Lack of common TCRA and TCRB clonotypes in CD8(+)/TCRαß(+) T-cell large granular lymphocyte leukemia: a review on the role of antigenic selection in the immunopathogenesis of CD8(+) T-LGL.

Clonal CD8(+)/T-cell receptor (TCR)αß(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vß and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαß(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRß (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαß(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCRαß(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαß(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.

A restricted clonal T-cell receptor αß repertoire in Sézary syndrome is indicative of superantigenic stimulation.

BACKGROUND: Sézary syndrome (SS) is a cutaneous T-cell lymphoma characterized by erythroderma, lymphadenopathy and malignant clonal T cells in the skin, lymph nodes and peripheral blood. A role for superantigens in the pathogenesis of SS has been postulated before. OBJECTIVES: To investigate a putative involvement of chronic (super-)antigenic stimulation in driving T-cell expansion in SS. METHODS: Antigenic specificity of the T-cell receptor (TCR) was assayed by molecular analysis of the TCRA (n=11) and TCRB (n=28) genes, followed by detailed in silico analysis. RESULTS: Sequence analysis of clonally rearranged TCRB genes showed over-representation of Vß8, Vß13, Vß17, Vß21 and Vß22, and under-representation of Vß2 and Jß1.1 when compared with healthy controls. No similarity was detected in amino acid motifs of the complementarity determining region 3 (CDR3). Analysis of TCRA rearrangements showed that there was no common Vα or Jα gene usage, and that TCRA CDR3 amino acid motifs were not highly similar. CONCLUSIONS: The lack of clear stereotypic TCRA and TCRB CDR3 amino acid motifs would argue against involvement of a single common antigen in the pathogenesis of SS. Nevertheless, the skewing of Vß and Jß gene usage does seem to point to a restricted TCR repertoire, possibly as a result of superantigenic selection prior to neoplastic transformation.

Human T-cell lines with well-defined T-cell receptor gene rearrangements as controls for the BIOMED-2 multiplex polymerase chain reaction tubes.

The BIOMED-2 multiplex polymerase chain reaction (PCR) tubes for analysis of immunoglobulin and T-cell receptor (TCR) gene rearrangements have recently been introduced as a reliable and easy tool for clonality diagnostics in suspected lymphoproliferations. Quality and performance assessment of PCR-based clonality diagnostics is generally performed using human leukemia/lymphoma cell lines as controls. We evaluated the utility of 30 well-defined human T-cell lines for quality performance testing of the BIOMED-2 PCR primers and protocols. The PCR analyses of the TCR loci were backed up by Southern blot analysis. The clonal TCRB, TCRG and TCRD gene rearrangements were analyzed for gene segment usage and for the size and composition of their junctional regions. In 29 out of 30 cell lines, unique clonal TCR gene rearrangements could be easily detected. Besides their usefulness in molecular clonality diagnostics, these cell lines can now be authenticated based on their TCR gene rearrangement profile. This enables their correct use in molecular clonality diagnostics and in other cancer research studies.

Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.