ScRNA-seq reveals novel immune-suppressive T cells and investigates CMV-TCR-T cells cytotoxicity against GBM

Abstract

Background: Glioblastoma (GBM) is a fatal primary brain malignancy in adults. Previous studies have shown that cytomegalovirus (CMV) is a risk factor for tumorigenesis and aggressiveness for glioblastoma. However, little is known about how CMV infection affects immune cells in the tumor microenvironment of GBM. Furthermore, there has been almost no engineered T-cell receptor (TCR)-T targeting CMV for GBM research to date.

Methods: We evaluated the CMV infection status of patients with GBM's tumor tissue by immune electron microscopy, immunofluorescence, and droplet digital PCR. We performed single-cell RNA sequencing for CMV-infected GBM to investigate the effects of CMV on the GBM immune microenvironment. CellChat was applied to analyze the interaction between cells in the GBM tumor microenvironment. Additionally, we conducted single-cell TCR/B cell receptor (BCR) sequencing and Grouping of Lymphocyte Interactions with Paratope Hotspots 2 algorithms to acquire specific CMV-TCR sequences. Genetic engineering was used to introduce CMV-TCR into primary T cells derived from patients with CMV-infected GBM. Flow cytometry was used to measure the proportion and cytotoxicity status of T cells in vitro.

Results: We identified two novel immune cell subpopulations in CMV-infected GBM, which were bipositive CD68⁺SOX2⁺ tumor-associated macrophages and FXYD6⁺ T cells. We highlighted that the interaction between bipositive TAMs or cancer cells and T cells was predominantly focused on FXYD6⁺ T cells rather than regulatory T cells (Tregs), whereas, FXYD6⁺ T cells were further identified as a group of novel immunosuppressive T cells. CMV-TCR-T cells showed significant therapeutic effects on the human-derived orthotopic GBM mice model.

Conclusions: These findings provided an insight into the underlying mechanism of CMV infection promoting the GBM immunosuppression, and provided a novel potential immunotherapy strategy for patients with GBM.

Keywords: Central Nervous System Cancer; Immunosuppression; T cell Receptor - TCR.

Figure 1

CMV infection triggers bipositive TAMs-driven remodeling of the GBM immune microenvironment. (A) Transmission electron microscopy images of CMV-infected GBMs. Scale bars respectively, 5 μm 1 µm 500 nm. The red arrows are pp65 at 10 nm. green arrows are IE1/2 at 4 nm, and the white box shows electron-dense particles. (B) The UMAP above shows the composition of the annotated cells, with different colors representing different cell populations. The below is the CMV infected map, and the dark blue is the CMV infected. (C) The dot plot shows the expression levels of different classical cell marker genes in annotated cell populations. (D) UMAP feature plot representation of marker gene expression within individually identified macrophage and cancer cell populations and phenotypic states. (E) Representative images of multiplex immunofluorescence staining in formalin-fixed paraffin-embedded tissues, indicating CD68⁺SOX2⁺ TAMs, in paired CMV⁺ and CMV⁻ samples. Scale bar, 200 µm and 50 µm. Boxplots illustrating the fraction of CD68⁺SOX2⁺ TAMs in CMV⁻ (light blue) and CMV⁺(deep blue), respectively. Box center lines, bounds of the box, and whiskers indicate medians, first and third quartiles, and minimum and maximum values within 1.5×IQR of the box limits, respectively. Significance was determined using a two-sided, unpaired Wilcoxon rank-sum test relative to CMV⁺ (n = 12) for CMV⁻ (n=8, p value<0.0001). (F) Representative flow cytometry plots and summary data showing the percentage of cellular staining for CD68⁺SOX2⁺ TAMs in CMV⁻ and CMV⁺ GBM (n=20). CMV, cytomegalovirus; GBM, glioblastomas; TAM, tumor-associated macrophages; UMAP, uniform manifold approximation and projection.

Figure 2

Bipositive TAMs significantly influence the clinical outcomes of GBMs. (A) UMAP plot of 236,575 cells from tumor tissue samples of patients with glioma from GSE182109 data. Each cluster is shown in a different color. (B) Violin plots showing the expression levels of different classical cell marker genes in the 8 cell clusters. (C) Radargram demonstrating Gene Ontology analysis associated with infection by pathogenic organisms. (D) The bar graph shows the percentage of each cell population in each sample in single-cell RNA sequencing (E) Box plot showing the percentage of different cell populations in low-grade gliomas (LGG), newly diagnosed glioblastomas (ndGBM), and recurrent glioblastomas (rGBM) (F) Comparison of the signatures of each cell type gene multivariable Cox regression was used to obtain the HRs (with Wald 95% CIs shown as horizontal bars, and p values given on the right) based on the cross-validated prognostic scores derived using the Cox model and applied to pairwise differences of expression of the genes. (G) Kaplan-Meier plot of cross-validated macrophage prognostic score from (F) at 50% cut-off, showing the genes with opposite effects selected by the model. GBM, glioblastomas; RNA-seq, RNA sequencing; TAMs, tumor-associated macrophages; t-SNE, t-distributed stochastic neighbor embedding; UMAP, uniform manifold approximation and projection.

