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Expansion of CD4+ cytotoxic T lymphocytes with specific gene expression patterns may contribute to suppression of tumor immunity in oral squamous cell carcinoma: single-cell analysis and in vitro experiments - PubMed

  • ️Sun Jan 01 2023

. 2023 Nov 23:14:1305783.

doi: 10.3389/fimmu.2023.1305783. eCollection 2023.

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Expansion of CD4+ cytotoxic T lymphocytes with specific gene expression patterns may contribute to suppression of tumor immunity in oral squamous cell carcinoma: single-cell analysis and in vitro experiments

Hu Chen et al. Front Immunol. 2023.

Abstract

Background: Cancer immunotherapy targeting CD8+ T cells has made remarkable progress, even for oral squamous cell carcinoma (OSCC), a heterogeneous epithelial tumor without a substantial increase in the overall survival rate over the past decade. However, the therapeutic effects remain limited due to therapy resistance. Thus, a more comprehensive understanding of the roles of CD4+ T cells and B cells is crucial for more robust development of cancer immunotherapy.

Methods: In this study, we examined immune responses and effector functions of CD4+ T cells, CD8+ T cells and B cells infiltrating in OSCC lesions using single-cell RNA sequencing analysis, T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing analysis, and multi-color immunofluorescence staining. Finally, two Kaplan-Meier curves and several Cox proportional hazards models were constructed for the survival analysis.

Results: We observed expansion of CD4+ cytotoxic T lymphocytes (CTLs) expressing granzymes, which are reported to induce cell apoptosis, with a unique gene expression patterns. CD4+ CTLs also expressed CXCL13, which is a B cell chemoattractant. Cell-cell communication analysis and multi-color immunofluorescence staining demonstrated potential interactions between CD4+ CTLs and B cells, particularly IgD- CD27- double negative (DN) B cells. Expansion of CD4+ CTLs, DN B cells, and their contacts has been reported in T and B cell-activated diseases, including IgG4-related disease and COVID-19. Notably, we observed upregulation of several inhibitory receptor genes including CTLA-4 in CD4+ CTLs, which possibly dampened T and B cell activity. We next demonstrated comprehensive delineation of the potential for CD8+ T cell differentiation towards dysfunctional states. Furthermore, prognostic analysis revealed unfavorable outcomes of patients with a high proportion of CD4+ CTLs in OSCC lesions.

Conclusion: Our study provides a dynamic landscape of lymphocytes and demonstrates a systemic investigation of CD4+ CTL effects infiltrating into OSCC lesions, which may share some pathogenesis reported in severe T and B cell-activated diseases such as autoimmune and infectious diseases.

Keywords: CD4+ cytotoxic T lymphocytes; CTLA-4; CXCL13; T cell dysfunction; double negative B cells; oral squamous cell carcinoma.

Copyright © 2023 Chen, Sameshima, Yokomizo, Sueyoshi, Nagano, Miyahara, Sakamoto, Fujii, Kiyoshima, Guy, Nakamura, Moriyama, Kaneko and Kawano.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1

The landscape of infiltrating immune cells. (A) IF staining of CD45+ cells in OSCC tissue. The staining showed that dense immune cells had infiltrated in OSCC of tongue tissue. Scale bars: 100 µm. (B) Overview of scRNA-seq. Resected tongues from three patients with OSCC underwent homogenization, from which CD45+ cells were extracted by magnetic isolation. GEX and V(D)J libraries were subsequently constructed using these cells for sequencing, and the sequencing data were analyzed by various methods. (C) UMAP visualization of infiltrating immune cells with assigned clusters. This displayed a variety of immune cells in OSCC tissues. ASC: antibody-secreting cell; B: B cell; CD4+ T: CD4+ T cell; CD8+: CD8+ T cell; γδ T: γδ T cell; Mφ: macrophage; Neu: neutrophil; NK: natural killer cell; pDC: plasmacytoid dendritic cell. (D) UMAP visualization of T cells colored by assigned T cell subsets. Various T cell subsets were identified by unsupervised clustering and assigned based on expression of specific marker genes. TCM-CD4: CD4+ central memory T cell; CD4+ CTL: CD4+ cytotoxic T lymphocyte; MAIT: mucosal associated invariant T cell; Treg: regulatory T cell; TCM-CD8: CD8+ central memory T cell; TEM-CD8: CD8+ effector memory T cell; TEX-CD8: CD8+ exhausted T cell; γδ T: γδ T cell; Proliferating T: proliferating T cell. (E) Violin plot of gene expression in T cell subsets. This plot showed the marker gene expression used to assign T cell subsets. (F) UMAP visualization of T cells combined with TCR clonal expansion. The specific gradient color denotes different degree of clonal expansion in TCRs. NA: αβ TCR sequence was undetected. (G) Chord diagrams of the unique and shared TCR clonotype counts in T cell subsets. The chords connecting two different subsets represent shared TCR clonotypes.

