Measuring B-Cell Depletion & Immune Repertoire Reconstitution at the Clonal Level

Learn how longitudinal AIRR sequencing helps to characterize how the adaptive immune repertoire rebuilds and changes after B-cell depletion.

Key Takeaways:

• Deep B-cell depletion is now achievable across antibody, CAR-T, in vivo CAR-T, and T-cell engager modalities. Characterizing immune repertoire reconstitution post-treatment is an important next step.
• Longitudinal, isotype-resolved AIRR sequencing can track clonal depletion and repertoire reconstitution at a resolution flow cytometry alone cannot reach.
• iRepertoire’s RepSeq+, RepSeq IQ, and ImmunoSight™ services support longitudinal characterization of B-cell depletion and immune repertoire reconstitution following treatment.

Beyond Immunosuppression: New Approaches to Treating Autoimmunity

B-cell depletion has been the standard protocol for treating autoimmune disease for two decades. Anti-CD20 antibodies such as Rituxan®, Ocrevus®, and Gazyva® showed that removing pathogenic B cells could suppress disease activity, but the effect faded as B cells returned, requiring repeat dosing and continuous immunosuppression.

The field has since moved toward deeper, more durable approaches: autologous CD19 CAR-T therapy, in vivo CAR-T delivered by lipid nanoparticle (LNP), and now in early clinical testing in lupus¹ T-cell engagers that have driven drug-free remission in refractory autoimmune disease with more than a year of follow-up², and RNA-based approaches that reprogram T cells inside the patient.

What Type of Repertoire Returns Following B-Cell Depletion?

Deep depletion is now achievable across nearly every therapeutic modality in development. Depth of depletion, measured by flow cytometry cell counts and time to repopulation, was once the primary readout of success. What remains difficult for developers is characterizing the adaptive immune repertoire that emerges post-treatment:

• Did the therapy eliminate the clonotypes of interest?
• How deep was the depletion?
• Are clones permanently eliminated, or do they persist and re-expand later?
• Does the B-cell repertoire regenerate as a more naïve population, or does it repopulate along the same pre-treatment clonotypic lines?
• Does the T-cell repertoire shift in parallel with B-cell changes, and what does that shift indicate about mechanism?
• Can immune diversity metrics or specific immune signatures serve as a biomarker for risk of relapse?

Cell counts confirm that a population has decreased, but they do not reveal which clones disappeared, whether the ones that return are new or persistent, or whether isotype and mutation patterns point toward a more naïve immune system. Techniques that profile the immune receptor repertoire can provide this level of information.

What Can We Learn From Longitudinal AIRR Profiling?

Adaptive immune receptor repertoire (AIRR) sequencing provides clone-level molecular measurements complementary to flow cytometry. Sequencing the BCR and TCR repertoire at the clonal level across pre- and post-treatment timepoints enables developers to directly track clonotype abundance and follow specific clonotypes of interest from baseline through subsequent timepoints. Longitudinal AIRR sequencing can reveal the degree of overlap with baseline clonotypes, and together with somatic hypermutation and isotype patterns, characterize whether reconstitution is consistent with a more naïve immune repertoire.

The choice of starting material matters here. gDNA-based profiling, the approach behind iRepertoire’s RepSeq IQ service, pairs an internal quantitative control with housekeeping genes to convert sequencing reads into actual cell and clone counts, making it well suited to answering how deep a depletion went and how many cells of a given clone remain. RNA is the only template that captures actively expressed, functional isotypes. iRepertoire’s RepSeq+ service uses RNA to capture isotype signal directly, providing information on isotype distribution and class switching, which can help characterize antigen experience and repertoire maturation. The same RNA-based data supports full V(D)J variable region sequencing, which enables clone tracking across timepoints and calculation of somatic hypermutation, both central to characterizing the extent and nature of repertoire reconstitution. Beyond repertoire sequencing, iRepertoire’s ImmunoSight panel adds targeted expression data across more than 150 immunophenotype genes, giving developers an additional layer of biomarker context tied to therapeutic response.

Data generated through clonotype and isotype tracking, rather than cell counts alone, are the type of evidence the field needs to establish a measurable research framework for characterizing immune reconstitution in longitudinal studies.

Looking Ahead

As autoimmune cell and gene therapies mature, understanding how the adaptive immune system rebuilds after treatment may become just as important as achieving the initial depletion. Developers of CAR-T, in vivo CAR-T, T-cell engager, or antibody treatments in autoimmune disease do not need to rely on cell counts alone to determine therapeutic success. Longitudinal, isotype-resolved AIRR profiling offers a reproducible way to track clonal depletion, characterize repertoire reconstitution, and may be useful in revealing biomarkers associated with risk for relapse.

If your program is investigating B-cell depletion and immune repertoire reconstitution, talk with an iRepertoire scientist about designing a longitudinal repertoire monitoring strategy for your indication and modality.

References

1. Wang, Q., et al. In Vivo CD19 CAR T-Cell Therapy for Refractory Systemic Lupus Erithematosus. N. Engl. J. Med. 2025 Oct 16;393(15):1542-1544. https://doi.org/10.1056/NEJMc2509522.
2. Bucci, L., et al. BCMA T-Cell Engager Therapy in Patients with Refractory Autoimmune Disease. N. Engl. J. Med. 2025 Oct 16;393(15):1544-1547. https://doi.org/10.1056/NEJMc2506740.