Figure 3

CMV infection amplifies a newly identified immunosuppressive FXYD6⁺ T cell, promoting the progression of GBM. (A) Distribution and expression of representative genes in lymphocyte clusters. (B) Violin plots showing the expression levels of different classical lymphocyte marker genes in the seven cell clusters. (C) Gene network diagram showing functional analysis of FXYD6⁺ T cells characterized genes. (D) Gene function network diagram demonstrating the analysis of the major functions of the FXYD6⁺ T cells signature gene cluster. (E) Over-represented GO terms of FXYD6⁺ T cells (pink) compared with other T cells (violet). (F) The inferred MDK signaling networks between cancer cells/ bipositive TAMs and T-cell clusters. (G) Expression score of the FXYD6⁺ T cells gene sets of non-responder and responder after neoadjuvant anti-PD-1 therapy. (H) Representative example of CMV⁻ (G15) and CMV⁺ (G3) GBM tumors. Tumor staining by multiplexed immunofluorescence shows the spatial distributions of FXYD6⁺ T cells, KI67⁺ cancer cells (tumor cell proliferation), and TNF-β (T-cell cytotoxicity). (I) Circos plots showing well-known ligand-receptor pairs from CellChat databases under the requirement that either the ligand or the receptor (or both) both being expressed. Arrows are pointing from the ligand toward the receptors. Different colors represent different cells, and the width is proportional to the number of events. Only selected example pairs are labeled and highlighted for the network. P values are adjusted for the number of cell types. CMV, cytomegalovirus; GBM, glioblastomas; PD-1, programmed death 1; TAM, tumor-associated macrophages; UMAP, uniform manifold approximation and projection.

Figure 4

T cells profile in glioblastomas with CMV infection. (A) The UMAP shows the composition of the T cells, with different colors representing different T-cell populations. (B) UMAP representation from colored based on patient ID. (C) The bar shows the percentage of T-cell subsets comparing CMV-infected and CMV-uninfected groups. (D) Bar graph showing the percentage of T-cell subsets in patients. (E) Distribution and expression of exhaustion-related genes in T cells in the CMV-uninfected group. (F) Distribution and expression of exhaustion-related genes in T cells in the CMV-infected group. (G) Slingshot trajectory analysis demonstrates the average expression pattern of each gene and signature across pseudotime (scaled from minimum to maximum average expression for each gene). CMV, cytomegalovirus; UMAP, uniform manifold approximation and projection.

Figure 5

Identification of clonally expanded T-cell receptors in patients with CMV-infected GBM. (A) UMAP showing the concentration of clonal cells in locations of T-cell clusters paired a-b TCRs were detected in T cells (n=9 subjects per group). (B) UMAP shows the composition of T cells colored by cluster. The deep blue dot shows CMV⁺ GBMs. (C) Grouped differential TCR-β sequences with genes in the horizontal coordinate and the frequency of use percentage of the V genes of the TCR in the vertical coordinate, with different colors indicating different groups. (D) Schematic diagram of Grouping of Lymphocyte Interactions with Paratope Hotspots 2 results, the larger and redder the nodes are the more core, and the thicker and redder the connecting lines between the nodes represent more similar CDR3 sequences. Note: The global pattern contains the “%!” symbol to indicate that variation is allowed at that position. (E) TCR substitutes CMV-TCR sequences for HDR templates of endogenous TCR with the CRISPR/Cas9 editing system. (F) Detection of gene editing in CMV-TCR by PCR-restriction fragment length polymorphism. CMV, cytomegalovirus; GBM, glioblastomas; HDR, homology directed repair; TCR, T-cell receptors; UMAP, uniform manifold approximation and projection.

Figure 6

CMV-TCR-T demonstrates efficacy against orthotopic glioblastomas in murine models. (A) CMV-TCR-T cells co-cultured with U251-GFP-Luci (HLA-A*0201) after flow cytometric detection of T-cell proliferation. (B) Flow cytometry cytotoxicity assay of UTD cells, sorted CMV-TCR-T cells against U251-GFP-Luci at indicated efficiency-to-target (E/T) ratios over 24 hours. (C) Flow cytometry detection of apoptotic tumor cells after co-culture of CMV-TCR-T cells and U251 after flow cytometric. (D) Cytokine production by primary human UTD, CMV-TCR-T cells when co-cultured overnight with U251 at an E/T of 1:3. (E) Schematic representation of experimental design in which a heterogeneous population (CMV⁺) of U251 glioma cells was implanted orthotopically into the brains of NSG mice. Both U251 were modified with luci allowing for visualization of total intracranial tumor burden by bioluminescent imaging. Mice were treated intraventricularly on day 30 postimplantation with UTD cells or CMV-TCR-T cells. (F) Bioluminescence analysis of mixed tumor growth over time, n= 5. (G) The mice body weight is displayed for individual mice and as average values (n=5, mean+SD is depicted; unpaired, two-tailed t-test, ***p< 0.001). (H) Hematoxylin and immunofluorescence staining for CD3 (T cells) and apoptotic cells identified by TdT-mediated dUTP nick end labeling (TUNEL) in tumor specimens from mice treated with intravenous CMV-TCR-T cells or UDT cells (scale bar, 20 µm). APC, allophycocyanin; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; IFN, interferon; PBS, phosphate-buffered saline; UTD, untransduced; TCR, T-cell receptor.