Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation

Abstract

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia

“…A total of 54 EDTA-anticoagulated whole peripheral blood (PB) samples (from an identical number of subjects) were studied. Forty-two of them corresponded to samples included in a previously reported study [ 13 ], while the remaining 12 samples were prospectively collected between July and September 2021. From all PB samples, 17 corresponded to adult HD, 5 to HDc (identified for the first time in this study by the TRBC1-based FCM assay), 8 were collected from subjects with reactive Tαβ lymphocytosis and 24 corresponded to patients with Tαβ-LGLL (18 TαβCD8-LGLL, 5 TαβCD4-LGLL and 1 Tαβ double-negative LGLL).…”

“…All samples were immunophenotyped using a direct immunofluorescence stain-and-then-lyse technique based on the EuroFlow standard operating procedures (SOP) [ 17 , 18 , 19 ]. The samples included in the previous publication [ 13 ] were stained with the anti-TRBC1 antibody (clone JOVI-1) in combination with monoclonal antibodies (Mab) recognizing maturation markers (e.g., CD27, CD28, CD45RA and CD62L; Table S1 Panel I ), strictly following the EuroFlow SOP. The other samples were stained with a total of 40 different Mab reagents, including: (i) a common backbone of 12 antibodies against TRBC1, maturation-associated molecules (CD27, CD28, CD45RA and CD62L), markers which are frequently aberrantly expressed by clonal LGL (i.e., CD2, CD5 and CD7), and a set of four markers for identification of T cells and their major subsets (CD3, CD4, CD8 and TCRγδ); (ii) 24 TCRVβ Mab reagents (IOTest ® Beta Mark TCRVβ Repertoire Kit—Beckman-Coulter, Brea, CA, USA); and (iii) CD45 conjugated with four different fluorochromes ( Table S1 Panel II ).…”

“…The percentage and absolute number of TRBC1 + and TRBC1 − cells within each of the different maturation-associated (i.e., naïve, central memory, transitional memory, CD28 + and CD28 − EM, EE and TE) populations of Tαβ cells and their major TCD8 and TCD4 subsets was investigated as previously described [ 13 ].…”

“…The evaluation of the pattern of expression of the constant region 1 of the T-cell receptor β chain (TRBC1) by flow cytometry (FCM) has been recently demonstrated to be a fast, simple, specific and accurate method for assessment of the clonal nature of Tαβ cells in patients suspected of having T-CLPD [ 12 , 13 , 14 , 15 ]. Thus, the monoclonal expansions of Tαβ cells typically show a monotypic/unimodal TRBC1 expression profile (either TRBC1 + or TRBC1 − ), whereas normal/reactive Tαβ cells consist of a (bimodal) admixture of both TRBC1 + and TRBC1 − cells [ 12 , 13 , 16 ], where TRBC1 + cells represent nearly 40% ± 6.7% (mean ± 1SD) of all Tαβ cells [ 13 ]. However, recent data also indicate that the percentage of TRBC1 + Tαβ cells may vary substantially among the different maturation-associated compartments of T cells.…”

“…However, recent data also indicate that the percentage of TRBC1 + Tαβ cells may vary substantially among the different maturation-associated compartments of T cells. Thus, more mature populations of EM (particularly within the CD28 − subset) early-effector (EE) and TE-T cells display significantly broader ranges of TRBC1 + and TRBC1 − cells, compared to both total Tαβ cells and their naïve and central/transitional memory compartments [ 13 ]. Of note, clonal cells from T-LGLL patients, as well as most small T-LGL clones detected in otherwise healthy donors (HDc) [ 14 , 15 ], frequently display phenotypic profiles that match those of EM/TE cells [ 5 , 8 ].…”