Flow Cytometry Evaluation of T-Cell Receptor Constant Beta Chain-1 (TRBC1) Expression and Characterization of T-Cell Clones of Uncertain Significance
Authors: George Giby V. , LaBarge Tia , Burack W. Richard , El Hussein Siba , Wallace Danielle S , Syposs Chauncey R. 
Published: December 2024

Background: Although T-cell clones may be observed as part of the normal immune response, the detection of an aberrant T-cell population often raises the possibility of a T-cell malignancy. Here, we describe the occurrence of T-cell clones of uncertain significance (T-CUS) in peripheral blood, tissue, and body fluids in patients with no documented evidence of T-cell malignancies.
Methods: Expression of cell surface markers was measured using the ClearLLab 10C 10-color B-cell, T-cell, M1, and M2 panels on the Navios, Beckman Coulter flow cytometer. TRBC1 expression was assessed in CD3/TCRaβ-positive T-cells and subpopulations with abnormal CD5, CD2, CD4, CD8, CD7, and CD57 expression. Clonality was defined as the percent positivity/negativity of TRBC1 ≥80% or ≤20% in CD3/TCRaβ-positive T-cells in each subset. Relevant clinical information was obtained for all patients. All statistical calculations were performed using JMP Pro 18.0.1. The association of T-CUS with age was assessed using an unpaired two-tailed t test, while its association with other variables was evaluated using Pearson and likelihood ratio chi-square tests.
Results: One hundred and seventeen samples from 108 patients (median age: 63 years [range: 2 - 96 years], 47% [51/108] female) were tested. Sample types included blood (N=72), bone marrow (N=15), lymph node (N=17), pleural fluid (N=5), cerebrospinal fluid (N=1), thyroid (N=1), breast (N=2), bone (N=2), and skin (N=2). Based on TRBC1 expression, flow cytometry identified phenotypically discrete clonal T-cell populations in 38.5% (30/78) of patients in the absence of a history of a T-cell malignancy (positive or ≥80% in six patients and negative or ≤20% in 24 patients). A median T-CUS size of 1.3% (range: 0.11-9.54%) was observed with a single clonal subset observed in the majority of patients. Polymerase chain reaction for T-cell receptor gamma rearrangement was only available for 10% (3/30) of these patients and was concordant with clonality observed by TRBC1 expression. Of the 30 patients, 40% (12/30) were diagnosed with a non-T cell malignancy, such as a B-cell lymphoma, a monoclonal B-cell population, or a myeloid malignancy. Aberrant T-cell clones were identified in 60% (18/30) of patients with non-malignant diagnoses. Of the 30 total T-cell clones, 18 (60%) were CD8-positive, 1 (3%) was CD4/CD8-double-negative, 5 (17%) were CD4/CD8-double-positive, and 6 (20%) were CD4-positive. Additional phenotyping revealed at least partial expression of CD56 (14) and/or CD57 (34) in the majority of cases. The most frequent T-cell antigens lost in this cohort were CD5 and CD7 (7 cases each, respectively). Interestingly, CD5 was more frequently lost in TRBC1-negative CD8-positive T-CUS populations, which may alternatively represent natural killer (NK) cells. A statistically significant association was observed between the presence and size of T-CUS and age (p&lt;0.0001), splenomegaly (p=0.0385), and lymphadenopathy (p=0.0385). We observed no association of T-CUS with sex, a history of transplant (either solid organ or allogeneic hematopoietic stem cell), or peripheral blood counts.
Conclusion: The evaluation of TRBC1 expression by flow cytometry offers a sensitive method for the identification of minute T-cell clones in patients with no evidence of T-cell malignancy. Although the overall immunophenotypic features of these clonal T-cell populations largely resemble that of T-large granular lymphocytic leukemia, we hypothesize that these clones comprise physiologically normal T-cell and NK-cell subsets that often arise due to chronic antigenic stimulation. Although short patient follow-ups limit our ability to assess whether any patients subsequently developed a T-cell malignancy, future studies will examine subtypes of T-CUS within various malignant and non-malignant settings and assess for any association.