Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.
"B. [a-f]: The bottom layer of reconstructs contained 1.8 × 105 fibroblasts in 450 μl type I collagen gel which was followed by addition of CRC cells (1 × 105) on top after 24 hr. After further 24 hr, a separating layer of fibroblasts in collagen gel (100 μl, 500 μm) was added on top of cancer cells, followed by the top layer containing CTL (1 × 105 ), or autologous PHA blasts (control lymphocytes [a, c, e]) mixed with 1 × 105 fibroblasts and collagen. Reconstructs were harvested on day 6 (3 days after adding T cells), fixed in buffered formalin and embedded in paraffin. "
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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' lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients.
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.
Journal of Translational Medicine 03/2011; 9(1):33. DOI:10.1186/1479-5876-9-33 · 3.93 Impact Factor
"Another implication of our results deals with anti-tumor immune responses in which both CTL and TCR γδ T cells play important roles. Attraction of these cells to the tumor mass is operated in part by CXCR3 ligands expressed in variable amounts in the tumor microenvironment by tumor cells or stromal cells. Since sHLA-G levels are strongly increased in many malignancies, dampened recruitment of CTL and TCR γδ T cells to the tumor site is likely to take place in vivo and provide a novel mechanism of sHLA-G related immunosuppression. "
[Show abstract][Hide abstract] ABSTRACT: In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.
T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4(+) T cells, ii) CXCR3 in CD8(+) T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vdelta2gamma9 T cells, and upregulated CXCR4 expression in TCR Vdelta2gamma9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4(+) T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8(+) T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vdelta2gamma9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T(FH)) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T(FH) and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T(FH) cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, beta-arrestin and SHP2 was modulated by sHLA-G treatment.
Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.
PLoS ONE 07/2010; 5(7):e11763. DOI:10.1371/journal.pone.0011763 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The majority of basic research until now is still performed using two-dimensional (2-D) cell culture studies with tumor cell
lines grown in monolayer as models for tissues. However, cancer biologists increasingly recognize that tumor cells kept in
monolayer culture poorly represent the behavior of tumors in vivo. Importantly, they do not adequately recapitulate the tumor microenvironment which critically determines tumor cell proliferation,
invasive growth, and metastatic spread. More sophisticated model systems are required not only to better understand the biological
basis of these processes but also to more effectively evaluate anticancer drug candidates. In Sect. 15.1 we will review the currently available complex in vitro cell culture model systems which are used in melanoma research. In Sect. 15.2 we will describe the spectrum of experimental mouse models that have been developed over the past decades and discuss some
of the relevant strengths and weaknesses of the individual approaches.
KeywordsMelanoma-Compex model systems-Organotypic skin culture-Spheroid-Transgenic mice
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