A recombinant antibody-lymphotoxin-alpha fusion protein induced an adaptive immune response protecting mice from melanoma. Importantly, this fusion protein elicited the formation of a lymphoid-like tissue in the tumor microenvironment containing L-selectin+ T cells and MHC class II+ antigen-presenting cells, as well as B and T cell aggregates. Furthermore, PNAd+/TCA4+ high endothelial venules were observed within the tumor, suggesting entry channels for naive T cell infiltrates. Over the course of therapy, a marked clonal expansion of certain TCR specificities occurred among tumor-infiltrating lymphocytes that displayed reactivity against melanoma cells and the TRP-2(180-188) peptide. Consequently, naive T cells may have been recruited to as well as primed and expanded in the lymphoid-like tissue induced by the lymphotoxin-alpha fusion protein at the tumor site.
Adoptive cell therapy (ACT) with ex vivo expanded tumour‐infiltrating lymphocytes (TILs) in combination with IL‐2 is an effective treatment for metastatic melanoma. Modified protocols of cell expansion may allow the treatment of most enrolled patients and improve the efficacy of adoptively transferred cells. The aims of this study were to establish and validate the novel ‘Young TIL’ method at our institution and perform a head‐to‐head comparison of clinical‐grade products generated with this protocol opposed to the conventional ‘Standard TIL’, which we are currently using in a pilot ACT trial for patients with melanoma. Our results confirm that ‘Young TILs’ display an earlier differentiation state, with higher CD27 and lower CD56 expression. In addition, CD8+ TILs expressing CD27 had longer telomeres compared with the CD27−. A recently described subset of NK cells, endowed with a high expression of CD56 (CD56bright), was detected for the first time in both types of cultures but at a higher frequency on Young TILs. Young and Standard TILs’ reactivity against autologous tumours was similar, with significant expression of TNF‐α/IFN‐γ/CD107a by CD8+ TILs detected in all cultures analysed. However, either slow expansion with high‐dose IL‐2 only or large numerical expansion with a rapid expansion protocol, which is required for current therapeutic protocols, significantly modified TIL phenotype by reducing the frequency of less differentiated, cancer‐specific TILs. These studies further support the adoption of the Young TIL method in our current ACT trial and highlight the importance of continuous quality control of expansion protocols.
Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identi®ed as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identi®ed in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/ STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identi®ed, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) a ecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.
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