Preferential usage of T‐cell receptor αβ variable regions among tumor‐infiltrating lymphocytes in primary human malignant melanomas

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Abstract
The usage of T-cell-receptor (TCR) αβ variable (V) regions among tumor infiltrating lymphocytes (TILs) in primary human malignant melanomas was characterized using a method based on the polymerase chain reaction (PCR). A panel of 57 different variable-region primers specific for the TCR Vα 1–29 and Vβ 1–28 was designed, and a semi-quantitative PCR method applicable to formalin-fixed, paraffin-embedded tissues was developed. This semi-quantitative method was demonstrated to be reproducible and to be useful for the assessment of Vα- and Vβ-gene-family usage in formalin-fixed, paraffin-embedded tissue samples. A total of 9 different histopathologically characterized primary tumors were analyzed in this study. The TILs in these tumors were found to preferentially express certain TCR Vα and Vβ regions. The differential usage of certain Vα regions was very pronounced as illustrated by Vα4, which was highly expressed in 3/8 tumors, and Vα22, which was highly expressed in 4/8 tumors. For comparison, specific highly expressed Vα regions in control samples of peripheral-blood lymphocytes rarely exceded 10%. The most highly expressed VB region was Vβ8, which was highly expressed in 2/8 tumors. For the highly expressed Vα4 and Vα22 and Vβ8 regions, the high levels may be explained by the in situ clonal or oligoclonal expansion of TIL. In one specific case, the high expression of Vβ8 was due to expansion of a single clone of TILs, as evidenced by a fully readable sequence of the CDR3 (V-D-J) region, determined by direct sequencing of the PCR product corresponding to Vβ8. In contrast, sequence analysis of Vα22, which was expressed in the same tumor sample at similar levels, demonstrated the simultaneous presence of 3 different CDR3 (V-J) sequences derived from Vα22 transcripts of exactly the same length. The observed preferential use of TCR Vα and Vβ regions suggests the in situ clonal expansion of specific populations of T-cells, possibly reactive with melanoma-associated peptides presented by HLA molecules. The preferential use of TCR Vα and Vβ regions may imply the involvement of a limited number of shared melanoma-associated peptides.

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