High expression of CCL3/CCL4/CCL5/CCR5 promotes exhausted CD8+ T cells terminal differentiation and is associated with poor prognosis in pediatric B-ALL patients

Abstract

This study aims to identify differentially upregulated ligand-receptor interactions between B-ALL cells and exhausted CD8⁺ T cells and to develop a multivariate Cox regression model for predicting the overall survival of pediatric B-ALL patients based on CCL3/CCL4/CCL5 expression levels. Pediatric B cell-acute lymphoblastic leukemia (B-ALL) is a hematopoietic malignancy. T cell exhaustion has an important impact on the prognosis of leukemia. The interaction between tumor cells and T cells can influence the degree of T cell exhaustion. However, the effects of B-ALL cells on exhausted T cell subpopulations and how the interaction influences the prognosis of B-ALL patients remain unclear. Single-cell RNA sequencing (scRNA-Seq) data from pediatric B-ALL patients were downloaded from GEO. Cell interaction analysis identified ligand-receptor pairs between B-ALL cells and exhausted CD8⁺ T cell. To confirm the function of CCL3/CCL4/CCL5/CCR5 in prognosis prediction, quantitative real-time polymerase chain reaction (qRT-PCR) was employed. We further developed an innovative stratified model that integrates CCL3, CCL4, and CCL5 through multi-Cox regression. Clustering of scRNA-Seq data revealed an increased proportion of exhausted CD8⁺ T cells in relapsed B-ALL, especially terminal exhausted CD8⁺ T cells (CD8_Ex), with increased exhaustion and decreased proliferation scores. Moreover, the CCL3/CCL4/CCL5-CCR5 axis was upregulated in interactions between B-ALL cells and terminal CD8_Ex. Transcriptome data from 221 pediatric B-ALL samples revealed that high CCL3/CCL4/CCL5/CCR5 levels correlate with low overall survival (OS). A multivariate Cox regression model incorporating CCL3/CCL4/CCL5 predicted prognoses. Finally, a model based on the adult B-ALL patients from our center also accurately predicted prognoses. We report for the first time the crucial role of the CCL3/CCL4/CCL5-CCR5 axis in the differentiation of terminal exhausted CD8⁺ T cells in B-ALL. High expression of CCL3, CCL4, CCL5, and CCR5 correlates with poor prognosis in B-ALL, suggesting potential biomarkers and therapeutic targets.

Keywords: B cell-acute lymphoblastic leukemia; cell interaction; exhausted CD8+ T cells; prognosis.

Figure 1.

Interactions between B-ALL cells and exhausted CD8⁺ T cell subpopulations in newly diagnosed and relapsed pediatric B-ALL patients based on scRNA-Seq. (a) UMAP projection of nine T cell clusters of newly diagnosed and relapsed pediatric B-ALL samples. Each dot corresponds to one single cell colored according to cell cluster. (b) Stacked bar chart showing the constitution of the nine T cell clusters in the de novo and relapsed groups. (c) Comparison of the frequency of exhausted CD8⁺ T cells in all T cells from the de novo (n = 7) and relapsed (n = 7) samples. Each dot represents one BM sample, while the center line indicates the median value. (d) UMAP plot showing three exhausted CD8⁺ T cells. (e) Heatmap showing the expression of marker genes of subclusters in exhausted CD8⁺ T cells. (f) Stacked bar chart showing the constitution of subclusters in exhausted CD8+ T cells in the de novo and relapsed groups. (g) Comparison of the frequency of progenitor and terminal exhausted CD8⁺ T cells in exhausted CD8⁺T cells from de novo (n = 7), relapsed (n = 7) samples. (h) Comparison of the proliferation score and exhaustion of terminal CD8_Ex between de novo and relapsed samples. (i) Bubble plots showing the intercellular communication between B-ALL cells and progenitor or terminal exhausted CD8⁺ T cells. (j) Boxplots showing the expression levels of the CCL3, CCL4, CCL5, and CCR5 genes in de novo and relapsed samples. Dx: newly diagnosed; Rel: relapsed. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 2.

Construction of a prognostic model based on the CCL3/CCL4/CCL5/CCR5 genes in the TARGET-P2-BALL dataset. (a–d) KM curves showing the OS of high and low expression of each gene in the TARGET-P2-BALL dataset. (a) CCL3. (b) CCL4. (c) CCL5. (d) CCR5. (e–g) KM curves showing the OS of two-gene co-expression of CCL3, CCL4, and CCL5. (e) CCL3/CCL4. (f) CCL3/CCL5. (g) CCL4/CCL5. (h–j) ROC curves showing the prognostic value of the two-gene co-expression of CCL3, CCL4, and CCL5. (h) CCL3/CCL4. (i) CCL3/CCL5. (j) CCL4/CCL5. (k) Forest plot showing multi-Cox regression analysis of B-ALL patients. (l) KM curve survival analysis of the low- and high-risk groups based on the multi-Cox regression model. (m) Radar plot showing the contribution of the three genes to OS, which was determined by the coefficients of the three genes in the multivariate COX regression model.