Single-cell immune repertoire sequencing enables the simultaneous detection of nearly ten thousand cells in a single experiment. It can concurrently obtain information on the 5' end mRNA expression profile, TCR, and BCR from one sample. By integrating gene expression profiles with V(D)J data, it facilitates the analysis of immune responses in complex tissue samples, monitors the efficacy of immunotherapies, and investigates the molecular mechanisms of disease onset and progression.

1.Extensive Experience: Over 1,000 TCR/BCR projects completed. A single experiment can construct libraries for 1,000 to 10,000 cells, yielding both transcriptome and immune repertoire data.
2.Rigorous Quality Control: Possess optimized immune repertoire sequencing and experimental quality control workflows to obtain paired full-length V(D)J sequence information.
3.Expert Data Analysis: Rich experience in immune repertoire sequencing and data analysis, enabling personalized data analysis services. Coupled with our single-cell analysis cloud platform, it allows for one-click generation of interactive and dynamic result reports.
4.Proven Track Record: Extensive immune repertoire sequencing experience, having contributed to the publication of high-impact SCI journals.

Sample Types:
Tissue, Blood, Cultured cell lines, Prepared single-cell suspensions
Note: If the client provides tissue samples and lacks the capability to perform tissue dissociation to obtain single-cell suspensions, Lieven Bio will endeavor to provide technical and experimental assistance. However, due to the specificity of different sample types, we cannot guarantee the applicability of our methods to all tissue types.

Quality Requirements:
1.Cell viability >70%.
2.Cell concentration: 500-2,000 cells/μl.
3.Volume not less than 200μl.
4.Cell culture media or buffers must not contain Ca2+ and Mg2+ ions.
5.Cell diameter less than 40μm.

Anti-BCMA CAR-T Offers New Hope for PMS Treatment
This study represents the world's first human trial (NCT04561557) of anti-BCMA CAR-T therapy for Progressive Multiple Sclerosis (PMS). Treatment of 5 patients (1 with primary PMS, 4 with secondary PMS) demonstrated three key efficacy and safety characteristics:
1.Excellent Safety Profile: Only Grade 1 Cytokine Release Syndrome (CRS) occurred, with no ≥Grade 2 CRS; ≥Grade 3 cytopenias recovered within 40 days, with no severe anemia or thrombocytopenia.
2.Strong Central Nervous System Targeting: CAR-T cells penetrated the blood-brain barrier, showed sustained expansion in the cerebrospinal fluid (CSF) with reduced exhaustion characteristics, and achieved deep clearance of plasma cells in both the central nervous system (CSF, bone marrow) and periphery.
3.Significant Neurological Improvement: All patients showed decreased EDSS scores, improved performance on the 9-Hole Peg Test (upper limb function) and Timed 25-Foot Walk (lower limb function), reduced levels of cerebrospinal fluid neurofilament light chain (NfL, a marker of neuroaxonal injury), and no new brain lesions

Multidimensional Profiling of Treatment Dynamics to Decode the Challenge of "CNS Targeting"
To elucidate how CAR-T cells exert their effects in the central nervous system (CNS), it is essential to precisely track their dynamic changes across the three key compartments—"blood, cerebrospinal fluid (CSF), and bone marrow"—and their regulatory impact on the immune microenvironment. The single-cell experimental design of this study was structured around three dimensions: "temporal, spatial, and multi-omics." Novel Bio was involved throughout the process, from protocol optimization to data interpretation.
1.Sample Design:
Covering Key Compartments + Full Treatment Cycle
To fully capture the in vivo trajectory of CAR-T cells, the study collected three core sample types from patients at baseline (pre-treatment), and at 10 days (peak expansion), 1 month, 3 months, and 6 months post-CAR-T infusion:

·Peripheral Blood (reflecting systemic CAR-T cell kinetics)
·Cerebrospinal Fluid (directly assessing therapeutic effects within the CNS)
·Bone Marrow (a site enriched for plasma cells, assessing targeted clearance)

CAR-T infusion product samples were also included, forming a complete data chain from "input - in vivo distribution - efficacy correlation".

2. Technology Platform:
Multimodal Integration Enables Panoramic "Transcriptome + Protein + Epigenome" Analysis
Single-cell multi-omics sequencing was performed using the 10x Chromium platform. Cell function and regulatory mechanisms were deciphered through integrated multiparameter assays. Novel Bio was responsible for the standardized analysis of all data:
Multimodal Integration: Data alignment was achieved using Seurat and Harmony to integrate scRNA-seq with CITE-seq data, while ArchR was employed to correlate scATAC-seq with transcriptome information, ensuring cross-technology data consistency.

