TCL1A-expressing B cells are critical for tertiary lymphoid structure formation and the prognosis of oral squamous cell carcinoma

Abstract

Background: Oral squamous cell carcinoma (OSCC) is a malignant tumor with a poor prognosis. Traditional treatments have limited effectiveness. Regulation of the immune response represents a promising new approach for OSCC treatment. B cells are among the most abundant immune cells in OSCC. However, the role of B cells in OSCC treatment has not been fully elucidated.

Methods: Single-cell RNA sequencing analysis of 13 tissues and 8 adjacent normal tissues from OSCC patients was performed to explore differences in B-cell gene expression between OSCC tissues and normal tissues. We further investigated the relationship between differentially expressed genes and the immune response to OSCC. We utilized tissue microarray data for 146 OSCC clinical samples and RNA sequencing data of 359 OSCC samples from The Cancer Genome Atlas (TCGA) to investigate the role of T-cell leukemia 1 A (TCL1A) in OSCC prognosis. Multiplex immunohistochemistry (mIHC) was employed to investigate the spatial distribution of TCL1A in OSCC tissues. We then investigated the effect of TCL1A on B-cell proliferation and trogocytosis. Finally, lentiviral transduction was performed to induce TCL1A overexpression in B lymphoblastoid cell lines (BLCLs) to verify the function of TCL1A.

Results: Our findings revealed that TCL1A was predominantly expressed in B cells and was associated with a better prognosis in OSCC patients. Additionally, we found that TCL1A-expressing B cells are located at the periphery of lymphatic follicles and are associated with tertiary lymphoid structures (TLS) formation in OSCC. Mechanistically, upregulation of TCL1A promoted the trogocytosis of B cells on dendritic cells by mediating the upregulation of CR2, thereby improving antigen-presenting ability. Moreover, the upregulation of TCL1A expression promoted the proliferation of B cells.

Conclusion: This study revealed the role of B-cell TCL1A expression in TLS formation and its effect on OSCC prognosis. These findings highlight TCL1A as a novel target for OSCC immunotherapy.

Keywords: B cells; Oral squamous cell carcinoma; TCL1A; Tertiary lymphoid structures.

Fig. 1

TCL1A is downregulated in OSCC tissues compared with normal tissues and is predominantly expressed in B cells. A Feature plot of single-cell RNA sequencing data showing that TCL1A is predominantly expressed in B cells and downregulated in OSCC tissues compared to normal tissues. B Dot plot showing that TCL1A is predominantly expressed in B cells. C UMAP plot showing that B cells were extracted and regrouped into different subsets. D Dot plot showing the differentially expressed genes among B-cell subsets. E, F Feature plot and dot plot showing that TCL1A is expressed mainly in germinal center B cells, followed by follicular B cells. G IF staining showing the expression levels of CD19 (red) and TCL1A (green) in OSCC tissues. Scale bars: 100 μm (upper); 10 μm (lower)

Fig. 2

TCL1A expression is related to favorable prognosis in OSCC patients. A Kaplan-Meier survival curves showing that TCL1A upregulation is significantly associated with improved OS in patients with OSCC (TCGA database). B Representative IHC images of tissue microarrays (TMAs). Scale bars: 500 μm (upper); 100 μm (lower). C Kaplan-Meier survival curve showing that TCL1A upregulation is significantly associated with improved overall survival in 146 OSCC patients according to IHC staining. D Cox regression analysis showing the multivariate analysis of factors related to overall survival in 146 OSCC patients in the TMA cohort

Fig. 3

TCL1A is highly expressed in tertiary lymphoid structure (TLS)-positive tissues from OSCC patients. A, B The expression of TCL1A is positively correlated with the enrichment of TLS signatures (EIF1AY, CETP, CCR6, CD79B, LAT, PTGDS, CD1D, RBP5, and SKAP1) in OSCC samples from the TCGA. C Representative images of H&E and immunohistochemistry (IHC) staining of OSCC tissues (white arrows indicate TLS). Scale bar: 500 μm (left); 100 μm (right). D Quantitative analysis showing the upregulation of TCL1A in TLS-positive tissues from OSCC patients according to the intensity of IHC staining. E The ROC curves for the TCL1A expression analysis validated its potential value in identifying TLS in OSCC patients. ****P < 0.0001. F mIHC staining showing the spatial localization of different cells in the TLSs of OSCC tissues

Fig. 4

CR2 expression in B cells increases after TCL1A expression is upregulated. A The expression of TCL1A is positively correlated with that of CR2 in OSCC samples from the TCGA database. B Violin plot showing that CR2 is highly expressed in TCL1A-expressing B cells. C IF staining showing the expression of TCL1A (red) and CR2 (green) in TLSs from OSCC tissue (scale bars: 100 μm). D, E Flow cytometry analysis showing that the proportions of TCL1A and CR2 cells increased after B cells were treated with estradiol. **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5

The trogocytosis effect of B cells is promoted after TCL1A expression is upregulated. A Flowchart illustrating the methodology employed to detect the trogocytosis effect of TCL1A⁺ B cells on dendritic cells. MACS: magnetic activated cell sorting. B, C Flow cytometry analysis demonstrated that B cells treated with estradiol promoted the trogocytosis-mediated transfer of CD11c and HLA-DR on dendritic cells

Fig. 6

CR2 expression is increased after TCL1A overexpression in BLCLs. A Construction of lentivirus for TCL1A overexpression. B RT-qPCR results showing that the expression level of TCL1A in the overexpression group (TCL1A-OE) was greater than that in the negative control group (TCL1A-NC). C-E Flow cytometry analysis revealing an increase in the expression of TCL1A, CR2, and KI67 after BLCLs were transduced with lentivirus. F Flow cytometry analysis showing that the percentage of fast-proliferating (CellTrace⁻) cells among BLCLs increased after TCL1A overexpression. G Flow cytometry analysis showing the cell cycle distribution of TCL1A-NC and TCL1A-OE cells. Representative results are shown (left). The results of the quantitative analysis of the population of BLCLs that proliferated quickly are shown (right). Each data point represents an individual subject. *P < 0.05; ***P < 0.001; ****P < 0.0001

Fig. 7

Working model: The role of TCL1A-expressing B cells in the tertiary lymphoid structures of OSCC