Figure 2
Figure 2

Gene expression and TCR repertoire analysis of conventional CD4+ T cells. (A) UMAP visualization of conventional CD4+ T cells. CD4+ CTL: CD4+ cytotoxic T lymphocyte; TCM: central memory T cell; TCM CXCL13+: CXCL13-expressing central memory T cell; TCM CXCR5+: CXCR5-expressing central memory T cell; TCM/TEM: central memory T cell and effector memory T cell. (B) Violin plot of gene expression in CD4+ T cell subsets. The plot depicts expression of genes associated with T cell functions in distinct subsets. (C) UMAP visualization of conventional CD4+ T cells integrated with TCR clonal expansion. The gradient color represents different degree of clonal expansion in distinct TCR clonotypes. NA: αβ TCR sequence was undetected. (D) Proportional stacked bar plot of clonal expansion in conventional CD4+ T cell subsets. A high level of clonal expansion was predominantly found in the CD4+ CTL subsets. (E) Chord diagrams of unique and shared TCR clonotype counts in conventional CD4+ T cell subsets. The chords connecting two different subsets represent shared TCR clonotypes. (F) UMAP visualization of conventional CD4+ T cells colored by pseudotime and integrated with single cell trajectories. The TCM subset was set as the root node of single cell trajectories. (G) Gene dynamic plot based on pseudotime. Cells were ordered in accordance with pseudotime, and the average expression of these genes in cells with identical pseudotime was calculated and visualized in this plot. (H) Heat map of the top 10 active regulons in T cell subsets. An extended regulon refers to a regulon that has been expanded to include both direct and indirect targets of a regulon. The counts of these genes are indicated by parenthesis following the regulons. (I) UMAP visualization integrated with RNA velocity analysis of conventional CD4+ T cells. The direction of the arrow represents the predicted direction of change in cell state, and the length of the arrow represents the predicted speed of change in cell state. (J) Multicolor IF staining of CD4+ CTLs and CXCL13+ CD4+ T cells in OSCC tissue. Scale bars: 50 µm (low magnification) and 5 µm (high magnification).

Figure 3
Figure 3

The CD4+ CTL subset may exert pleiotropic effects in tumor immunity. (A) Violin plot of gene expression related to cytotoxicity in several cytotoxic T cell subsets. (B) Multicolor IF staining of CD4+ CTLs and HLA-DR+ tumor cells. Conjugation between these two cell types is depicted by the yellow arrow and line. GZMA: granzyme A; panCK: pan-cytokeratin; c-Casp3: cleaved caspase-3. Scale bars: 50 µm (low magnification) and 5 µm (high magnification). (C) Multicolor IF staining of CD4+ CTLs and apoptotic tumor cells. The white arrow indicates CD4+ CTLs, and the dotted box indicates apoptotic tumor cells. Scale bars: 50 µm (low magnification) and 5 µm (high magnification). (D) Visualization of hierarchical clustering analysis in T cell subsets. (E) Visualization of genes in module 3 (M3). The top 10 kME value genes are displayed in this plot. (F) Feature plot of M3 hMEs. Expression of M3 hMEs was calculated in single cells and visualized on UMAP. (G) Violin plot of M3 hMEs. Expression of M3 hMEs was calculated in single cells and visualized in various T cell subsets. (H) Dot plot of the top 10 terms of biological processes in GO enrichment analysis. The enrichment p-values were calculated using the hypergeometric distribution test and adjusted by the Benjamini–Hochberg method. (I) Dot plot of hMEs in all modules. Expression of hMEs in all modules was calculated in single cells and visualized in various T cell subsets. The dotted box indicates the specific T cell subsets. (J) Heat map of significant receptor–ligand interactions. The gradient color represents the quantity of significant interactions. (K-L) Dot plot of specific receptor–ligand pairs. The permutation test was used to calculate the p-value. (M) Multicolor IF staining of CXCL-13-producing CD4+ CTLs (left), CTLA-4-expressing CD4+ CTLs (upper right), and conjugation with B cells (lower right). Conjugation between these cells is depicted by the yellow arrow and line. Scale bars: 20 µm (low magnification) and 5 µm (high magnification).

