Fine specificity of monoclonal antibodies directed at human T cell receptor variable regions: Comparison with oligonucleotide‐driven amplification for evaluation of Vβ expression

Abstract

Seven distinct anti-human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR V beta genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide-driven amplification and cDNA sequencing. Four mAb were found to delineate the V beta 3, V beta 8, V beta 17 and V beta 19 subfamilies, respectively. The remaining reagents recognize subsets within the V beta 2, V beta 5 and V beta 13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide-driven amplification for evaluation of V beta 3, V beta 8, V beta 17 and V beta 19 subfamily expression in the peripheral blood. Although the V gene subfamily-specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR beta chain. The present data confirm the necessity to establish a complete set of well-characterized monoclonal reagents to study human T cell responses.

Increased usage of vβ2 and vβ6 in rheumatoid synovial fluid t cells

“…Second, previous studies of RA synovial T cells have shown that T cell oligoclonality is primarily found in IL-2 receptor-positive cells, which are likely to have been activated in vivo (10,ll). Third, when $ expression has been measured with MAb and PCR methods on the same polyclonal cells, the results have differed (36). One explanation for this discrepancy is that MAb measure relative numbers of cells bearing a particular V, , but do not take into account the activation status of the cells that are identified.…”

Expression and activity of IL‐17 in cutaneous T‐cell lymphomas (mycosis fungoides and sezary syndrome)

“…It was selected after TCR repertoire analysis performed by a semiquantitative PCR assay using the 24 different BV family-specific oligonucleotides. 26 The monoclonality of these Sezary T-cell lines was assessed by flow cytometry with anti-CD4 and anti-TCR V␤ mAb (Beckman-Immunotech) as well as V␥-J␥ multiplex-based analysis. The purity of recovered cells ranged from 97% to 99%.…”

Direct evidence to support the immunosurveillance concept in a human regressive melanoma.

“…A polyclonal biotinylated sheep antibody to mouse Ig (reactive with all mouse isotypes) and a streptavidin-biotinylated peroxidase complex (all obtained from Dako, Glostrup, Denmark) served as a detection system for the primary antibody. 3-Amino-9-ethylcarbazole (Dako) was used ( 18 ). In particular, purified TA-37+ cells from peripheral blood of normal donors were shown to express Vf1l6 transcripts exclusively while displaying a highly polymorphic TCR Va repertoire (data not shown).…”