Fig 2: PD-1 blockade enhanced adoptive T-cell therapy and was associated with decreased tumor infiltration by transferred T cells and increased tumor infiltration by endogenous T cells. (A) Tumor response to treatment with adoptive T-cell therapy with or without PD-1 blockade. C57BL/6J (CD45.2+) mice with established B16-K-OVA tumors were treated with host conditioning followed by the treatment indicated in the symbol legend. ‘OT-I’ indicates OT-I T cell (CD45.1+) administration. ‘Anti-PD-1’ indicates anti-PD-1 antibody administration. Statistical significance was determined with a mixed model two-way analysis of variance with repeated measures. The data shown are representative of three independent experiments. (B) Absolute numbers (cells/g tumor) of transferred OT-I T cells (CD45.1+, red) and endogenous CD8 T cells (CD45.2+, gray) infiltrating tumors on day 7 after treatment. Statistical significance was determined with an unpaired t-test. (C) Proportion of transferred OT-I T cells (CD45.1+, red) and endogenous CD8 T cells (CD45.2+, gray) infiltrating tumors on day 7 after adoptive cell transfer. (D) Absolute numbers (cells/g tumor) of endogenous CD4+ T cells infiltrating tumors on day 7 after adoptive cell transfer. Statistical significance was determined with an unpaired t-test. All data have five mice per group and are represented as mean±SEM. * represents p<0.05, ** represents p<0.01. See also online supplemental figure S1. PD-1, programmed cell death protein-1.

Fig 3: Tracking lung-resident memory B cells(A) Schematic of BAT reporter mice.(B) FACS plots of naive (B220+ tdTomatoneg mVenusneg GL7neg) and memory (tdTomato+ mVenusneg GL7neg) cells from spleens and lungs of BAT mice 6 weeks postinfection. In the lung, plots are also pregated on parenchymal cells (in vivo CD45neg).(C) Frequencies of cells expressing the indicated markers gated as in (B). Data represent one of 3 independent experiments. Statistical analysis was made using an ordinary one-way ANOVA. Error bars represent SD. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(D–F) Bulk RNA-seq of naive B cells and HA+ memory B cells from lung and spleen derived from BAT mice. Lung B cells were also pre-gated on parenchymal CD69+ cells. Gating shown in Figure S1D. (D) Heatmap of key migration and residency genes. (E) Full heatmaps of all differentially expressed genes with an adjusted p value (FDR) < 0.05. (F) PCA plots, applied to the top 500 most variable genes. Shown are data from 3 samples collected in 3 independent experiments (n = 6 per experiment).(G) HA specific memory B cell, as defined as in (E and F) and PC numbers (gating shown in Figure S1D) in BAT mice, quantified over time. Data in (G) are pooled from 6 independent experiments with 4–6 mice per group. See also Figure S1.

Fig 4: VRC01-class BGC cell kinetics in response to MD39-GT3.1(A) Schematic of the experiment. Day 21 corresponds to either day 21 or day 22, depending on the replicate. Control: N = 3, n = 5; HYCAP1: N = 1, n = 5; HYCAP3: N = 3, n = 5.(B) Representative flow plot showing BGC VRC01gHL gating on day 10, from a HYCAP3 T cell recipient mouse. Cells were gated as in Figure 1C.(C) Representative flow plot showing the AIM gating strategy from a HYCAP1 T cell recipient mouse on day 10. Cells are gated on CD4+ T cells. Frequencies shown in the AIM+ (CD40L+CD69+) gates shows the percentage of AIM+ cells among total CD4 T cells. Control: N = 2, n = 5; HYCAP1: N = 1, n = 5; HYCAP3: N = 2, n = 5.(D) Frequency of total BGC cells.(E) Percentage of VRC01gHL cells among BGC cells; gated as CD45.1+ or CD45.1-CD45.2+, depending on the congenic marker of transferred VRC01gHL cells.(F) Number of BGC VRC01gHL cells in the spleen, back-calculated using the number of total splenocytes.(G) Total MD39-specific CD4 T cell responses, gated as in (C).(H) MD39-specific TFH responses, gated as in (C).(I) Percentage of HYCAP (CD90.1+TVß12+) cells among total MD39-specific TFH cells.(J) Area under the curve (AUC) analysis of total serum response to the trimer with and without RM19R competition.Pooled data from 3 experiments are shown in (D–F). Pooled data from 2 experiments are shown in (G–I). Mean and standard deviation are shown for graphs on linear scales. Geometric mean and geometric standard deviation are shown for data graphed in log scale. Two-tailed Student’s t test: ns, p > 0.05; *p <0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.