Reprogramming CAR-T Cell Fate: SeekOne™ DD Single Cell Multiome Reveals How Proteasome Inhibition Preserves Memory and Restrains Exhaustion
Release date : Sep 07,2026
Classification : Blog
Reprogramming CAR-T Cell Fate: SeekOne™ DD Single Cell Multiome Reveals How Proteasome Inhibition Preserves Memory and Restrains Exhaustion
For Research Use Only. Not for use in diagnostic procedures.
T cell exhaustion remains a major barrier to durable cancer immunotherapy. Chronically stimulated CD8⁺ T cells progressively lose effector function and stem-like capacity, limiting the persistence of tumour-infiltrating lymphocytes and engineered CAR-T cells. Although damaged mitochondria are known to accumulate during exhaustion, the molecular route connecting mitochondrial stress to long-term T cell fate has remained unclear.
In a 2026 Nature study, Xu and colleagues identified a proteasome-guided haem signalling axis that promotes terminal exhaustion. Depolarized mitochondria increased proteasome activity and mitochondrial protein degradation, releasing regulatory haem that disrupted BACH2-controlled transcription. The study then showed that low-dose bortezomib treatment during CAR-T manufacturing could improve therapeutic performance.
Why SeekOne™ DD Single Cell Multiome (ATAC+RNA) Matters
- 1 Paired modalities in the same cell. RNA and ATAC profiles connect cell identity, functional programmes and regulatory accessibility without relying on separate-cell integration.
- 2 State-specific resolution. Memory-like, exhausted and other CAR-T populations can be resolved before accessibility changes are assigned to the relevant cellular context.
- 3 Treatment comparisons across complex conditions. The study compared vehicle- and bortezomib-pretreated CAR-T cells both before and after co-culture with Nalm6 target cells, separating baseline effects from responses revealed by repeated antigen challenge.
- 4 An integrated analysis workflow. SeekArcTools generated gene-count and peak-count matrices, while weighted nearest-neighbour analysis combined transcriptomic and chromatin information into a unified view of CAR-T heterogeneity.
From Observation to Intervention
The authors first analysed public scRNA-seq datasets from tumour-infiltrating CD8⁺ T cells and found that proteasome activity increased with terminal exhaustion across cancer types. Proteomic, reporter, perturbation and animal experiments connected mitochondrial depolarization to CBLB-dependent mitochondrial protein degradation, regulatory haem release, altered BACH2–BLIMP1 control and impaired stemness.
Clinical scRNA-seq of 30,392 CAR-T cells from six patients with B-ALL (performed on a 10x Genomics platform) identified memory-like and cytotoxic populations. Persistent complete responders were enriched for memory-like cells, whereas rapid-relapse patients showed more Cytot-GZMB cells and higher proteasome scores. Public pre-infusion datasets from 94 additional patients supported the same association. These observations motivated a functional intervention.
SeekOne™ DD in Action: Multiome Evidence
Finding 1 — Bortezomib improves CAR-T performance
Adding 0.1 nM bortezomib during CD19 CAR-T manufacturing reduced the PD-1⁺TIM3⁺CD39⁺ exhausted population after repeated Nalm6 challenge. In leukaemia-bearing C-NKG mice, bortezomib-pretreated CAR-T cells significantly prolonged survival compared with control CAR-T cells. These functional experiments established that transient proteasome modulation could improve therapeutic performance, but left open whether the treatment preserved a distinct molecular state.
Finding 2 — Single-cell multi-omics resolves treatment-responsive states
The authors applied the SeekOne™ DD Single Cell Genome Multi-omics (ATAC+RNA) Kit to bortezomib- and vehicle-pretreated CAR-T cells from healthy human donors, both before and after Nalm6 co-culture. Matched RNA and ATAC profiles, integrated by weighted nearest-neighbour analysis, resolved six clusters. The memory-like CAR-T_2 cluster increased after bortezomib treatment, while CAR-T_4 represented an exhausted state — the intervention changed the balance of molecular states rather than affecting all CAR-T cells uniformly.
Finding 3 — RNA programmes show preserved function after antigen challenge
Within exhausted CAR-T_4 cells, co-cultured controls had the lowest proliferation, cytotoxicity, interferon and interleukin scores. Bortezomib-pretreated cells retained stronger cytotoxicity and cytokine programmes after challenge — a partial functional rescue within a vulnerable population that an averaged measurement could obscure.
Finding 4 — Chromatin accessibility reveals coordinated regulatory reprogramming
The ATAC layer showed reduced accessibility at PRDM1 and PDCD1 in bortezomib-treated CAR-T_4 cells after target-cell challenge. Differentially accessible regions favoured memory- and stemness-associated motifs, including KLF2, EGR2, RUNX2 and FOXO1, while terminal-exhaustion motifs were reduced.
Together with the RNA findings, these data support coordinated transcriptional and chromatin remodelling rather than a transient expression-only response. Accessibility and motif enrichment nominate regulatory programmes but do not prove direct binding or causality; the paper's perturbation and in vivo experiments provide the broader mechanistic evidence.
Why It Matters
Single-cell RNA sequencing and single-cell ATAC sequencing each capture essential but partial views of cell state. The SeekOne™ DD Single Cell Multiome (ATAC+RNA) platform delivers both modalities from the same cell, enabling researchers to ask whether a treatment has reshaped both transcription and chromatin — not just one or the other.
In this study, the multiome evidence complemented the functional and mechanistic data: it showed that bortezomib pretreatment produced coordinated transcriptional and epigenetic changes that persisted after repeated antigen stimulation, distinguishing durable reprogramming from transient expression shifts. This complementary framework — where functional assays establish causality and multiome data characterize the molecular state — illustrates how paired modalities add depth to cell-therapy research.
Xu Y, Shangguan Y, Chuang Y-M, Chang T-H, Liu W, Peng J-J, et al. Proteasome-guided haem signalling axis contributes to T cell exhaustion. Nature (2026). https://doi.org/10.1038/s41586-026-10250-y
Corresponding authors: Yingxi Xu, Jianxiang Wang, Ping-Chih Ho
Note: Volume and page numbers should be verified against the published version.
Contact
For more information about SeekOneTM DD scATAC+RNA-seq, please contact us at:
info@seekgene.com
For Research Use Only. Not for use in diagnostic procedures.
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