From Data to Mechanism: Decoding the "Black Box" of CAR-T Therapy
Why is anti-BCMA CAR-T effective in the central nervous system (CNS)? How do these cells survive and expand across different tissues? Through targeted analysis, Novel Bio systematically addressed these critical questions, delivering six key analytical outcomes for the study:
1. Single-Cell Clustering and Precise CAR-T Cell Identification
By integrating transcriptomic and protein data (CAR protein signals from CITE-seq), CAR-T cells were accurately identified from 498,114 cells and further annotated into 17 T cell subsets (e.g., CD8+ effector T cells, CD4+ effector memory T cells). CAR-T cells in the cerebrospinal fluid (CSF) exhibited lower exhaustion markers (reduced oxidative stress and mitochondrial dysfunction) and demonstrated delayed clonal expansion at the 1-month mark, providing evidence for their long-term CNS residency and sustained functionality.

Supporting Details: The study combined scRNA-seq, scTCR/BCR-seq, CITE-seq, and scATAC-seq to analyze blood, CSF, bone marrow, and infusion products. After quality control, 498,114 high-quality cells were classified into 17 major cell populations. From these, 217,820 T cells were extracted and reclustered into 17 T cell subsets, with TCR sequences detected in 199,712 (91.7%) cells. CAR-T cells were precisely identified across all three anatomical sites using CAR transgene alignment and CITE-seq CAR protein signals. The expansion phase was dominated by CD8+ effector/effector memory cells, while the CSF showed relative persistence of CD4+ Tem cells. Gene module scoring and multi-omics results consistently indicated that CSF CAR-T cells exhibited "comparable cytotoxicity but lower exhaustion," linked to reduced oxidative stress and mitochondrial dysfunction. Temporally, while CAR-T cells peaked in the periphery (blood/bone marrow) around day 10, the CSF displayed delayed clonal expansion at 1 month, with long-term detectability, providing direct evidence of CAR-T CNS residency and sustained action.

2. T Cell Clonal Kinetics: Revealing Cross-Compartment Migration Patterns
Using scTCR-seq data, the clonal expansion dynamics of CAR-T cells were tracked across blood, CSF, and bone marrow:

·In peripheral blood and bone marrow, CAR-T cells reached peak clonal expansion by day 10.

·In the CSF, CAR-T cells showed higher clonal diversity, with significant expansion occurring only at 1 month.
·Clonal overlap analysis confirmed that expanded CAR-T clones (at day 10) could migrate between all three compartments, demonstrating their ability to penetrate the blood-brain barrier—directly supporting the "CNS targeting" capability of CAR-T cells.

Supporting Details: The study revealed a sequential expansion trajectory: peripheral dominance followed by central migration. High-frequency clones were common in blood and bone marrow at peak expansion (day 10), while the CSF initially showed higher diversity and limited expansion. Significant amplification in the CSF occurred around 1 month, accompanied by an increase in S/G2/M phase clones and high-frequency clones. Clonal overlap analysis confirmed shared clones across blood, CSF, and bone marrow at peak expansion, with persistent shared clones in the CSF at 1 month, indicating enhanced cross-compartment migration during maximal expansion. This supports CAR-T cell ability to cross the BBB and establish CNS residency. The expansion phase was dominated by CD8+ effector/effector memory clones, while CD4+ effector memory clones showed relative persistence in the CSF during the contraction phase, forming a temporal sequence of "peripheral rapid burst – delayed central expansion – CSF persistence."

3. B Cell Lineage Analysis: Confirming "Deep Plasma Cell Clearance" Mechanism
The core pathology of PMS involves CNS plasma cells secreting autoantibodies that drive inflammation. Novel Bio analyzed B cell lineage changes by integrating BCR sequencing and transcriptomic data:

·At baseline, the Replacement-to-Silent mutation (R/S) ratio in the CDR3 region of CSF plasma cells exceeded 1.5 (compared to ~1 in healthy individuals), indicating strong antigenic selection and pathological involvement.

·By 3 months post-treatment, plasma cells were significantly reduced across all three compartments. Residual plasma cells showed normalized BCR mutation frequencies and R/S ratios, accompanied by reduced CSF oligoclonal bands (OCB) and kappa free light chain (KFLC) levels—directly confirming that anti-BCMA CAR-T eliminates pathogenic plasma cells.

