Co-expression of B7-H3 and LAG3 represents cytotoxicity of CD4+ T cells in humans

Abstract

Recent studies have highlighted the potential contribution of CD4⁺ T cells with cytotoxic activity (CD4 CTLs) to anti-tumor immunity. However, their precise roles remain elusive, partly due to the absence of specific markers defining CD4 CTLs with target-killing potential in humans. We previously demonstrated that Epstein-Barr virus (EBV)-driven immortalized B cell lines efficiently induce human CD4 CTLs with cytotoxic functions comparable to cytotoxic CD8⁺ T cells (CD8 CTLs). Here we show that EBV-driven CD4 CTLs exhibit prolonged proliferation and sustained cytotoxicity compared with CD8 CTLs, although their cytotoxic function markedly decreased during long-term culture. Comparative transcriptomic analysis of CD4 CTLs with varying cytotoxic activities identified B7-H3 and LAG3 as surface molecules associated with highly cytotoxic CD4 CTLs. Co-expression of B7-H3 and LAG3 correlated with CD107a expression and was observed on CD4⁺ T cells with enhanced cytotoxic potential in a target-dependent manner but not on CD8 CTLs. Furthermore, B7-H3⁺LAG3⁺ CD4⁺ T cells were induced during co-culture with bone marrow cells from pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL). These findings suggest that B7-H3 and LAG3 co-expression represents a characteristic feature of functional CD4 CTLs in humans, providing valuable insights into the role of CD4 CTLs in tumor immunity.

Keywords: B7-H3; CD4+ cytotoxic T cells; EB virus; LAG3; leukemia; lymphoma; tumor immunity.

Figure 1

CD4 CTLs proliferate longer than CD8 CTLs but lose their activity upon repeated stimulation (A) Schematic representation: CD4⁺ and CD8⁺ T cells were two-way-sorted from the peripheral blood T cells from healthy donors and independently stimulated weekly with irradiated autologous LCLs generated from the same donor. IL-7 and IL-2 were added to the culture medium from day 0 and day 4, respectively. (B) Representative flow cytometry plots for the expression of Granzyme B (GZMB) and Perforin on day 0 (upper panels) and day 16 (lower panels) of stimulation with autologous LCLs in the CD45RO⁺CD4⁺ compartment (left panels) and CD45RO⁺CD8⁺ compartment (right panels). Percentage in the gate is shown. (C) Percentage of cytotoxic T cell population defined by the co-expression of GZMB and Perforin at day 16-30 of CD4⁺ and CD8⁺ T cells (n=3). (D) Percentage of specific killing activity at day 16-30 of CD4⁺ and CD8⁺ T cells (n=4) against autologous LCLs. Results of the 10:1 effector to target (E/T) ratio are shown. (E) Accumulated cell number of CD4⁺ (filled circles) and CD8⁺ (open circles) T cells from 6 independent experiments involving 4 healthy donors (n=6). (F) Proliferative duration of CD4⁺ and CD8⁺ T cells (n=6). (G) Representative cytotoxic activity of CD4⁺ T cells on day 30 (4CTL-ST) and day 68 (4CTL-LT) at the indicated E/T ratio. (H) Cumulative analysis of killing activity for CD4⁺ T cells from days 16-30 (4CTL-ST) and days 56-105 (4CTL-LT) from 6 independent experiments involving 4 donors (n=6). Results of the 10:1 effector to target (E/T) ratio are shown. Data are mean ± SD with statistical significance determined by unpaired t-test (C, D, F, H) and paired t-test (I–K). The p-values are represented as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Transcriptome analysis identifies B7-H3/CD276 and LAG3 discriminating functional CD4 CTLs (A) PCA plots of unstimulated CD4⁺ T cells, 4CTL-ST (at days of 23-30), and 4CTL-LT (at days of 58-105) from three healthy donors. The top 10,000 genes with the highest variance were used to plot the PCA. (B, C) Heatmap of gene expression related to helper CD4⁺ T cell subsets (B) and cytotoxicity of lymphocytes (C) in indicated CD4⁺ T cell fractions. Color code values indicate relative expression levels shown as TPM. (D) Venn diagrams of DEGs upregulated in 4CTL-ST to unstimulated CD4⁺ T cells, and DEGs downregulated in 4CTL-LT to 4CTL-ST. The numbers of genes are indicated in each compartment. (E) The distribution of expression levels (measured as TPM) of all RNA-sequenced genes (in gray) and genes encoding the surface membrane proteins (in red) in the indicated samples. Log₁₀ TPM values greater than 0 in 4CTL-ST are enclosed. (F) Fold change in gene expression of the indicated molecules, normalized to β2 microglobulin, in 4CTL-ST as measured by quantitative real-time PCR (n=3). Bars show relative gene expression compared to CD4⁺ T cells stimulated with CD3/CD28 for 4 days.

