Integrated single-cell RNA sequencing and TCR profiling identifies immune drivers of severe early toxicity in CLL patients treated with idelalisib

Abstract

Abstract PI3K inhibitors, such as idelalisib and duvelisib, have demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia (CLL) but severe autoimmune toxicities limit their clinical use. We have previously shown that idelalisib toxicity is driven by immune dysregulation, with a decline in CD4+ regulatory T cells (Tregs) and a shift toward effector T cells and Th17 pathway activation. However, these informative CyTOF studies did not address three critical questions: what underlies Treg dysfunction, how does idelalisib affect effector cells, and what drives Th17 pathway hyperactivation. We therefore performed single-cell RNA sequencing (scRNAseq) with TCR sequencing (scTCRseq) on T-cell-enriched PBMCs from 8 CLL patients with and without idelalisib-induced toxicity (n=4 for each group, matched pre- and on treatment samples). Differential abundance analysis revealed significantly higher baseline mucosal-associated invariant T (MAIT) and CD8+ naive cells in toxicity patients (tox pts), with lower MAIT, CD8+ effector memory (TEM) and CD4+ TEM cells on treatment. Supporting a pathogenic role for MAIT cells, differential expression (DE) analysis showed idelalisib increased IL7R, FOS, and STAT1 expression, suggesting increased Th17 differentiation in tox pts. GSEA demonstrated that Tregs in toxicity patients displayed upregulated ZEB1 and BACH2 (transcriptional repressors of Treg function) alongside downregulated EGR1 (a FOXP3 activator) on treatment, leading to impaired critical TNFA signaling via NFKB. Additionally, underexpression of key T cell migration markers (CFL1, RAC2, ACTB, CORO1A, and TMSB4X) in Tregs from tox pts suggested reduced migratory ability to inflammation sites thus impairing their immunity suppression capacity. Integration of scRNAseq and scTCRseq data revealed that T cells in toxicity-associated samples exhibited a greater proportion of unique clones at both pre- (tox vs nontox: 87 vs 72%) and on-treatment (tox vs nontox: 88 vs 68%) timepoints and higher clonal diversity compared to non-toxicity controls, with minimal changes following idelalisib exposure. Patient-specific clonal analysis revealed that idelalisib treatment induced expansion of new T cell clones primarily from CD8+ naive, CD8+ TEM, CD4+ central memory (TCM), and MAIT cells in both groups. In tox pts, major clones on treatment were new clones, whereas non-tox patients showed expansion of pre-existing clones. DE analysis revealed these expanded clones express distinct transcriptional programs between the two cohorts. In toxicity patients, emergent clones of CD8+ naive cells showed TH17/TH1 polarization (TBX21 and IFNG) with higher levels of and coexpression of cytotoxic (GZMB) and migratory (CXCR3) markers compared to new clones of nontox pts, suggesting these emergent clones may contribute to toxicity. CD4+ TCM new clones in the tox pts exhibited metabolic reprogramming activating the JAK-STAT pathway, as evidenced by enrichment of IFN-γ/α response (STAT1, JAK2, IRF1, TBX21 and IFNG) and allograft rejection pathways (CXCR3, PRF1 and IFNGR1). Key metabolic drivers of this expansion including MTHFD2, LDHA, and ACLY were co-expressed with cytotoxic and migratory phenotype markers in toxicity samples on treatment but not at baseline. These toxicity-associated CD4+ TCM clones also showed enrichment in TH17/TH1 differentiation and glycolysis pathway genes compared to pre- and other on-treatment clones. Lineage trajectory analysis revealed that a small subset of these CD4+ TCM new clones exhibited TH17/TH1 differentiation in tox pts while co-expressing cytotoxic and migratory markers, coinciding with relatively activated PI3K-AKT-mTOR signaling. Notably, this pattern was absent in non-toxicity controls, highlighting toxicity-specific metabolic reprogramming that leads to aberrant effector functions linked with autoimmune-like early immune toxicity. This aligns with prior findings that CD4+ T cells reprogram metabolism and promote Th17 responses via HIF-1α and GLUT1 in rheumatoid arthritis, an autoimmune disease. Overall, our findings suggest idelalisib-induced toxicity develops in patients with a predisposed immune microenvironment with sustained higher clonal diversity and metabolic-immune dysregulation, leading to the expansion of CD8+ and CD4+ T cell clones with TH17/TH1 polarization, cytotoxicity, and migratory phenotypes. These insights into PI3Kδ inhibitor-induced toxicity may suggest potential targets to mitigate adverse effects.