Single-cell transcriptomic and spatial analysis reveal the immunosuppressive microenvironment in relapsed/refractory angioimmunoblastic T-cell lymphoma

Abstract

Angioimmunoblastic T-cell lymphoma (AITL) is a kind of aggressive T-cell lymphoma with significant enrichment of non-malignant tumor microenvironment (TME) cells. However, the complexity of TME in AITL progression is poorly understood. We performed single-cell RNA-Seq (scRNA-seq) and imaging mass cytometry (IMC) analysis to compare the cellular composition and spatial architecture between relapsed/refractory AITL (RR-AITL) and newly diagnosed AITL (ND-AITL). Our results showed that the malignant T follicular helper (Tfh) cells showed significantly increased proliferation driven by transcriptional activation of YY1 in RR-AITL, which is markedly associated with the poor prognosis of AITL patients. The CD8⁺ T cell proportion and cytotoxicity decreased in RR-AITL TME, resulting from elevated expression of the inhibitory checkpoints such as PD-1, TIGIT, and CTLA4. Notably, the transcriptional pattern of B cells in RR-AITL showed an intermediate state of malignant transformation to B-cell-lymphoma, and contributed to immune evasion by highly expressing CD47 and PD-L1. Besides, compared to ND-AITL samples, myeloid-cells-centered spatial communities were more prevalent but showed reduced phagocytic activity and impaired antigen processing and presentation in RR-AITL TME. Furthermore, specific inhibitory ligand-receptor interactions, such as CLEC2D-KLRB1, CTLA4-CD86, and MIF-CD74, were exclusively identified in the RR-AITL TME. Our study provides a high-resolution characterization of the immunosuppression ecosystem and reveals the potential therapeutic targets for RR-AITL patients.

Fig. 1. Comparison of the TME composition between ND-AITL and RR-AITL at the single-cell level.

A Overview of sample collection, computational analysis and experimental validation workflow. B UMAP plot of 55482 cells obtained from scRNA-seq profiles from 9 samples, colored by four main cell types. The cell counts and cell proportions for each cell type are labeled on the graph. C Dot plot depicting the expression of representative markers for four distinct cell types. Dot size is indicative of the proportion of cells within each cell type expressing these markers, and dot color represents the mean expression levels of these markers within each cell type. D MDS analysis demonstrates the inter- and intragroup differences among different ROIs. The dots were color-coded to indicate groups, with red representing the RR-AITL group and blue representing the ND-AITL group. The size of the dots was proportional to the number of cells within the ROIs. E The heatmap illustrates the protein levels of classical marker genes within individual cell. For the sake of clarity, a random sample of 5000 cells was selected for visualization. F The UMAP plot illustrates the identified cell types within the IMC dataset. G The cell types were visually represented in the images, with different colors denoting distinct cell types. One representative ROI was shown for each sample. The upper panel comprises four images from ND-AITL, while the lower panel displays three images from RR-AITL.

Fig. 2. Differences in the cellular states of malignant Tfh cells between ND-AITL and RR-AITL.

A UMAP visualization of various T/NK cell clusters (left) and subtypes (right). B Boxplot showing enrichment scores of AITL signature and stemness inferred by CytoTRACE across different T cells clusters. The boxplot represents the mean value and the error bar represents the standard error value. C Principal component analysis (PCA) of the clusters according to the mean transcription factor activity of each cluster. D Volcano plot of gene expression changes of Tfh cells between ND-AITL and RR-AITL. Upregulated (red) and downregulated (blue) genes in RR-AITL are highlighted. E Violin plot showing expression levels of CXCL13 in Tfh cells from ND-AITL and RR-AITL samples (upper panel). Violin plot showing enrichment score of AITL signatures in Tfh cells from ND-AITL and RR-AITL samples (bottom panel). P values were determined by two-sided unpaired Wilcoxon test. The boxplot represents the mean value and the error bar represents the standard error value. F GSEA plot showing the hallmarks that are significantly enriched in RR-AITL and ND-AITL, respectively. G Boxplot showing enrichment score of proliferation signature and PI3K/AKT pathway. P values were determined by two-sided unpaired Wilcoxon test. H Boxplot showing intensities of pAKT in IMC data. P values were determined by two-sided unpaired Wilcoxon test. I The IMC images showing the protein activity of pAKT within malignant Tfh cells. The colors represent protein activity levels, ranging from lower (blue) to higher (red). J, K Immunofluorescence analysis for ND-AITL and RR-AITL samples stained by an-ti-DAPI (blue), anti-CXCL13 (green), anti-pAKT (red) antibodies. Scale bar, 100 μm.

