The identification of specific lymphocyte populations that mediate
tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with
cancer. To this end, mutant
hypoxanthine-guanine phosphoribosyltransferase (
HPRT) deficient (
HPRT-) T-cells were used as probes to detect T-cell clonal amplifications and trafficking in vivo in patients with advanced
melanoma. Mutant T-cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of
6-thioguanine (TG) selection and from
tumor-bearing lymph nodes (LNs) or metastatic
melanoma tissues by TG-selected mass cultures. Nonmutant (wild-type) cells were obtained from all sites by analogous means, but without TG selection.
cDNA sequences of the
T-cell receptor (TCR) beta chains (TCR-beta), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous
biomarkers of in vivo clonality of mature T-cell clones. Clonal amplifications, identified as repetitive TCR-beta V-
region, complementarity determining region 3 (CDR3), and J-region gene sequences, were demonstrated at all sites studied, that is, peripheral blood, LNs, and metastatic
tumors. Amplifications were significantly enriched among the mutant compared with the wild-type T-cell fractions. Importantly, T-cell trafficking was manifested by identical TCR-beta
cDNA sequences, including the hypervariable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T-cell fractions from
melanoma patients are enriched for proliferating T-cells that infiltrate the
tumor, making them candidates for investigations of potentially protective immunological responses.