Characteristics of premanufacture CD8+T cells determine CAR-T efficacy in patients with diffuse large B-cell lymphoma

Abstract

Although chimeric antigen receptor (CAR) T cells have become an important treatment option for patients with relapsed/refractory B-cell malignancies, more than 60% of patients with diffuse large B-cell lymphoma (DLBCL) treated with CAR-T cell therapies fail to achieve a durable response. To reveal changes in CAR-T cell therapy and identify response biomarkers, we conducted a retrospective analysis of pre-manufacture source T cells and CAR-T cell products and their association with outcome in 58 patients with r/rDLBCL who received tandem CD19/CD20 CAR-T cell therapy. We performed bulk RNA-Seq, single-cell RNA-Seq, and paired T cell receptor sequencing on CAR-T cell products and pre-manufacture T cells from DLBCL patients. We note that a CD8⁺ stem cell-like memory T cell population with a higher proportion and enhanced activating capacity of the CAR-T cell products was key to achieving durable clinical response. By analysing autologously-derived, pre-manufacture T cells, our data suggest that heterogeneity in the cellular and molecular features of pre-manufacture T cells contribute to the variation in efficacy after CAR-T cell therapy in DLBCL. The differences in anti-tumour efficacy of CAR-T cells among patients with different clinical outcomes appear to be due to the loss of CCR7 gene expression, coupled with increased expression of activation- and inhibitor-related genes in the CD8⁺ naïve-T cell populations among the apheresis T cells from patients with a poor molecular response. These findings significantly advance our understanding of the underlying molecular determinants of pre-manufacture T cell function.

Fig. 1

Evaluation of the clinical efficacy and anti-tumour function of tandem CAR-T cells in patients with r/rDLBCL. a Duration of response and survival after receiving the tandem CAR-T cell infusion. CR complete response, NE could not be evaluated, PD progressive disease, PR partial response. b Kaplan–Meier estimates of overall survival (up) and duration of response (down). c List of grouped in all 58 r/rDLBCL patients. d The CytoTox® 96 Non-Radioactive Cytotoxicity assay was employed to assess the cytotoxicity of CAR-T cells infused into Nalm6 cells at E:T ratio of 1:10. e Cytokine production by infused CAR-T cells co-cultured with Nalm6 cells at an E:T ratio of 1:1 for 24 h was measured using Luminex assays. f After coculturing CAR T-cells with Nalm6 cells at a 1:1 E:T ratio for 1 h, the expression of CD107a was observed. g Histogram plots of IFNγ, TNFα and GZMB expressed in infused CAR-T cells stimulated with small molecule mimics of TCR signalling. d–g CR patients n = 32; PD patients n = 11. h Serum cytokines, including TNFα, IL-2 and IFNγ, were measured in all evaluable patients, and their profiles are presented in this section. Response patients n = 45; Resistant patients n = 11. PD patients are resistant patients in this study

Fig. 2

Correlation analysis of multiple factors of the CAR-T cell products with the treatment efficacy of CAR-T cells in DLBCL. a Analysis of the CD4/CD8 ratio of CAR-T cell products in the patients according to the response status. b Ratio of CD4⁺ and CD8⁺ CAR-T cells in blood measured in all patients at the indicated times. a, b Response patients n = 45; Resistant patients n = 11. c Analysis of CD8⁺ TSCM of infused CAR-T cells in patients by response status (left), quartile map of objective response among the 56 evaluable patients (right). d ROC curve for response prediction based on CD8⁺ TSCM population in CAR-T cell products consisting of frequency in the validation cohort (n = 56). e, f Kaplan–Meier analysis of PFS in response patients stratified by high versus low levels of the appointed index (n = 45). The dotted lines are the error bars. 0.15 is the quartile of the ratio of the CD8⁺ TSCM population in CAR-T cell products to the SPD (tumour burden). g ROC curve for recurrence prediction based on CD8⁺ TSCM population in CAR-T cell products normalised to tumour burden consisting of frequency in the validation cohort (n = 45)

