SummaryIn polymyositis (PM), CD8 + T cell receptor (TCR) all3 + cells invade and destroy major histocompatibility complex class I-positive muscle fibers. We combined polymerase chain reaction (PCR) and double-fluorescence immunocytochemistry to analyze the T cell receptor (TCR) repertoire expressed in muscle of PM patients. In patient 1, inverse PCR revealed a preferential usage of TCR Vot33.1, V313.1, and V35.1. Six of six TCR Vot33.1 § clones and five of seven V/313.1 + clones had identical nucleotide sequences. In contrast, the VB5.1 + TCRs were more heterogeneous. Similar results were obtained with an independent PCR method using primers specific for TCR Vot33, V/313, or VBS. No TCR sequences could be amplified from noninflammatory control muscle. Furthermore, none of the TCR sequences found in PM muscle could be detected in blood from the same patient or from a normal control subject. Immunohistochemistry confirmed that V135.1 and Vr were overrepresented in the muscle lesions of this patient. 32% of all CD8 + T cells were V/313.1 +, and 16% were V/35.1 +. However, "~60% of the CD8 § T cells that invaded muscle fibers were V/313.1 +, whereas 10% were V/35.1 +. In patient 2, 50% of the T cells were V/35.1+, and as in patient 1, these T cells were mainly located in interstitial areas. In patient 3, >75% of the autoinvasive T cells stained with an anti-V~3 mAb. Sequence analysis of 15 PCR clones amplified with a V~3-specific primer showed that 9 (60%) sequences were identical. The results suggest that (a) a strikingly limited TCR repertoire is expressed in PM muscle; (b) there is a dissociation between the TCR usage of autoinvasive and interstitial T ceils; and (c) the autoinvasive T cells are clonally expanded.

Skin mast cells are typically located in the perivascular or perineural connective tissue. We observed that HMC-1 mast cells growing in suspension adhered efficiently to (> 90% of cells) and spread on top of fibroblast monolayers and to a lesser degree on purified extracellular matrix proteins. Since adhesive interactions determine cell migration and tissue localization we studied the mechanism. It was found that HMC-1 cells attach to collagen I and fibronectin, laminin, collagen IV and vitronectin, but not to collagens III and VI or hyaluronic acid. Adhesion to fibronectin, collagen I and laminin was completely inhibited by mAbs blocking beta 1-integrins, whereas adhesion of HMC-1 cells to vitronectin was inhibited by anti-alpha v-chain mAbs. However, attachment of HMC-1 cells to fibroblasts was not influenced by mAbs blocking beta 1- or alpha v-chain function, by RGD peptides or by mAbs interfering with other receptors, most notably c-kit. Identical results were obtained with normal mast cells isolated from human foreskin. These results indicate that human mast cells attach to fibroblasts independently of beta 1- or alpha v-integrins as well as of c-kit receptor-mediated mechanisms. The functional characteristics observed (i.e. only partial sensitivity to trypsin and EDTA, no increase in trypsin sensitivity by pretreatment with EDTA) suggest that cadherin receptors were not involved, and it is likely that the adhesion process observed involved not-yet-defined heterotypic cell-cell adhesion receptors.

IL‐10 mRNA expression and protein production in established melanoma cell lines and freshly cultured primary and metastatic melanoma cells was examined. The in situ distribution of IL‐10 in native melanoma tissue was also investigated by immunohistochemistry in primary tumors, metastases, benign melanocytic nevi and normal skin of healthy persons and melanoma patients. IL‐10 mRNA, but not IL‐10 protein in the culture supernatant, was found in 1 of 4 cultured melanoma cells of primary tumors, while 3 of 6 melanoma‐metastasis‐derived cultures expressed both IL‐10 mRNA and protein. No IL‐10 was detected in skin biopsies of healthy volunteers or in the healthy skin of melanoma patients; nor was IL‐10 found in congenital nevi. In only 1 of the 11 examined primary malignant melanomas was IL‐10 immunoreactivity detected within the cytoplasm of cells in the tumor. On the other hand, 4 of 9 metastases clearly displayed scattered IL‐10+ cells. In all sections with IL‐10‐positive cells, the cells were positive for HMB‐45. No co‐expression of CD3 and IL‐10 was observed. The data suggest that melanoma cells themselves are the main origin of IL‐10 in tumor specimens in vivo. The preferential expression of IL‐10 in metastatic lesions and in cultured cells from metastases might indicate an increased spreading potential of IL‐10‐secreting melanoma‐cell clones. © 1996 Wiley‐Liss, Inc.