CD8(+), HLA-unrestricted, cytotoxic T-lymphocyte line against malignant melanoma

A CD8(+ )cytotoxic T lymphocyte (CTL) line was derived from the peripheral blood mononuclear cells of a patient with primary melanoma. The CD8(+ )CTL line specifically lysed the autologous primary melanoma cells and not the natural killer cell-sensitive K562 cells or lymphokine activated killer cell-sensitive DAUDI cells. When a large panel of human leukocyte antigen (HLA)-matched and -unmatched allogeneic melanoma, glioma, breast and colorectal carcinoma cells was tested as targets in cytolysis assays, 4 HLA-matched and two HLA-unmatched allogeneic metastatic melanoma lines were lysed by the CD8(+ )CTL. Lysis of autologous and allogeneic melanoma cells was dependent on the effector-to-target cell ratio. Lysis of autologous melanoma cells was not blocked by anti-HLA class I or class II antibodies, confirming that the cytolytic activity of the CD8(+ )CTL was HLA-unrestricted. CTL lysis of autologous melanoma cells was CD3 (T cell receptor) dependent and FAS-FAS-L, and CD1 independent. Identification of the melanoma-associated antigen recognized by the HLA-unrestricted CTL may provide a vaccine for a broad population of melanoma patients

In vitro migration of cytotoxic T lymphocyte derived from a colon carcinoma patient is dependent on CCL2 and CCR2

BACKGROUND: Infiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells. METHODS: To identify chemokine and chemokine receptors involved in T-cell migration toward CRC cells, we have used our previously published three-dimensional organotypic CRC culture system. Organotypic culture was initiated with a layer of fetal fibroblast cells mixed with collagen matrix in a 24 well tissue culture plate. A layer of CRC cells was placed on top of the fibroblast-collagen layer which was followed by a separating layer of fibroblasts in collagen matrix. Anti-CRC specific cytotoxic T lymphocytes (CTLs) mixed with fibroblasts in collagen matrix were placed on top of the separating layer. Excess chemokine ligand (CCL) or Abs to chemokine or chemokine receptor (CCR) were used in migration inhibition assays to identify the chemokine and the receptor involved in CTL migration. RESULTS: Inclusion of excess CCL2 in T-cell layer or Ab to CCL2 in separating layer of collagen fibroblasts blocked the migration of CTLs toward tumor cells and in turn significantly inhibited tumor cell apoptosis. Also, Ab to CCR2 in the separating layer of collagen and fibroblasts blocked the migration of CTLs toward tumor cells and subsequently inhibited tumor cell apoptosis. Expression of CCR2 in four additional CRC patients\u27 lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients. CONCLUSIONS: Our data suggest that CCL2 secreted by tumor cells and CCR2 receptors on CTLs are involved in migration of CTLs towards tumor. Gene therapy of tumor cells with CCL2 or CCL2/anti-tumor Ab fusion proteins may attract CTLs that potentially could inhibit tumor growth

SECTM1 Produced by Tumor Cells Attracts Human Monocytes via CD7-Mediated Activation of the PI3K Pathway

Tumor-associated macrophages (TAMs) have essential roles in tumor progression and metastasis. Tumor cells recruit myeloid progenitors and monocytes to the tumor site, where they differentiate into TAMs; however, this process is not well studied in humans. Here we show that human CD7, a T-cell and NK cell receptor, is highly expressed by monocytes and macrophages. Expression of CD7 decreases in M-CSF-differentiated macrophages and in melanoma-conditioned medium–induced macrophages (MCMI/Mφ) in comparison to monocytes. A ligand for CD7, SECTM1 (secreted and transmembrane protein 1), is highly expressed in many tumors, including melanoma cells. We show that SECTM1 binds to CD7 and significantly increases monocyte migration by activation of the PI3K (phosphatidylinositol 3′-kinase) pathway. In human melanoma tissues, tumor-infiltrating macrophages expressing CD7 are present. These melanomas, with CD7-positive inflammatory cell infiltrations, frequently highly express SECTM1, including an N-terminal, soluble form, which can be detected in the sera of metastatic melanoma patients but not in normal sera. Taken together, our data demonstrate that CD7 is present on monocytes and tumor macrophages and that its ligand, SECTM1, is frequently expressed in corresponding melanoma tissues, possibly acting as a chemoattractant for monocytes to modulate the melanoma microenvironment

Acquired Resistance to BRAF Inhibitors Mediated by a RAF Kinase Switch in Melanoma Can Be Overcome by Cotargeting MEK and IGF-1R/PI3K

SummaryBRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAFV600E melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.(VLID)460218

CD8⁺, HLA-unrestricted, cytotoxic T-lymphocyte line against malignant melanoma

Abstract A CD8+ cytotoxic T lymphocyte (CTL) line was derived from the peripheral blood mononuclear cells of a patient with primary melanoma. The CD8+ CTL line specifically lysed the autologous primary melanoma cells and not the natural killer cell-sensitive K562 cells or lymphokine activated killer cell-sensitive DAUDI cells. When a large panel of human leukocyte antigen (HLA)-matched and -unmatched allogeneic melanoma, glioma, breast and colorectal carcinoma cells was tested as targets in cytolysis assays, 4 HLA-matched and two HLA-unmatched allogeneic metastatic melanoma lines were lysed by the CD8+ CTL. Lysis of autologous and allogeneic melanoma cells was dependent on the effector-to-target cell ratio. Lysis of autologous melanoma cells was not blocked by anti-HLA class I or class II antibodies, confirming that the cytolytic activity of the CD8+ CTL was HLA-unrestricted. CTL lysis of autologous melanoma cells was CD3 (T cell receptor) dependent and FAS-FAS-L, and CD1 independent. Identification of the melanoma-associated antigen recognized by the HLA-unrestricted CTL may provide a vaccine for a broad population of melanoma patients.</p