Supporting Details: Integrated BCR-seq and transcriptome analysis revealed that pre-treatment plasmablasts/plasma cells in patient CSF were under intense antigenic selection pressure. Post-treatment, within 3 months, plasmablasts/plasma cells were significantly reduced across all compartments, with mutation frequencies and R/S ratios in residual cells returning to near-normal levels. This was accompanied by reduced CSF OCB, decreased KFLC and its index, and lower immunoglobulin levels, collectively proving that BCMA-targeted CAR-T cells can deeply clear pathogenic plasma cells and reverse central immune imbalance.

4. Cell-Cell Interaction Analysis: Deciphering "Microglial Activation" Regulation
Microglial overactivation is a key driver of neuroaxonal injury. CellChat analysis revealed:

·At baseline, strong interactions existed between B cells and microglia in the CSF, with B cells driving microglial inflammation via the TNF-α/NF-κB pathway.

·Post-treatment, following B cell clearance, microglial inflammatory pathways (e.g., cytokine production, antigen presentation) were significantly downregulated, and intercellular interaction strength decreased—explaining the reduction in the neuroaxonal injury marker (NfL) observed in patients.

Supporting Details: CellChat inference identified significantly enhanced B cell-microglia communication axes in pre-treatment CSF. Following CAR-T treatment, microglia-related inflammatory and antigen presentation pathways were downregulated, overall ligand-receptor interaction strength weakened, and a shift toward a more homeostatic, less inflammatory phenotype was observed. This network-level remodeling aligned with decreased NfL, directly supporting the mechanistic chain of "B cell clearance → alleviation of microglial overactivation → reduced neuroaxonal injury."

5. Epigenetic Regulation Analysis: Identifying Exhaustion Drivers in CAR-T Cells
Integrating scATAC-seq data, chromatin accessibility was analyzed in CAR-T cells from different compartments:

·Bone marrow CAR-T cells showed enrichment of oxidative stress-related transcription factor motifs (e.g., SMAD4, ID3), leading to faster exhaustion and shorter survival.
·CSF CAR-T cells lacked these epigenetic features and expressed lower levels of exhaustion markers (e.g., PD-1)—providing epigenetic targets for optimizing CAR-T cell persistence.

Supporting Details: Integrated scATAC-seq and transcriptome analysis revealed an oxidative stress-centric epigenetic fingerprint in bone marrow CAR-T cells. In contrast, CSF CAR-T cells lacked this enrichment, had lower oxidative stress/mitochondrial dysfunction scores, reduced exhaustion phenotypes, and lower expression of canonical exhaustion markers. This suggests that targeting pathways like SMAD4/ID3 could enhance CAR-T persistence and functional longevity in the CNS environment.

6. Clinical Data Integration: Bridging Molecular Mechanisms and Efficacy
Correlating single-cell analysis results with clinical indicators (EDSS scores, NfL, imaging) revealed:

·Patients with higher CAR-T clonal diversity in the CSF and more complete plasma cell clearance showed greater EDSS score improvements.
·The degree of microglial inflammatory pathway downregulation positively correlated with reduced MRI lesion volumes—providing molecular biomarkers for predicting clinical efficacy.

Supporting Details: Aligning multi-omics data with clinical readouts revealed a consistent mechanism-effect loop. The low-exhaustion phenotype, persistent expansion, and higher clonal diversity of CSF CAR-T cells accompanied functional scale improvements. Reductions in KFLC, OCB, and plasmablasts/plasma cells indicated effective removal of pathogenic plasma cells. Longitudinal improvements in EDSS, 9HPT, T25FW, and NfL aligned with this molecular remodeling, supporting the causal chain of "BCMA+ plasma cell clearance → CNS inflammation alleviation → functional improvement."

Pioneering CAR-T Therapy for Autoimmune Diseases, Novel Bio Drives Innovation
This study not only confirms anti-BCMA CAR-T as a potential curative therapy for PMS but also pioneers the field of "CAR-T therapy for CNS autoimmune diseases." Novel Bio’s "integrated single-cell multi-omics analysis capabilities" supported the study throughout, from experimental design optimization and data quality control to mechanistic interpretation:
1.Expertise in processing challenging samples (e.g., low-cell-count, high-impurity CSF), ensuring data reliability.
2.Ability to integrate multi-dimensional data (transcriptome, proteome, epigenome) to deeply explore cellular functions and regulatory networks.
3.Proficiency in correlating single-cell mechanisms with clinical efficacy, providing end-to-end solutions for translational research.
Looking ahead, Novel Bio will continue to focus on applying single-cell multi-omics technologies in areas such as cancer immunology, autoimmune diseases, and neurological disorders, delivering precise and efficient experimental and data analysis support for groundbreaking research and accelerating clinical translation.

Original Article Link:https://doi.org/10.1016/j.cell.2025.09.020