Figure 4
Figure 4

Gene expression and TCR repertoire analysis of CD8+ T cells. (A) UMAP visualization of CD8+ T cells. TCM CD8: central memory CD8+ T cell; TEM CD8: effector memory CD8+ T cell; TEX CD8: exhausted CD8+ T cell; CD8+ CTL: CD8+ cytotoxic T lymphocyte. (B) Violin plot of gene expression in CD8+ T cell subsets. (C) Multicolor IF staining of CD8+ exhausted T cells in OSCC tissue samples. The white arrow indicates CD39+ PD-1+ CD8+ exhausted T cells. Scale bars: 20 µm (low magnification) and 5 µm (high magnification). (D) UMAP visualization of CD8+ T cells integrated with TCR clonal expansion. The gradient color represents the different degree of clonal expansion in distinct TCR clonotypes. NA: αβ TCR sequence was undetected. (E) Proportional stacked bar plot of clonal expansion in CD8+ T cell subsets. (F) Chord diagrams of unique and shared TCR clonotype counts in CD8+ T cell subsets. The chords connecting two different subsets represent shared TCR clonotypes. (G) UMAP visualization of CD8+ T cells colored by pseudotime and integrated with single cell trajectories. (H) Gene dynamic plot based on pseudotime. Cells were ordered in accordance with the pseudotime, and the average expression of these genes in cells with identical pseudotime was calculated and visualized in this plot. (I) UMAP visualization integrated with RNA velocity analysis of CD8+ T cells. The direction of the arrow represents the predicted direction of the change in cell state, and the length of the arrow represents the predicted speed of change in cell state.

Figure 5
Figure 5

Gene expression and BCR repertoire analysis of B cells. (A) UMAP visualization of B cells. Cells on the UMAP are colored in accordance with the subsets. DN1: double negative 1 B cells; DN3: double negative 3 B cells; ASC: antibody-secreting cell; GC B: germinal center B cell. (B) Dot plot of gene expression in B cell subsets. (C) UMAP visualization of B cells integrated with BCR clonal expansion. The gradient color represents different degree of clonal expansion in distinct BCR clonotypes. NA: BCR sequence was undetected. (D) Circos plot of VJ germline gene usage in DN3 and ASC cells. This plot provided a circular visualization of how V and J germline genes were combined. (E) Visualization of the SHM frequency in B cell subsets. The black line on the dots represents the mean value. P-values were calculated by the Kruskal–Wallis test and adjusted by the Benjamini–Hochberg method, *P < 0.05, *****P < 0.00001, ns: not significant. (F) HE staining and multicolor IF staining of DN B cells and conjugation with CD4+ CTLs in two consecutive OSCC sections. Conjugation between these cells is depicted by the yellow arrow and line. Scale bars: 100 µm (low magnification) and 5 µm (high magnification).

Figure 6
Figure 6

Multicolor IF staining and survival analysis. (A) Multicolor IF staining of CD4+ T cell subsets in OSCC tissue. The white arrow indicates cells in distinct subsets. Th1: T helper 1 cell; Th2: T helper 2 cell; Th17: T help 17 cell; Tfh: T follicular helper cell; Treg: regulatory T cell. Scale bars: 10 µm (low magnification) and 5 µm (high magnification). (B) Box plots of the frequency of CD4+ T cell subsets (n = 20) among CD4+ cells (left) and the density of these subsets (right). The TEM subsets included Th1, Th2, Th17, and Tfh subsets. (C) Progression-free survival analysis using Kaplan–Meier models. The p-value was calculated by the log-rank test.

Figure 7
Figure 7

Estimated schema of CD4+CTL and other T cells immunological processes in oral squamous cell carcinoma (OSCC). (Top left) Upon tumor antigen presented by antigen-presenting cells, activated CD4+ cytotoxic T lymphocytes (CD4+CTL), CD4+ TCM and CD8+ TCM expanded within tumor lesions. CD8+ TCM subsequently differentiates into effector memory T cell (CD8+ TEM) or CD8+CTL and expresses strong cytotoxicity. (Bottom right) Both CD4+CTLs and CD8+CTLs can induce tumor cell death; CD4+CTLs recognize the antigens presented via MHC class II molecules (MHC II) and induce the tumor cell death through granzyme B (GzmB) and perforin (PFN) release or TRAIL release, while CD8+CTLs also express Fas ligand (FasL) and recognize antigens presented via MHC class I molecules (MHC II). (Top right) Activated CD4+ TCM and CD4+CTLs produce CXCL13, attracting CXCR5+ B cells and interacting with them via TCR/MHC class II and costimulators such as CD28/B7, thereby initiating extrafollicular responses and promoting the differentiation of B cells into antibody secreting cells (ASCs) or double negative B cell 3 (DN3). IFN-γ produced by CD4+CTLs can also promote the responses. With high antigen load and prolonged antigen exposure, CD8+CTLs differentiate into CD8+ exhausted T cells (CD8+ TEX) and become dysfunctional, marked by upregulation of inhibitory receptor (IR) genes, ENTPD1 and TOX, and the downregulation of TCF7. Although CD4+CTLs display similar gene expression patterns, whether they can become dysfunctional remains controversial (broken arrows). These CD4+CTLs also express CTLA-4, competitively binding with B7 molecules and potentially limiting immune responses between CD4+ T cells and B cells.

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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. NK was supported by JSPS KAKENHI (Grant numbers: 21KK0163, 22H03290, and 23K18362), the JST FOREST PROGRAM (JPMJFR220M), Astellas Foundation for Research on Metabolic Disorders, the Takeda Foundation, the Nakajima Foundation, and the QR program (SENTAN-Q project) of Kyushu University.