Oral communication, CS18 / C82

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Functional cloning of a gp100-reactive TCR from depigmenting vitiligo skin

In vitiligo, progressive melanocyte loss is accompanied by skin infiltrating T cells. As T cells effectively eliminate melanocytes in vitiligo but fail to clear melanoma tumors, TCRs specific for vitiligo antigens may offer superior therapeutic efficacy for clearance of melanoma. Thus, we proposed isolating, cloning and characterizing T cells infiltrating vitiligo skin. T cells were sorted using peptide-pulsed dimers, reactivity towards melanoma cells was compared with TIL, and cells were cloned by limiting dilution. Peptide reactivity was confirmed by ELISPOT analysis. Clones were amplified to > 1×10(6) cells and subjected to RNA isolation. Rearranged TCR genes were identified by 5’RACE and subsequent DNA sequencing of cloned variable subunit fragments. T cells amplified from depigmenting skin of an HLA-A*0201+ patient were >16% gp100-209-217-reactive and demonstrated superior avidity compared with melanoma-derived T cells. TCR subunit analysis revealed a TCRalpha8,TCRbeta17 and a TCRalpha21,TCRalpha17 expressing clone. The former was linked by a P2A slippage sequence, cloned into the SAMEN construct under the CMV promoter, transfected into Phoenix packaging cells and transduced into Jurkat cells. Resulting Jurkat-SILv44 cells produced 20 pg/ml IL-2 in response to peptide-pulsed T2 cells, even in the absence of phorbol ester stimulation. These studies represent the first cloning of a vitiligo skin-derived, HLA-A2+, gp100-reactive TCR. Whereas previous studies have implicated the alpha subunit in defining peptide binding specificity of MART-1-reactive T cells, both our gp100-reactive clones share a common beta subunit with gp100-reactive, melanoma-derived T4H2, suggesting the defining subunit may be peptide-dependent. Importantly, SILv44 Jurkat cells display high TCR affinity as demonstrated by successful peptide-mediated activation without the need for CD8 co-stimulation.

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