Fig. 3. The distinct transcriptional regulatory mechanisms of malignant Tfh cells in RR-AITL.

A Dot plot shows TFs that significantly activated in Tfh cells from RR-AITL compared to ND-AITL. Y axis represents -log10 transformation of adjusted P values of the activity of TFs inferred by pySCENIC, x axis represents -log10 transformation of adjusted P values of the expression of TFs. B Density plot shows the activity of YY1 across Tfh from NC (yellow), ND-AITL (blue) and RR-AITL (red). The vertical line represents the median of YY1 activity. C The intensities of YY1 between Tfh cells from ND-AITL and RR-AITL. The y axis represents the protein levels of YY1 in IMC dataset. D The protein activity of YY1 within malignant Tfh cells shown on IMC images. The colors represent protein activity levels, ranging from lower (blue) to higher (red). E, F Immunofluorescence analysis for ND-AITL (E) and RR-AITL (F) samples stained by anti-DAPI (blue) and anti-YY1(red) antibodies. Scale bar, 100 μm. G The spearman correlation between activity of YY1 and the enrichment score of pathways in malignant Tfh cells. Each data point represents an individual cell. H The functional enrichment analysis of YY1 target genes inferred by pySCENIC. The spearman correlation between YY1 expression and enrichment scores of proliferation (I), G2M checkpoint (J), TNFα signaling via NFκB (K) in GSE51521 dataset. Each data point represents individual sample. L Overall survival of AITL patients in our in-house cohort through Kaplan–Meier analysis. All patients were categorized into two groups based on the median YY1 expression level estimated by IHC. The significance was evaluated by the log-rank test. M Kaplan–Meier analysis was performed to assess overall survival in PTCL patients within the GSE58445 dataset. Only samples with available follow-up data were included in the analysis. Patients were stratified into four quartiles based on YY1 expression levels. For further comparison, the top 25% and bottom 25% of patients were specifically analyzed. The statistical significance of survival differences between these two groups was evaluated using the log-rank test.

Fig. 4. The exclusion and dysfunction of CD8⁺ T cells in RR-AITL.

A T/NK cells from NC, ND-AITL and RR-AITL illustrated in UMAP plots, respectively. B Group preference of T cells subtypes, with color encoded by odds ratio estimated by one-sided Fisher’s exact test. *P value < 0.05, **P value < 0.01,***P value < 0.001. The orange color represents enrichment of subpopulation in the sample, while green color represents depletion of subpopulation in the sample. C Immunofluorescence analysis for ND-AITL (left) and RR-AITL (right) samples stained by anti-DAPI (blue), anti-CD8 (green), anti-FOXP3(red) antibodies. Scale bar, 100 μm. D The percentage of selected cell types within IMC dataset between ND-AITL and RR-AITL. E The representative markers for Tfh cells (ICOS; green) and CD8⁺ T cells (CD8α; red) stained in each ROI. F Enrichment scores of pathways related to T cell migration capacity in CD8⁺ T cells from ND-AITL and RR-AITL samples. P values were determined by two-sided unpaired Wilcoxon test. G Enrichment scores of cytotoxic and exhausted signatures of CD8⁺ T cells from ND-AITL and RR-AITL samples. ***P value < 0.001. P values were determined by two-sided unpaired Wilcoxon test. H Violin plots depict the intensities of immune checkpoint molecules in CD8⁺ T cells between seven and eight ROIs from RR-AITL and ND-AITL, respectively. P values were determined by two-sided unpaired Wilcoxon test. I Violin plots showing the intensities of PD-L1 in malignant Tfh cells across seven and eight ROIs in RR-AITL and ND-AITL, respectively. P values were determined by two-sided unpaired Wilcoxon test. J The spearman correlation between YY1 expression and the percentage of CD8⁺ T cells estimated by CIBERSORTx in GSE51521 dataset. Each data point represents individual sample.

