also Figures S4, S5, and S6 and Table S1.