Fig. 3

Bulk RNA-seq analysis of infused CD8⁺ CAR-T cells and premanufacture apheresis CD8⁺ naïve T cells. a Schematic of validation Bulk RNA-sequencing experimental design. Comparing resistant patients with DR patients, the volcano plot shows fold changes in gene expression in infused CD8⁺ CAR-T cells (b) apheresis CD8⁺ naïve T cells (c). Genes that have been upregulated are displayed in the colour red, while genes that have been downregulated are displayed in the colour blue. Genes that have not shown a statistically significant difference in expression are displayed in the colour grey. The log2 of the tag counts represents the values. The p value was reported as less than 0.05, indicating statistical significance. GO functional clustering of genes that were regulated in infused CD8⁺ CAR-T cells (d) apheresis CD8⁺ naïve T cells (e) of DR patients versus resistant/PD patients. Representative GSEA enrichment plot in infused CD8⁺ CAR-T cells (f) apheresis CD8⁺ naïve T cells (g) of DR patients versus resistant patients. h The Venn diagram illustrates the intersection of various TF genes (p < 0.05) among infused CD8⁺ CAR-T cells, apheresis CD8⁺ naïve T cells, and apheresis CD8⁺ non-naïve T cells of CR and PD patients. down: TFs in CD8⁺ naïve T cells significantly differentially expressed in CR versus PD patients

Fig. 4

Single-cell RNA-seq analysis of infused CAR-T cell products. a Overview of all cells passing quality control for subsequent analysis in this study. In the tSNE plot, cells are colour coded on the left according to the corresponding patient origin (patient ID) and on the right according to the cluster number. b A bar graph showing the distribution of cells from DR and resistant patients among clusters. c The GO term dot plot illustrates the top ten differentially regulated pathways upon comparison of CAR-T cell infusions between DR patients and resistant patients. Differentially upregulated genes and pathways are in red; downregulated genes and pathways are in blue. d Expression of selected genes overlaid onto the tSNE plot. e The expression of T cell memory-associated markers is shown for each cluster. The size of the bubbles is proportional to the percentage of cells within a cluster that express a specific gene, while the colour corresponds to the average expression level of that gene within each cluster. f Quantification of the proportion of cells with different gene expression characteristics in DR and resistant patients. The heatmap displays the genes that are differentially expressed between CD8⁺TCF7⁺ CAR-T cells (g) CD8⁺CCR7⁺ CAR-T cells (h) obtained from patients with DR and those from patients with PD

Fig. 5

Analysis the composition and function of initial apheresis T cells. a Analysis of the cell population of apheresis T cells in DR and resistant patients. b Analysis of multiple indexes in CD8⁺ apheresis T cells (up) and CD4⁺ apheresis T cells (down) in DR and resistant patients. c Histogram plots of IFNγ, TNFα and GZMB expressed in apheresis T cells stimulated using small molecule mimics of TCR signalling. a–c DR patients, n = 26; PD patients n = 11. d The percentage of TSCM and CD8⁺ TSCM cell populations in CAR-T cells from different groups. e Cytotoxic activity of infused CAR-T cells and CAR-T cells cultured with naïve T cell of apheresis T cells with Nalm6 cells at an E:T ratio of 1:10. P-number indicated the unique number of patients in clinical trial (NCT03097770); P-number (PD) indicated CAR-T cultured with apheresis T cell from resistant patients; P-number (Naïve) indicated CAR-T cultured with naïve T cell from resistant patients. P-number (Naïve) indicated CAR-T cultured with naïve T cell from resistant patients. DR- number (Naïve) indicated CAR-T cultured with naïve T cell from DR patients. f Statistical analysis of cytotoxic activity of infused CAR-T cells and CAR-T cells cultured with naïve T cells. d–f DR patients n = 6; PD (resistant) patients n = 7

Fig. 6

Single-cell RNA-seq analysis of apheresis T cells that share TCRs with CAR-T cell products. a On the right, cells are colour coded according to the corresponding patient origin (patient ID), while on the left, cells are colour coded according to the cluster number. The function of different clusters was defined according to differentially expressed genes. b A bar graph showing the distribution of cells from DR and PD patients among clusters. c Expression of selected genes overlaid onto the tSNE plot. d Violin plots showing selected gene expression in the CD4⁺ naïve (top) and CD8⁺ naïve (down) T cell populations. Stars indicate significance (p < 0.05). e The heatmap displays the genes that are differentially expressed between apheresis T cells obtained from patients with DR and those from patients with PD. f Quantification of the proportion of apheresis T cells with different gene expression characteristics in DR and PD patients. g The expression of T cell memory-associated genes and activation/inhibitory associated-markers are shown for different cells. The size of the bubbles is proportional to the percentage of cells within a cluster that express a specific gene, while the colour corresponds to the average expression level of that gene within each cluster. h Heatmap showing the differentially expressed genes between CD8⁺TCF7⁺ T cells of apheresis cells from DR and resistant patients. i Characterisation of functional/molecular differences of infused CAR-T cells and apheresis T cells between DR and resistant patients. Image (i) created with BioRender.com