Fig 2: Design of a PBMC-based LN model. (A and B) Lymphocytes were isolated from paired PB and LN samples and characterized in treatment-naïve CLL patients (n = 24). (A) Cells were gated based on the following surface markers: CLL (CD5+/CD19+), CD4+ T cells (CD3+/CD4), CD8+ T cells (CD3+/CD4+), and NK cells (CD56+). Healthy B cells (CD5−/CD19+) were excluded from the analysis due to very small populations. Error bars represent the mean ± SEM. (B) Detailed analysis of the T cell subsets. The dashed line represents the >10% CD4+ T cell cutoff we applied in selection of PB biobank samples. Error bars represent the mean ± SEM (n = 24), *P < 0.05, **P < 0.01 (2-way repeated measures ANOVA). (C) CLL sample after spheroid formation. The sample was stained for DAPI (blue), CD4 (green), CD8 (yellow), CD14 (red), and CD19 (magenta) and analyzed by confocal microscopy. (D) CLL samples were stimulated as indicated and cultured in ULA plates for 5 d. Cells were gated based on the following surface markers: CLL (CD5+/CD19+), CD4+ T cells (CD3+/CD4+), CD8+ T cells (CD3+/CD4+), and NK cells (CD56+). Healthy B cells (CD5−/CD19+) were excluded from the analysis due to very small populations. Error bars represent the mean ± SEM (n = 8). CLL = chronic lymphocytic leukemia; IL = interleukin; LN = lymph node; NK = natural killer; PB = peripheral blood; PBMCs = peripheral blood mononuclear cells; ULA = ultra-low attachment.

Fig 3: TC1 tumors enrich CD8+ T cell-suppressive PD-L1+ macrophages(A) Volcano plot of gene expression between MC38 and TC1 tumors (GEO: GSE85509).(B) Uniform manifold approximation and projection (UMAP) of untreated TC-1 tumor scRNA-seq data (GSM7103827).(C) Inferred cell-cell communication by CellChat from dataset in (B) (bandwidth indicates intensity of cell-to-cell communication).(D) Macrophages as density over expression of indicate gene levels from dataset in (B).(E) CD45+ cell fraction from TC1 tumors (day 23 after injection). Frequency of TAMs (n = 6; two-tailed paired t test).(F) Flow cytometry analysis of PD-L1+, CSF1R+, CD206+ (gating on unstained samples) on TAM from TC1 tumors isolated on day 23 post injection.(G) UMAP of TC1-tumor scRNA-seq data indicating normalized Cd274 expression (log1p-transformed reads per 10,000).(H–J) Flow cytometry of T cell recovery after cocultures with TAMs from TC1 tumors (day 23 post injection), pre-incubated with/without anti-PD-L1 for 48 h, together with paired spleen-derived T cells. (H) TAM/T cell coculture experimental setup. (I and J) Frequency of (I) CD8+ T cells and (J) CD4+ T cells (n = 3; two-tailed paired t test).(K) Relative information flow (CellChat) of Cd274+ and Cd274− macrophages (1,006 cells) from dataset in (B).(L) TNF, TRAIL, FASLG expression of indicated TAMs from TC1 tumors (isolated on day 23 post injection) (n = 5; two-way ANOVA; Sidak’s multiple comparisons test).(M) Flow cytometry analysis of live T cell recovery, as per experimental setup in (H) and (J) but pre-incubated with/without anti-TNF/anti-TRAIL for 48 h, together with paired spleen-derived T cells (n = 5; two-way ANOVA).(N) Flow cytometry analysis of Efluor 780+ dead/dying cell in untreated/anti-PD-L1-treated TC1-derived TAMs (isolated on day 23 post injection) (n = 4; two-tailed paired t test).(O) DGE of Cd274+ macrophages (blue) vs. Cd274− macrophages (red) from dataset in (B). The x axis: log2 fold change of PD-L1+ to PD-L1−. Size of circles: −log10-transformed p values.(P) Percentage TAM survival from TC1 tumors (day 23 post injection) treated with different inhibitors (n = 4; one-way ANOVA; Dunnett’s multiple comparisons test). Here, “n” represents biological replicates. See also Figure S4.

Fig 4: Increased levels of CD81 and CD82 expression within T-cell clusters boost their cytotoxic potential. (A) Overview of the experimental method and materials used. (B) Schematic of the bispecific T-cell engager, incorporating binding regions for CD3 and CEA. (C) Flow cytometry to assess transduction efficiency. Cells were separated into CD4+ and CD8+ T-cell subsets. Gray graph represents the baseline expression level, red graph illustrates the expression of CD81 after transduction with CMV-CD81, and blue graph shows the expression of CD82 after transduction with CMV-CD82. Data was analyzed 3 days after transduction. (D) Cytolysis graph comparing naive CD8+ T cells with CD81 and CD82 overexpression. Statistical significance was calculated at 48 h. The Effector : Target (E:T) ratio is 5:1, and target cells were seeded at 1E4, with bispecific engager added at 25 nM. (E) Statistical significance calculated for the area under the curve of the cytolysis data presented in (D). (F) Cytolysis graph comparing naive CD4+ T cells with CD81 and CD82 overexpression. Statistical significance was calculated at 48 h. The E:T ratio is 5:1, and target cells were seeded at 1E4, with bispecific engager added at 25 nM. (G) Statistical significance calculated for the area under the curve of the cytolysis data presented in (F). Statistical significance between groups was calculated using two-way ANOVA followed by Tukey’s multiple comparison test (**p ≤ 0.01, ****p ≤ 0.0001).

Fig 5: T cells displaying heightened levels of CD81 and CD82 expression demonstrate higher levels of activation markers. (A) T cells were stratified into CD81lowCD82low and CD81highCD82high subsets. The blue square denotes CD81lowCD82low, whereas the orange square indicates CD81highCD82high. (B) Comparison of the expression levels of activation markers, CD25 and CD69, within the CD81lowCD82low and CD81highCD82high regions across the total number of CD3+ T, CD8+ T, and CD4+ T cells. Blue region signifies gating for low CD81 and CD82 expression, whereas the orange region designates gating for the simultaneous high expression of CD81 and CD82. This comparison is presented as a histogram and bar graph to display fold changes. In the bar graph, mean fluorescence intensity (MFI) values under baseline conditions are represented using white bars, and MFI values following IL-2 treatment are indicated using black bars. Data is derived from three independent wells. (C) Comparison of the expression of exhaustion markers PD-1 and LAG-3 within the CD81lowCD82low and CD81highCD82high regions across the total number of CD3+ T, CD8+ T, and CD4+ T cells. Blue region depicts gating for low CD81 and CD82 expression, whereas the orange region signifies gating for the simultaneous high expression of CD81 and CD82. This comparison is displayed as a histogram and bar graph to demonstrate fold changes. In the bar graph, MFI values without stimulation are denoted using white bars, and MFI values after IL-2 treatment are indicated using black bars. Data is based on three independent wells. Statistical significance between groups was assessed using two-way ANOVA followed by Tukey’s multiple comparison test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).