Prolonged cytopenia following CD19 CAR T cell therapy is linked with bone marrow infiltration of clonally expanded IFNγ-expressing CD8 T cells

Abstract

Autologous anti-CD19 chimeric antigen receptor T cell (CAR T) therapy is highly effective in relapsed/refractory large B cell lymphoma (rrLBCL) but is associated with toxicities that delay recovery. While the biological mechanisms of cytokine release syndrome and neurotoxicity have been investigated, the pathophysiology is poorly understood for prolonged cytopenia, defined as grade ≥3 cytopenia lasting beyond 30 days after CAR T infusion. We performed single-cell RNA sequencing of bone marrow samples from healthy donors and rrLBCL patients with or without prolonged cytopenia and identified significantly increased frequencies of clonally expanded CX3CR1^hi cytotoxic T cells, expressing high interferon (IFN)-γ and cytokine signaling gene sets, associated with prolonged cytopenia. In line with this, we found that hematopoietic stem cells from these patients expressed IFN-γ response signatures. IFN-γ deregulates hematopoietic stem cell self-renewal and differentiation and can be targeted with thrombopoietin agonists or IFN-γ-neutralizing antibodies, highlighting a potential mechanism-based approach for the treatment of CAR T-associated prolonged cytopenia.

Keywords: B cell lymphoma; CAR T cell; cytopenia; interferon.

Graphical abstract

Figure 1

Single-cell RNA-seq of healthy donor and post-CAR T rrLBCL bone marrow (A and B) Uniform manifold approximation and projection (UMAP) plots of all bone marrow mononuclear cells surviving QC criteria (92,676 cells) colored by patient (A) or cluster ID (B). (C and D) Stacked bar graphs show the number of cells falling within each cluster for each patient (C) and the number of cells from each patient falling within each cluster (D). (E) A bubble plot shows cell lineage and subtype markers for all bone marrow mononuclear cell clusters shown in (B). (F) A UMAP plot, showing clusters from (B), colored by the origin of the cell from either rrLBCL (pink) or healthy donor (green) bone marrow aspirates. (G) scCODA analysis of the frequency of cells across each cell cluster comparing rrLBCL (n = 14) and healthy donor (n = 5) bone marrow aspirates. Bars and error bars represent mean +/- standard error. ∗scCODA FDR < 0.05.

Figure 2

Comparison of cell frequencies and signatures between bone marrow aspirates of CAR T patients with or without prolonged cytopenia (A) scCODA analysis of the frequency of cells within each cluster in CAR T-treated rrLBCL patients with prolonged cytopenia (n = 9) compared to those without (n = 5). Bars and error bars represent mean +/- standard error. ∗scCODA FDR < 0.05. (B) A bubble plot shows SCPA for mSigDB hallmark gene sets across clusters of bone marrow mononuclear cells, comparing CAR T-treated rrLBCL patients with prolonged cytopenia (n = 9) to those without (n = 5). Gene sets with significant enrichment in any analyzed cluster from patients with prolonged cytopenia compared to those without are shown. Results for all pathways can be found in Table S5. (C) A volcano plot shows SCPA for mSigDB hallmark gene sets in hematopoietic stem cells (HSCs), comparing CAR T-treated rrLBCL patients with prolonged cytopenia (n = 9) to those without (n = 5). (D and E) Enrichment scores of IFN-γ-induced (IFNγ_Up) or suppressed (IFNγ_Down) genes from Zeng et al. (G) and Debruin et al. (H) comparing HSCs from CAR T-treated rrLBCL patients with prolonged cytopenia (125 cells from nine patients) to those without (87cells from five patients). Dot and bars represent median and first to third quartile. (F) IFNG-expressing cells across each cluster of bone marrow mononuclear cells, comparing cells from CAR T-treated rrLBCL patients with prolonged cytopenia (n = 9) to those without (n = 5). Bars and error bars represent mean +/- standard error. ∗Wilcoxon test FDR < 0.05.

Figure 3

Characteristics of bone marrow CD8 T cell subsets associated with prolonged cytopenia (A and B) UMAP plots of all CD8 T cells colored by patient (A) or cluster ID (B). (C) Bubble plot of cluster markers for CD8 T cell clusters shown in (B). (D) scCODA analysis of the frequency of cells within CD8 T-each cluster in CAR T-treated rrLBCL patients with prolonged cytopenia (n = 9) compared to those without (n = 5). Bars and error bars represent mean +/- SEM. ∗scCODA FDR < 0.05. (E) TCR clonotype frequency (left) and density of expanded TCR clonotypes (right) for CD8 T cells from CAR T patients with prolonged cytopenia (11,790 cell from nine patients) and those without (3,176 cells from five patients, excluding cells from control samples). (F) Comparison of clonotype proportion for all CD8 T cells (left, 14,966 cells) and for expanded clonotypes (right, 8,477 cells) in patients with prolonged cytopenia (n = 9) compared to those without (n = 5). Box represents median (bold line) and interquartile range, whiskers represent 1.5 x interquartile range, dots represent outliers. Statistical significance was assessed by Wilcoxon rank-sum test. (G) Comparison of clonotype proportion for all CX3CR1^hi T_EFF cells (left, 12,822 cells) and for expanded CX3CR1^hi T_EFF cell clonotypes (right, 8,416 cells) in patients with prolonged cytopenia (n = 9) compared to those without (n = 5). Box represents median (bold line) and interquartile range, whiskers represent 1.5 x interquartile range, dots represent outliers. Statistical significance was assessed by Wilcoxon rank-sum test. (H) Density of IFNG⁺ cells shown over a UMAP of CD8 T cell clusters. Only CD8 T cells from rrLBCL patients (n = 14) are shown; healthy donor cells are excluded. (I) The fraction of IFNG-expressing cells is shown comparing patients with prolonged cytopenia (n = 9) compared to those without (n = 5) for all CD8 T cells (left, 1,732 IFNG⁺ cells), and CX3CR1^hi T_EFF cells (right, 1,367 IFNG⁺ cells). Bars represent the mean; error bars represent standard error of the mean. Bars and error bars represent mean +/- standard error. Statistical significance was assessed by Wilcoxon rank-sum test. (J) Density of TBX21-expressing cells shown over a UMAP of CD8 T cell clusters. Only CD8 T cells (17,826 cells) from rrLBCL patients (n = 14) are shown; healthy donor cells are excluded. (K) The fraction of TBX21-expressing cells is shown comparing patients with prolonged cytopenia to those without for all CD8 T cells (left) and CX3CR1^hi T_EFF cells (right). Bars represent the mean; error bars represent standard error of the mean. Bars and error bars represent mean +/- standard error. Statistical significance was assessed by Wilcoxon rank-sum test. (L) Spectral flow cytometry of residual cryopreserved bone marrow cells from patients with PC (n = 6) and those without PC (n = 3) showing the sub-sampled (1,000 cells per sample) MFIs for IFN-γ in CD3⁺CD8⁺GZMB⁺ cells. (M) IFN-γ gMFI for all CD3⁺CD8⁺GZMB⁺ cells in each sample from patients with (n = 6) or without (n = 3) PC. Bars and error bars represent mean +/- standard error. p value represents a one-sided Wilcoxon rank-sum test.