Single-Cell Analysis of Anti-BCMA CAR T Cell Therapy in Patients with Central Nervous System Autoimmunity

Central Nervous System (CNS) autoimmune diseases, such as Neuromyelitis Optica Spectrum Disorders (NMOSD), are inflammatory demyelinating conditions characterized by relapses of optic neuritis and myelitis. Despite significant progress in the treatment of NMOSD, a subset of patients responds poorly to current therapies, with recurrent and difficult-to-treat disease courses. This may be due to current treatments not effectively targeting the immune dysregulation within the CNS. Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) induces CNS demyelinating damage via various pathways, including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated potential in long-term control of hematological malignancies and has shown preliminary efficacy in the treatment of some refractory autoimmune diseases. However, the mechanism of action of CAR T cells in autoimmune diseases is not yet clear. This article aims to explore the characteristics and roles of CAR T cells in autoimmune diseases by performing single-cell multi-omics analysis on NMOSD patients who have received anti-B cell maturation antigen (BCMA) CAR T cell treatment.

Source of the Study

This study was led by the Department of Neurology at Tongji Hospital affiliated with Huazhong University of Science and Technology, in collaboration with Beijing Tongren Hospital, Nanjing IASO Biotechnology Co., Ltd., Mayo Clinic, and several other institutions. The study results were published in the journal “Science Immunology” on May 10, 2024 (Qin et al., Sci. Immunol. 9, eadj9730 (2024)).

Research Procedure

Study Design

In this study, researchers generated and analyzed single-cell transcriptomic, T cell receptor (TCR), and B cell receptor (BCR) data from peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells of five anti-AQP4 IgG-positive refractory/recurrent NMOSD patients and five age- and sex-matched healthy controls. A total of 113,766 immune cell single-cell transcriptomic data (from 10 blood samples) and 34,098 CSF cell single-cell transcriptomic data (from 10 CSF samples) were obtained.

Data Processing

Through multi-omics analysis of these samples, researchers identified and analyzed paired sequences of T cells and B cells. The results showed that refractory NMOSD patients had an increase in B cells in the CSF but not significantly in the blood. Conversely, both PBs and PCs were significantly elevated in these two fluids in NMOSD patients. Further analysis indicated that the B cell lineage in the CSF was highly clonal, suggesting proliferation of antigen-specific clones within the CSF.

CAR T Cell Analysis

The study conducted a detailed investigation of the evolutionary trajectories of CAR T cells in five NMOSD patients before and after infusion. It was found that anti-BCMA CAR T cells peaked around 10 days after infusion, then decreased significantly within one month. Detailed clustering and gene expression analysis of these CAR T cells were performed through single-cell TCR sequencing and CITE-seq analysis. The results showed that pre-infusion CAR T cells were mainly proliferating T cells with CD4+ and CD8+ phenotypes. After infusion, these cells were mainly replaced by effector memory T cells (TEM).

Experimental Results

The study found that CAR T cells showed high levels of inflammatory and cytotoxicity-related gene expression early after infusion (within 7 to 14 days), which significantly declined later (21 to 28 days). CD4+ CAR T cells displayed gradually increasing memory-related gene expression, while CD8+ CAR T cells showed a gradual increase in natural killer (NK)-like receptor expression. Through TCR clone tracking, researchers discovered that these cells mainly exhibited a CD8+ proliferative phenotype in the early and late stages, indicating that these cells play an important role in the treatment of NMOSD.

CAR T Cell Penetration in CSF

Using imaging flow cytometry, researchers observed that anti-BCMA CAR T cells made direct contact with CD138+ PCs in the CSF, indicating that CAR-mediated PC killing can occur in both blood and CSF. The results showed that sBCMA and Ig in blood and CSF significantly decreased within 3 months after infusion, indicating PCs depletion and suppression of humoral immunity. Further single-cell RNA sequencing results showed that pre-infusion CAR T cells containing chemotaxis-related genes had better proliferation after infusion.

CAR T Cell Reversal of CSF Immune Dysregulation

The study demonstrated that after CAR T cells eliminated abnormal PB/PC in the CSF, the transcriptomic features of immune cells in the CSF showed significant improvement in terms of immune responses, inflammation activation, and chemotaxis. This suggests that after CAR T cell therapy, inflammatory characteristics of immune cells in the CSF are suppressed, and immune dysfunction is reversed.

Conclusion and Significance

This study revealed the mechanism of anti-BCMA CAR T cells in the treatment of NMOSD through detailed single-cell multi-omics analysis. These findings provide important scientific evidence for further optimization of CAR T cell therapy in autoimmune diseases. The study suggests that CAR T cells rich in CXCR3 expression have higher therapeutic effects in CNS autoimmune treatment, possibly by improving the proliferation of CAR T cells in the CNS and the efficiency of PB/PC depletion.

Highlights of the Study

  1. CAR T cells rich in CXCR3 expression showed significant penetration and proliferation effects in the CNS. This provides a new approach for further optimization of CAR T cell therapy.
  2. Through single-cell multi-omics analysis, the detailed mechanism of CAR T cells in NMOSD treatment was revealed. Results indicate that CAR T cells can reverse immune dysregulation in the CSF, thereby suppressing inflammatory responses.
  3. The study discovered the dynamic changes of CAR T cells after infusion, including early proliferation and inflammatory activation, and later memory phenotypes and NK-like characteristics. These findings provide important clues for understanding the behavior of CAR T cells in vivo.

Future Research Directions

Researchers suggest that further exploration of CAR T cells rich in CXCR3 expression in other CNS autoimmune diseases could be conducted. Additionally, there is a need to validate these findings in a larger group of patients and explore how to optimize the characteristics of CAR T cells through means such as gene editing, to improve their persistence and effectiveness in the treatment of autoimmune diseases.

In summary, this study provides new insights and directions for the application of CAR T cells in the treatment of CNS autoimmune diseases, with significant scientific value and clinical implications.