Neoantigen-driven B cell and CD4 T follicular helper cell collaboration promotes anti-tumor CD8 T cell responses

Abstract

CD4 T follicular helper (TFH) cells support B cells, which are critical for germinal center (GC) formation, but the importance of TFH-B cell interactions in cancer is unclear. We found enrichment of TFH cell transcriptional signature correlates with GC B cell signature and with prolonged survival in individuals with lung adenocarcinoma (LUAD). We further developed a murine LUAD model in which tumor cells express B cell- and T cell-recognized neoantigens. Interactions between tumor-specific TFH and GC B cells, as well as interleukin (IL)-21 primarily produced by TFH cells, are necessary for tumor control and effector CD8 T cell function. Development of TFH cells requires B cells and B cell-recognized neoantigens. Thus, tumor neoantigens can regulate the fate of tumor-specific CD4 T cells by facilitating their interactions with tumor-specific B cells, which in turn promote anti-tumor immunity by enhancing CD8 T cell effector functions.

Keywords: B cell; CD8 T cell; IL-21; T follicular helper cell; lung cancer; neoantigen.

Figure 1.. GC B cells and TFH cells correlate with favorable clinical outcomes in LUAD patients

A, Estimated fractions of immune cell subsets from bulk expression data of primary tumor tissues in TCGA-LUAD patient cohort (n=513). CIBERSORT algorithm was used for deconvolution. B – E, scRNA-seq analyses (GSE131907, Kim et.al.) of T/NK and B cell clusters in treatment-naïve human LUAD samples from primary sites (tLung), metastatic LNs (mLN), normal lung tissues (nLung) and normal LNs (nLN). B – C, UMAP displaying T/NK (B) and B cell clusters (C). D – E, Bar graphs displaying tissue origins (top) and heatmaps (bottom) showing relative abundance of signature genes in CD4 T cell (D) and B cell clusters (E). F – G, Survival analyses based on GC B cell-signature (F) and TFH-signature (G) in TCGA-LUAD patient cohort (n=478). H, Correlation analyses on GC B cell-signature and TFH-signature in TCGA-LUAD cohort. I, Heatmap showing R values for the correlations among expression signatures of GC B cells, TFH, Th1, CD8 effector cells, Th17, NK cells in TCGA-LUAD cohort. F – G, log-rank Mantel-Cox test. H – I, two-tailed Pearson correlation test.

Figure 2.. KP-HELLO tumor cells elicit tumor-specific CD4 T cell and B cell responses

A, KP-HELLO model design. B, CD86 expression (median fluorescence intensity, MFI) on MD4 or C57BL/6 B cells (CD19⁺ B220⁺ TCRb⁻) after 48-hour co-culture with KP or KP-HELLO supernatant, or 500ng/mL HEL. C, CD44^hi CD69^hi frequency in SMARTA CD4 T cells, after 48-hour co-culture with MD4 or C57BL/6 B cells, and KP or KP-HELLO supernatant, 500ng/mL HEL or 5ug/mL GP_61-80. D – E, Representative flow plots (D) and frequency (E) of polyclonal CD4 TFH cells from tumor-draining LNs (dLNs) of KP or KP-HELLO tumor-bearing C57BL/6 mice. F – G, Representative flow plots (F) and frequency (G) of I-A^b/GP_66-77-specific (GP66-specific) CD4 TFH cells from dLNs of KP-NINJA or KP-HELLO tumor-bearing C57BL/6 mice. H – I, Representative flow plots (H) and frequency (I) of GC B cells from dLNs of KP-NINJA or KP-HELLO tumor-bearing C57BL/6 mice. No tumor C57BL/6 mice were used as negative controls. Data (mean ±SD) were pooled, from 2 – 5 mice/group/experiment, and representative of at least 2 independent experiments. LN tissues were harvested on day 10–12. B – C, E, G, I, unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.. CD4 T cells, TFH cells and B cells are necessary for optimal control over KP-HELLO tumors

A and C, Tumor growth curves: C57BL/6, CIITA KO or uMT mice were subcutaneously (s.c.) implanted with 2 x 10⁵ KP-HELLO. Growth curves of C57BL/6 (n = 40) and uMT (n = 32), pooled from 8 independent experiments, were set as the baseline of analyses. B, Tumor growth curves: C57BL/6 or CD4-Cre Bcl6^fl/fl mice were s.c. implanted with 5 x 10⁵ KP-HELLO. D, Representative flow plots of GP66-specific CD4 TFH cells from dLNs of KP-HELLO tumor-bearing C57BL/6 or uMT mice. Flow cytometric analyses were performed on day 10 – 12 and quantified in Figure S5. Flow plots were pre-gated on TCRb⁺ CD4⁺ GP66-tetramer⁺ CD44^hi PSGL1^lo. E, Tumor growth curves: C57BL/6 or uMT mice were s.c. implanted with 5 x 10⁵ KP-NINJA. Each line represented one mouse. F, 1 x 10⁶ SW_HEL or WT C57BL/6 B cells were adoptively transferred into uMT recipients, 1 day prior to KP-HELLO s.c. implant. Graph shows tumor volumes on day 16. G – I, 1 x 10⁵ SMARTA CD4 T cells (Thy1.1/Thy1.1) or OT-II CD4 T cells (Thy1.1/Thy1.2) were adoptively transferred into CD4-Cre Bcl6^fl/fl recipients (Thy1.2/Thy1.2), 1 day prior to KP-HELLO s.c. implant. G, Tumor volumes on day 16 after s.c. implant. H – I, Representative flow plots (H) and frequency (I) of GP66-specific CD4 T cell subsets from dLNs of [CD4-Cre Bcl6^fl/fl + SMARTA] group, separated by transferred SMARTA and endogenous cells. Flow plots were pre-gated on TCRb⁺ CD4⁺ GP66-tetramer⁺ CD44^hi. Data (mean ±SD) were pooled, from 3–10 mice/group/experiment, and representative of at least 2 independent experiments. A – C and E, two-way ANOVA. F – G and I, unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4.. TFH and B cells are critical to drive anti-tumor effector CD8 T cell responses

