CD44 variant isoform v10 is expressed on tumor-infiltrating lymphocytes and mediates hyaluronan-independent heterotypic cell-cell adhesion to melanoma cells.

CD44 is a family of cell-surface receptors on human lymphocytes that act as co-stimulatory molecules leading to the induction of effector functions in T cells. We have analyzed primary cutaneous malignant melanomas with clinical and histologic signs of tumor regression using immunohistochemistry and observed the predominant expression of the CD44 variant isoform v10 on CD3 CD4/CD8 co-expressing tumor-infiltrating lymphocytes (TIL). We further analyzed the role of CD44v10 in adhesion of lymphocytes to human melanoma cells. In contrast to CD44- lymphatic cells, CD44v10+ lymphatic cells strongly bound to cultured human melanoma cells and to frozen tissue samples of melanomas. Antibody blocking studies revealed a hyaluronan-, integrin-, and selectin-independent pathway of adhesion. Furthermore, CD44v10+ lymphatic cells exhibited significantly higher invasiveness in three-dimensional collagen matrices as compared with CD44H+ and CD44-negative lymphocytes. These results indicate that expression of CD44v10 on TIL may mediate adhesion to melanoma cells and result in gain of novel invasive properties.

Downregulation of tapasin expression in progressive human malignant melanoma.

Tumor cells with alterations in the MHC class I peptide-loading complex are unable to load peptide antigens onto MHC class I molecules. These alterations result in destabilization of MHC class I expression on the tumor cell surface and thus play a critical role in escape from immunological recognition by the acquired cellular immune system. By forming physical links between class I heavy chains and TAP molecules, a component of the class I peptide-loading complex, tapasin, plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. In the present study, we compared 104 human melanoma lesions representing different stages of tumor progression for their expression of tapasin in tumor cells by immunohistochemistry. Tapasin downregulation was significantly associated with tumor progression. Whereas 100% of melanomata in situ and 96.2% of primary melanomas with a Breslow index of 0.75 mm as well as in 21.1% metastatic melanoma lesions. The downregulation of tapasin in advanced stages of human melanoma may reflect the accumulation of alterations in the antigen-presenting/processing machinery associated with neoplastic progression. These alterations may lead to failure of the acquired cellular immune system to control progression and metastatic spread of melanoma cells in vivo, and thus contribute to the immune escape phenotype of human melanoma cells.

Hyaluronan-independent adhesion of CD44H+ and CD44v10+ lymphocytes to dermal microvascular endothelial cells and keratinocytes.

We have recently shown the CD44 variant isoform 10 (CD44v10) to be expressed on reactive as well as malignant cutaneous lymphocytes; however, the functional consequences of CD44v10 expression on lymphocytes are not elucidated. By using appropriately transfected lymphatic cells we analyzed the role of CD44v10 on lymphocytes in cell-matrix adhesion and homotypic and heterotypic cell-cell adhesion assays. Despite a low binding affinity to hyaluronan, CD44v10-expressing lymphocytes exhibited heterotypic cell-cell adhesion to inflamed dermal microvascular endothelium and keratinocytes, as indicated by Stamper-Woodruff assays on tissue sections of delayed type hypersensitivity reactions and adhesion assays with cultured keratinocytes and cytokine-stimulated human dermal microvascular endothelial cells. Antibody-blocking assays excluded interaction of CD44v10 with the principal CD44 ligand hyaluronan as well as involvement of selectins or integrins in these heterotypic cell-cell adhesion assays. In contrast, cellular aggregation assays with fluorescence-labeled CD44v10- and CD44H-expressing lymphocytes revealed homotypic CD44v10/CD44v10 binding as well as binding of CD44v10 with CD44H. Heterotypic cell-cell adhesion assays with ultraviolet-A-irradiated CD44v-negative cytokine-stimulated endothelial cells demonstrated binding kinetics of CD44v10-expressing lymphocytes paralleling those of endothelial CD44H expression. These results imply that a hyaluronan-independent CD44v10/CD44H-mediated pathway is involved in lymphocyte infiltration into the dermis and epidermis of inflamed skin and suggest modulation of CD44H expression on inflamed dermal microvascular endothelium as a mechanism of ultraviolet-A-induced therapeutic effects on the skin.

Intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 induces T cell-mediated tumor protection in vivo.

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000