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The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) β-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαβ+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vβ repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

Objective The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. Materials and Methods We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Results Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Conclusion Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods. [ABSTRACT FROM AUTHOR]

Objectives Flow cytometry immunophenotyping is limited by poor resolution of T-cell clones. A newly described antibody was recently used to distinguish normal peripheral blood T cells from malignant T-cell clones. Here, we evaluate this antibody as a new diagnostic tool for detecting T-cell clonality in mature peripheral T-cell lymphomas. Methods Immunostaining for the T-cell receptor β chain constant region 1 (TRBC1) along with routine T-cell markers was performed on 51 peripheral blood and two bone marrow samples submitted to the flow cytometry laboratory for suspected T-cell malignancy. Results TRBC immunophenotyping identified malignant T-cell clones with 97% sensitivity and 91% specificity. Findings correlated with molecular T-cell clonality testing. In cases with equivocal molecular results, TRBC1 immunophenotyping provided additional diagnostic information. Conclusions TRBC1 flow cytometric immunophenotyping is a robust and inexpensive method for identifying T-cell clonality that could easily be incorporated into routine flow cytometric practice. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (GVHD), leukemia relapse, and immune deficiency remain the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor natural killer cells (NK) and cytokines have good potential in GVHD prevention and immune reconstitution enhancement. Improved survival after allo-HSCT therefore requires effective prophylaxis to reduce GVHD and strategy to mediate graft-versus-leukemia (GVL) effect. We studied the administration of expanded donor NK cell infusion and interleukin-2 (IL-2) and IL-15 mixture treatment in a murine allo-HSCT model for its effects on GVHD, immune reconstitution and leukemia relapse. In the GVHD model, recipient mice were reconstituted with bone marrow (BM) cells and splenocytes via vein. In the leukemia model, recipient mice were inoculated with EL9611 leukemia cells via vein 8 d prior to transplant. NK cell infusion mice group had lower clinical GVHD scores and suffered less severe GVHD-associated weight loss than control mice group. 90% of control mice died of leukemia relapse within 52 d post-transplant. NK cell infusion and IL-2 and IL-15 treatment recipient mice had improved survival compared with control mice. NK cell infusion and IL-2 and IL-15 treatment recipient mice had also accelerated lymphoid immune reconstitution compared with control mice group. Expanded donor NK cell infusion and IL-2 and IL-15 treatment could promote lymphoid immune reconstitution, mitigate GVHD, and reduce leukemia relapse in allo-HSCT recipients. The findings may have important significance for complication prevention in clinical allo-HSCT. [ABSTRACT FROM AUTHOR]

Objective: Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T-cell immunity. Measurement of T-cell receptor excision circles (TRECs) and T-cell receptor β (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T-cell production and reflecting antigen-specific T-cell clones proliferation. Methods: We used real-time quantitative PCR to detect TRECs from 43 Chinese patients who underwent three kind of allo-HSCT without T-cell depletion. RT-PCR was performed to amplify 24 subfamily genes of TCRBV in 24 patients of them. Results: For haploidentical-D group, the TRECs numbers were lower up to 24 months. For matched-sibling donor (MSD) group, the recovery of TRECs was faster than those of other two groups. TRECs values in matched-unrelated donor (MUD) were in the middle. During 2–19 months after transplantation, there were 6–16 BV subfamilies expressed and 33–48% of them were polyclones. The usage rate of TCRBV and percentage of polyclones in haploidentical-D were less than those of other two groups. Twenty-three CDR3 molecules were obtained from nine patients who were potentially associated with GVHD or CMV infection. Conclusions: Analyzing the changes of TCRBV repertoire and measuring TRECs during immune reconstitution would be useful to determine the host’s current immune status and ability of T-cell immune reconstitution and also to find antigen-specific T-cell clones in the three kinds of HSCT. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies to specific families of TCR variable domains serve as highly useful immunochemical tools for basic research in T-cell biology and diagnosis of autoimmune diseases. Monoclonal antibody MEM-262 characterized in this communication recognizes β chains of the TCR expressed by HPB-ALL cell line (carrying Vβ5.3) and a small subset of peripheral blood T cells. This subset is larger than that recognized by a previously described Vβ5.3-specific mAb. MEM-262 potently stimulates selective expansion of the T-cell subset, efficiently immunoisolates native TCR complexes as well as free β chains and uniquely recognizes denatured TCRβ chains under the conditions of Western blotting. [Copyright &y& Elsevier]