Fig. 5. Integrative analysis for B cells from ND-AITL, RR-AITL and DLBCL.

A Volcano plot of expression changes between B cells from ND-AITL and RR-AITL samples. Upregulated genes in RR-AITL (red) and ND-AITL (blue) are highlighted. B The enrichment analysis of upregulated genes in B cells from RR-AITL compared to ND-AITL. The Cancer Cell Line Encyclopedia gene set is derived from Enrichr website. C The UMAP plot illustrates B cells colored according to sample classification, including B cells from ND-AITL (green), RR-AITL (orange), and DLBCL (blue). D Barplot showing the fraction of B cells from ND-AITL (green), RR-AITL (orange) and DLBCL (blue) samples in each cluster. E The violin plot showing IGKC fraction in ND-AITL (green), RR-AITL (orange) and DLBCL (blue) samples. The IGKC fraction, IGKC/(IGKC + IGLC2), was calculated for each B cell. F The trajectory plots showing the pseudotime (left) and group (right) of each cell. G Density plot (upper panel) of cell number along the pseudotime from ND-AITL (green), RR-AITL (orange) and DLBCL patients (blue). Heatmap (bottom panel) showing gene expression alterations along the pseudotime. H The plot showing expression of MYC, TCL1A, STMN1, VPREB3, CD24 and PCNA along the pseudotime. Line indicates smoothed mean expression of genes in the state across pseudotime. I The protein levels of cMyc in B cells. P values were determined by two-sided unpaired Wilcoxon test. J The IMC images showing the protein activity of cMyc within B cells. The colors represent protein activity levels, ranging from lower (blue) to higher (red).

Fig. 6. The dysfunctional myeloid cells in RR-AITL.

A The CNs were depicted in the representative images from RR-AITL and ND-AITL, with colors indicating different CNs. B Pie chart showing the CNs distribution within ND-AITL and RR-AITL. C Boxplots depict the percentage of selected CNs within all cells between ROIs from ND-AITL and RR-AITL. D The heatmap illustrates the scaled percentage of cell types in each cellular neighborhood. The predominant cell type in each CN is presented in the right. E The percentage of selected cell types between ROIs from ND-AITL and RR-AITL. P values were determined by two-sided unpaired Wilcoxon test. F four markers representing B cells, Tfh cells, myeloid cells, and CD8⁺ T cells were visualized in the representative images. G The enrichment score of phagocytosis and antigen presentation in myeloid cells based on scRNA-seq data. H Violin plot showing the protein levels of CD47 in malignant Tfh cells and B cells. P values were determined by two-sided unpaired Wilcoxon test. I The heatmap illustrates significant pairwise cell-cell interactions (in orange) or avoidance (in green) between different cell types. Boxes highlight significant interactions (in red) or avoidance (in blue) between RR-AITL and ND-AITL. P-values were determined using a two-sided unpaired Wilcoxon test. *P values < 0.05, **P values < 0.01.

Fig. 7. The alterations of cell-cell communications in RR-AITL compared to ND-AITL.

A Circos plots showing the interaction numbers among various cell types in ND-AITL(left) and RR-AITL(right) samples. These interactions were predicted based on scRNA-seq data. B The number of significant ligand-receptor pairs between the RR-AITL and ND-AITL, as depicted in a lollipop plot. C, D Heatmap depicting the intricate ligand-receptor interactions between Tfh cells and other cell types. The ‘Common’ category (green) signifies the presence of ligand-receptor pairs in both ND-AITL and RR-AITL. The ‘ND-specified’ category (blue) and ‘RR-specified’ category (red) indicate the occurrence of ligand-receptor pairs exclusively in ND-AITL or RR-AITL, respectively. Numbers enclosed in red boxes indicate the interacting pairs displayed in Figure E. E Sankey plot illustrating the essential ligand-receptor pairs exclusive to RR-AITL, with ligands expressed on Tfh cells. The color scheme indicates different cell types expressing the receptors. F The IMC images showing the protein activity of CD20, CD86 within B cells and ICOS, CTLA4 within Tfh cells in ND-AITL and RR-AITL samples. The colors represent corresponding proteins. The solid line frame represents a magnified version of the dashed line frame. Scale bar, 100 μm. G Schematic representation of TME alterations between RR-AITL and ND-AITL.