Mini Review

TRBC1, an Addition Makes Rapid and Easy T Cell Clonality Assessment for Your Assay

T cell clonality testing has important clinical and research value. The gene rearrangement of the TCR β chain locus involves the selection of one of two mutually exclusive TRBC genes, and one of 52 TCR-Vβ genes. TCR β chain constant region is encoded by two genes, namely T-cell receptor β chain constant region 1 (TRBC1) and T-cell receptor β chain constant region 2 (TRBC2). Nonpathological polyclonal T cells express a mixture of TRBC1 and TRBC2, while malignancy T cells are usually monoclonal for one β chain constant region variant ¹. Both NGS-based TCR γ PCR and Vbeta repertoire analysis are highly complex, expensive, and labor-intensive. Due to poor specificity or lack of immunophenotypic clonal characterization, it provides ambiguous information in solving clonality ².

JOVI.1 is an antibody developed for CAR-T treatment of T-cell lymphoma, showing that the expression of TRBC1 in healthy peripheral blood samples and peripheral T-cell lymphoma is significantly different ³. Studies have also shown that 25% to 47% of peripheral blood T cells express TRBC1. Recently, several leading clinical laboratories have shown important data for clonality evaluation using JOVI.1 antibody for TRBC1 assay ^4-7, proving that, compared with NGS-based TCR γ PCR or Vbeta repertoire analysis, flow cytometric TRBC1 assay can provide comparable sensitivity and specificity, but faster and lower cost. The TRBC1 test provides real-time information about the T cell clonality of T cell subsets defined by the immunophenotyping. In all tested specimens, including peripheral blood and bone marrow ^4,5,7, lymph nodes⁷, as well as tissues and body fluids ⁶, the neoplastic T cell population with restricted (monotype) TRBC1 expression can be distinguished from non-neoplastic T cells.

The expression of TRBC1 in all healthy donor blood is polytypic. The ratio of TRBC1+ to TRBC1– T cells is approximately in in the range of 1:1 to 1:2 (Figure 1). According to the observations from multiple laboratories, the cut-off value of neoplasms (monotypic TRBC1 expression) on which the phenotypic distinction from background benign T-cells is >85% or <15% ⁵Figure 1. Comparison of clonality of reactive T cells and neoplastic T cells by TRBC1 (clones JOVI.1) FITC. Left: Reactive T cells show a polytypic pattern with 42.6% of cells positive for TRBC1 and 57.4% of cells negative for TRBC1. Right: Neoplastic T cells show a monotypic pattern with 8.7% of cells positive for TRBC1. Evaluated by Wei Wang from MD Anderson Cancer Center.

Application:

1. The expression of TRBC1 can only be assessed in CD3-positive, TCRaβ-positive T cells, therefore accurate gating of immunophenotypically distinct T-cell subsets is critical; CD4/CD8-double negative populations should be divided into subsets of α/β and γ/δ-expressing T cell.
2. Flow cytometric assessment of TRBC1 expression may be used to distinguish subsets of tumors that have obvious morphological overlap with other reactive or neoplastic processes.

Limitation of TRBC1 assessment

• Surface CD3-negative neoplasms and/or TCR γ/δ positive T cells
• B cells or NK cells and Benign thymocytes

TRBC1 assessment provides an opportunity for a simplified immunophenotypic evaluation of T-cell clonality, which can be easily implemented in the clinical workflow of routine flow cytometry practice.

Panel and Materials:

T cell Panel with TRBC1

mFluor450	mFluor540	iFluor488/FITC	PE	PerCP-Cyanine5.5	PE-Cyanine7	iFluor647/APC	APC-iFluor700	APC-Cyanine7
CD7	CD45	TRBC1	TCRγ/δ	CD2	CD3	CD5	CD4	CD8
1030146	1016166	113315	—-	100266	105386	112146	1120176	110996

Reference:

 

1              Mahe, E., Pugh, T. & Kamel-Reid, S. T cell clonality assessment: past, present and future. J Clin Pathol 71, 195-200, doi:10.1136/jclinpath-2017-204761 (2018).

2              Rosati, E. et al. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol 17, 61, doi:10.1186/s12896-017-0379-9 (2017).

3              Maciocia, P. M. et al. Targeting the T cell receptor beta-chain constant region for immunotherapy of T cell malignancies. Nat Med 23, 1416-1423, doi:10.1038/nm.4444 (2017).

4              Novikov, N. D. et al. Utility of a Simple and Robust Flow Cytometry Assay for Rapid Clonality Testing in Mature Peripheral T-Cell Lymphomas. Am J Clin Pathol 151, 494-503, doi:10.1093/ajcp/aqy173 (2019).

5              Shi, M. et al. T-cell clones of uncertain significance are highly prevalent and show close resemblance to T-cell large granular lymphocytic leukemia. Implications for laboratory diagnostics. Mod Pathol, doi:10.1038/s41379-020-0568-2 (2020).

6              Berg, H. et al. Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms. Cytometry B Clin Cytom, doi:10.1002/cyto.b.21881 (2020).

7              Shi, M. et al. Single Antibody Detection of T-Cell Receptor alphabeta Clonality by Flow Cytometry Rapidly Identifies Mature T-Cell Neoplasms and Monotypic Small CD8-Positive Subsets of Uncertain Significance. Cytometry B Clin Cytom 98, 99-107, doi:10.1002/cyto.b.21782 (2020).