Multiomic correlates of ex vivo αCD19 chimeric antigen receptor T-cell (CART) cytotoxicity in venetoclax-treated patients with chronic lymphocytic leukemia (CLL)
Notice bibliographique
Résumé
Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) results in the expansion of immunosuppressive B-cells leading to risk for secondary malignancies, infection, and suboptimal T-cell based immunotherapeutic responses. Treatments that restore immune function (e.g. ibrutinib) may improve the efficacy of CD19 CAR T-cell (CART) therapies. We previously demonstrated that venetoclax (Ven) causes complex immunologic changes which broadly improve circulating immune cell (including T-cell) function. While we found that Ven appears to improve CD19 CART cytotoxicity in most patients, some CD19 CAR T-cells manufactured after Ven treatment did not perform as well in ex vivo cytotoxicity assays. We performed a multiomic correlation analysis to elucidate germane molecular features that may help predict and optimize Ven-enhanced CD19 CART cytotoxicity. Methods: We collected peripheral blood mononuclear cells (PBMCs) and serum from 13 consented CLL patients initiating standard of care Ven-based therapy (Ven-monotherapy N = 9, Ven+ɑCD20 N = 4; BDR-164122). Specimens were collected before treatment (D0) and after 30 days (D30) of therapy. CD19 CART were manufactured for 6 patients at both timepoints using an ɑCD19 construct (scFv/CD28/TCRζ via retroviral transduction). Cytotoxicity was measured against Raji-Luciferase cells. D0 and D30 specimens were analyzed by flow cytometry (including T-cell subsets and exhaustion markers) and RNA-seq (with xCell for cell-type deconvolution, rMATS for splicing, LISA for master regulator analysis). Enrichment analysis identified biological processes linked to cytotoxicity with statistical significance calculated as False Discovery Rate (FDR) adjusted P value. Metabolomics were performed with the Biocrates MxP Quant 500 XL assay. For each multiomic feature, magnitude change from D0 to D30 was calculated. These changes were correlated with changes in CD19 CART ytotoxicity across 7 effector-to-target ratios (1:8 through 8:1) using Spearman's correlation. Results: Enhanced CD19 CART cytotoxicity correlated with increased NK-cell frequency (CD56+/CD57+, CD56-/CD57+) determined by flow cytometry and cell-type deconvolution. In addition, RNA sequencing studies demonstrated that CD19 CART activity correlated with gene expression changes in interferon-ɑ (IFNA1, FDR<0.001) and interferon-ɣ (IFNG, FDR<0.002) response pathways. Specifically, an increase in key interferon signaling genes (GZMA, STAT1, CASP1) were associated with improved CD19 CART cytotoxicity. Moreover, a decrease in some interferon signaling genes (JAK2, STAT2, IFNAR2) was associated with a decrease in CD19 CART activity. LISA analysis identified POU2F2 and HDAC1 as key transcriptional regulators of genes associated with enhanced post-Ven CD19 CART killing. Elevated anti-inflammatory metabolites (cortisol, cortisone), as well as cysteine and phosphatidyl lipids correlated with improved CD19 CART cytotoxicity. Diminished CD19 CART cytotoxicity correlated with increased CD4+ LAG3+ T-cells coexpressing TIM3/TIGIT, and total CD3+ T-cells. Cell-type deconvolution demonstrated an inverse correlation between regulatory T-cells (T-regs) and CD19 CART cytotoxicity. Increase in NFκB signaling and splicing factor genes (e.g. RELA, RELB, NFKB1, NFKB2, TNF, U2AF1, U2AF2) were associated with a decrease in CD19 CART cytotoxicity (FDR<0.001). Alternative splicing by rMATS analysis identified a MAP3K7 (TAK1) event resulting in a TAK1 variant lacking its negative regulatory domain. This alternative splice form of TAK1 enhances NFκB signaling and impacts CD19 CART activity. Elevated serum triacylglycerols, GABA, spermine, and taurine detected by metabolic studies correlated with diminished CD19 CART cytotoxicity. Conclusion: Using a comprehensive multiomic approach we uncovered signaling pathways associated with improved CD19 CART activity following Ven-based therapy in CLL patients. The activation of the interferon-ɑ, interferon-ɣ and glucocorticoid signaling pathways enhance CART activity. In contrast, NFκB signaling and spliceosome-related pathways contribute to sustained immune activation and negative impacts on CAR T-cell function. Venetoclax treatment appears to alter T-cell (especially T-reg) and NK-cell subtypes, which influence the anti-tumor activity of CD19 CAR T-cells. Our work provides a foundation for further mechanistic inquiry into Ven-related immunologic impacts, which can improve synergistic combination treatments using CART for CLL patients.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».