Figure 3

B7-H3 and LAG3 co-expression correlates with functional CD4 CTLs (A) Representative FACS plots for B7-H3 and LAG3 expression on CD4⁺ T cells on day 9 after stimulation with autologous LCLs. (B, C) Percentages of B7-H3 ⁺LAG3⁺ CD4⁺ T cells (B) and CD107a⁺ CD4⁺ T cells (C) on day 9 and day 16. Each line represents independent experiments from 5 healthy donors. (D) Correlation plot showing the percentages of CD107a⁺ CD4⁺ T cells and B7-H3⁺LAG3⁺ CD4⁺ T cells on day 9 and day 16. (E) Representative contour plot showing B7-H3 and LAG3 expression on CD4⁺ T cells (left panel) and histograms depicting CD107a expression in B7-H3/⁺LAG3⁺ (blue) and B7-H3^- LAG3^- (light gray) compartment (right panel) on day 16. (F) Percentage of CD107a⁺ population in the indicated compartments from 8 independent experiments from 5 healthy donors (n=8). (G) Representative contour plots of GZMB and Perforin expression in the B7-H3⁺LAG3⁺ (right upper panel) and B7-H3^-LAG3^- (right lower panel) compartments gated on CD4⁺ T cells (left panel). (H) Percentage of GZMB⁺Perforin⁺ population (from Figure 3G ) in the indicated compartments (n=8 from 5 healthy donors). (I) % specific killing on day 26-30 at the indicated E/T ratio of sorted B7-H3⁺LAG3⁺ CD4⁺ T cells (blue) and B7-H3^-LAG3^-CD4⁺ T cells (light gray).(4 independent assays from 3 donors.) (J) Representative contour plots of B7-H3 and LAG3 on CD4⁺ T cells 48 hours after stimulation with CD3/28 antibodies (left panel) or re-stimulation with autologous LCLs (right panel). (K) Percentages of CD107a⁺ population (open bars) and B7-H3⁺LAG3⁺ population (blue filled bars) are determined in indicated T cells from Figure 3J (n=3). (L) Percentages of B7-H3⁺LAG3⁺ population in indicated T cell fractions at 48 hours after re-stimulation with autologous LCLs (n=3). Data are mean ± SD with statistical significance determined by paired t-test (B, C), unpaired t-test (F, H, L), two-way ANOVA (I), or one-way ANOVA (K). The p-values are represented as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Pearson’s correlation analysis was used to calculate correlation coefficients (D).

Figure 4

B7-H3⁺LAG3⁺ CD4⁺ T cells with enhanced cytotoxic capacity expand ex vivo from the bone marrow of pediatric B-ALL patients (A–G) Bone marrow mononuclear cells from 12 patients were cultured ex vivo with IL-2 and IL-7. Cells were analyzed by flow cytometry on day 1 and 14. Each line or dot represents an individual patient. (A) Percentages of CD107a-expressing cells in CD4⁺ T cells on day 1 and day 14 (B) Percentages of B7-H3⁺LAG3⁺ cells in CD4⁺ T cells on day 1 and day 14. (C) Correlation plot showing the percentages of CD107a⁺ CD4⁺ T cells and B7-H3⁺LAG3⁺ CD4⁺ T cells on day 1 and day 14. (D) Percentage of CD107a⁺ population in CD4⁺ and CD8⁺ T cells on day 14 (n=12) (E) Percentage of B7-H3⁺LAG3⁺ population in CD4⁺ and CD8⁺ T cells on day 14 (n=12) (F) Representative histograms for CD107a expression in B7-H3⁺LAG3⁺ (blue) and B7-H3-LAG3- (light gray) compartments on day 14. (G) Percentage of CD107a⁺ population in the indicated compartments. Samples with B7-H3⁺LAG3⁺ population at day 14 (from panel B) were analyzed (n=8). (H) Representative contour plots of GZMB and Perforin expression in the B7-H3/CD276⁺LAG3⁺ (right upper panel) and B7-H3/CD276^-LAG3^- (right lower panel) compartments gated on CD4⁺ T cells (left panel). (I) Percentage of GZMB⁺Perforin⁺population (from panel H) in the indicated compartments. Samples with B7-H3⁺LAG3⁺ population at day 14 (from panel B) were analyzed (n=8). Data are mean ± SD with statistical significance determined by paired t-tests (A, B) and unpaired t-tests (D, E, G, I). The p-values are represented as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Pearson’s correlation analysis was used to calculate correlation coefficients (C).