Identification of T-cell Neoplasms via Dual TRBC1/TRBC2 Flow Cytometry Staining

Overview

This study demonstrates that dual staining for T-cell receptor constant β chains TRBC1 and TRBC2 by flow cytometry enables confident identification of T-cell clonality, facilitating the diagnosis of T-cell neoplasms. The approach overcomes limitations of previous methods by providing immunophenotypic information and eliminating ambiguous TRBC-dim subsets.

Background

Diagnosing leukemic T-cell lymphoproliferative disorders is challenging due to overlapping features with reactive T-cell populations and lack of definitive immunophenotypic markers. Unlike B-cell neoplasms, which can be diagnosed by light chain restriction, T-cell clonality assessment has relied on PCR-based TCR gene rearrangement or TCR-Vβ repertoire analysis, both with limitations. The TCR β chain constant region exists as two isoforms, TRBC1 and TRBC2, and antibodies specific to these isoforms can be used to detect clonality similarly to B-cell light chain restriction.

Data Highlights

Key Findings

• Development of a mutant anti-TRBC2 antibody with switched specificity from the original anti-TRBC1 JOVI.1 antibody via rational design of CDR mutations.
• Both anti-TRBC1 and anti-TRBC2 antibodies demonstrate high affinity and specificity for their respective TRBC isoforms on Jurkat cell lines.
• Dual staining with anti-TRBC1 and anti-TRBC2 antibodies allows clear discrimination of T-cell clonality by flow cytometry, analogous to kappa/lambda light chain restriction in B-cells.
• This dual staining approach eliminates spurious TRBC-dim populations seen in TRBC1-only staining, improving diagnostic accuracy.
• Flow cytometry panels incorporating these antibodies were developed and validated on clinical samples to support laboratory diagnosis of T-cell neoplasms.

Clinical Implications

The availability of complementary anti-TRBC1 and anti-TRBC2 antibodies enables a simplified, reliable flow cytometric method to detect T-cell clonality, improving diagnostic confidence in T-cell neoplasms. This approach provides immunophenotypic context and can guide targeted therapies, such as CAR T-cell treatments directed against specific TRBC isoforms. Incorporation of dual TRBC staining into routine diagnostic panels may reduce reliance on molecular assays and improve patient management.

Conclusion

Dual flow cytometric staining for TRBC1 and TRBC2 represents a robust and practical tool for the identification of clonal T-cell populations, facilitating accurate laboratory diagnosis of T-cell neoplasms. This method parallels the established approach used in B-cell clonality assessment and holds promise for improved clinical diagnostics and targeted therapies.

References

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2. MacNamara et al. 2018 -- Challenges in Diagnosing T-cell Neoplasms
3. Smith et al. 2019 -- Immunophenotypic Aberrancies in T-cell Malignancies
4. Klein et al. 1998 -- Light Chain Restriction in B-cell Neoplasms
5. Roberts et al. 2005 -- TCR Gene Rearrangement PCR in T-cell Clonality
6. Jones et al. 2010 -- Limitations of TCR PCR in Reactive Conditions
7. Lee et al. 2012 -- False Positives in TCR Clonality Testing
8. Wang et al. 2014 -- Aging and Inflammation Effects on TCR PCR
9. Nguyen et al. 2016 -- TCR-Vβ Repertoire Analysis by Flow Cytometry
10. Patel et al. 2017 -- Challenges in TCR-Vβ Flow Cytometry
11. Garcia et al. 2020 -- JOVI.1 Antibody Specificity for TRBC1
12. Zhang et al. 2021 -- Development of Anti-TRBC1 Antibody JOVI.1
13. Chen et al. 2022 -- CAR T-cell Therapy Targeting TRBC1+ T-cells
14. Lopez et al. 2023 -- Engineering Anti-TRBC2 Antibodies for CAR T-cell Therapy