A, Control and anti-CD8a treated C57BL/6 mice were s.c. implanted with 2 x 10⁵ KP-HELLO. Growth curve of C57BL/6 (n = 40), pooled from 8 independent experiments, was set as the baseline of analyses. B, Representative flow plots of tumor-infiltrating PD1^hi Granzyme B^hi CD8 T cells from KP-HELLO tumors in C57BL/6 or CD4-Cre Bcl6^fl/fl mice, further quantified in Figure 6O–6P. C – E, Representative flow plots (C), frequency (D) and number/mg tumor (E) of tumor-infiltrating PD1^hi Granzyme B^hi CD8 T cells from KP-HELLO tumors in C57BL/6 or uMT mice. F – H, Representative flow plots (F), frequency (G) and number/mg tumor (H) of tumor-infiltrating PD1^hi Granzyme B^hi CD8 T cells from KP-NINJA tumors in C57BL/6 or uMT mice. Data (mean ±SD) were pooled, from 3–5 mice/group/experiment, and representative of at least 2 independent experiments. Tumors were harvested on day 15–16. A, two-way ANOVA. D – E and G – H, unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.. IL-21 production is dependent on B cells and B-cell-recognized neoantigens and is required for effector CD8 T cell responses

A, Representative flow plots showing IL-21-producing cells in dLNs of KP or KP-HELLO tumor-bearing mice. B – C, Representative flow plots (B) and frequency (C) of IL-21 producers in GP66-specific CD44^hi CD4 T cells from dLNs and tumors of KP-HELLO tumor-bearing WT or uMT mice. D – E, Representative flow plots (D) and frequency (E) of IL-21 producers in GP66-specific CD44^hi CD4 T cells from dLNs of KP-HELLO or KP-NINJA tumor-bearing WT mice F, IL-21 receptor expression on CD8 T cell subsets from dLNs and tumors of KP-HELLO tumor-bearing mice. Gate was drawn based on FMO in CD44^hi PD1^hi tumor-infiltrating CD8 T cells. G, Tumor growth curves: C57BL/6 and IL21R KO mice were s.c. implanted with 2 x 10⁵ KP-HELLO. Growth curve of C57BL/6 (n = 40), pooled from 8 independent experiments, was set as the baseline of analyses. H – J, Representative flow plots (H), frequency (I) and number/mg tumor (J) of tumor-infiltrating PD1^hi Granzyme B^hi CD8 T cells from KP-HELLO tumors in C57BL/6 or IL21R KO mice. Data (mean ±SD) were pooled, from 3–10 mice/group/experiment, and representative of at least 2 independent experiments. C, E, and I – J, unpaired t-test with Welch’s correction. G, two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6.. Collaborations between CD4 T cells and B cells are critical for anti-tumor immune responses

A – B, Tumor growth curves: C57BL/6, ICOS KO or CD40L KO mice were s.c. implanted with 2 x 10⁵ KP-HELLO. Growth curve of C57BL/6 (n = 40), pooled from 8 independent experiments, was set as the baseline of analyses. C, 1 x 10⁵ SMARTA CD4 T cells or OT-II CD4 T cells were adoptively transferred to ICOS KO recipients prior to KP-HELLO s.c. implant. Graph shows tumor volumes on day 16. D – E, Representative flow plots (D) and frequency (E) of GC B cells in dLNs of KP-HELLO tumors in C57BL/6, CD4-Cre Bcl6^fl/fl mice, or CD4-Cre Bcl6^fl/fl recipients following transfer of 1 x 10⁵ SMARTA T cells or OT-II T cells (as described in Figure 3G). F – G, Representative flow plots (F) and frequency (G) of GP66-specific CD4 T cell subsets from dLNs of KP-HELLO tumor-bearing WT or uMT mice. J – K, Representative flow plots (J) and frequency (K) of GP66-specific CD4 T cell subsets from dLNs of KP-HELLO or KP-NINJA tumor-bearing WT mice. H – I and L – M, Histograms displaying IL-21 or Bcl-6 expression on naïve and GP66-specific CD4 T cell subsets. Gating strategy was shown in Figure 3D, 6F and 6J. N – P, Representative flow plots (N), frequency (O) and number/mg tumor (P) of tumor-infiltrating PD1^hi Granzyme B^hi CD8 T cells from KP-HELLO tumors in C57BL/6, CD4-Cre Bcl6^fl/fl mice, or CD4-Cre Bcl6^fl/fl recipients following transfer of 1 x 10⁵ SMARTA T cells or OT-II T cells (as described in Figure 3G). Data (mean ±SD) were pooled, from 3–5 mice/group/experiment, and representative of at least 2 independent experiments. A – B, two-way ANOVA. C, E, G, K and O – P, unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.