In situ cytokine therapy: redistribution of clonally expanded T cells.

Immunity to tumors relies on recirculating antigen-specific T cells. Whilst induction of antigen-specific T cells by immunotherapy has been convincingly proven, direct evidence for recirculation of such cells is still lacking. Here, employing a recently established in situ immunotherapy model for murine melanoma we directly demonstrate the redistribution of clonally expanded T cells. In this model IL-2 is targeted to the tumor microenvironment by means of specific antibody-IL-2 fusion proteins resulting in the expansion of T cells. The therapeutic effect of the fusion protein is not restricted to tumors expressing the targeted antigen, but extends to antigen negative variants of the tumor if present in the same animal. Analysis of the T cell infiltrate by quantitative reverse transcription-PCR revealed the presence of highly expressed TCR BV regions in both tumor variants. TCR clonotype mapping revealed that the high expressions of these regions were caused by clonal expansions and, notably, that these specific clonotypic TCR transcripts were identical in both tumors. Thus, T cell clones activated locally by targeted IL-2 therapy recirculate and mediate eradication of distant tumor sites not subjected to in situ cytokine therapy.

Comparative delineation of T cell clonotypes in coexisting syngeneic B16 melanoma.

B16 is a murine melanoma of C57B1/6 origin, which rapidly develops as a tumor when inoculated into syngeneic immunocompetent hosts. Nevertheless, B16 tumors are considered to be immunogenic since tumor regression can be induced by means of immunotherapeutic intervention. Furthermore, B16 melanoma cells express several melanoma-associated antigens that may serve as targets for autologous T cells. To study the in vivo T cell response against B16, with particular emphasis on diversity and systemic involvement, we examined the spectra of T cell clonotypes in coexisting B16 melanoma lesions in C57B1/6 mice. Three tumors from each animal (n = 8) were examined for the presence of clonotypic T cells using the highly sensitive T cell receptor (TCR) clonotype mapping technology. Systematic analysis of the TCRB variable regions 1-16 revealed from 19 to more than 30 clonotypic TCR transcripts in each tumor. To study intra- and inter-individual variations in the T cell response further, more than 600 clono-typic TCR transcripts were compared for sequence identity. Overall, approximately 2% of the T cell clonotypes were detected in more than one tumor from the same animal. Furthermore, none of the detected clonotypes was present in more than one animal, arguing against recurrent or "public" T cell responses against B16 melanoma. Our data strongly suggest that anti-melanoma T cell responses in this murine model encompass mainly localized T cells, and that systemic involvement is limited.