Laura Caputo

Migration of cytotoxic T lymphocytes toward melanoma cells in three‐dimensional organotypic culture is dependent on CCL2 and CCR4

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor‐positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patients CTL toward autologous tumor cells has been studied in a novel three‐dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti‐CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2‐transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.

CXC Chemokine Ligand 12 (Stromal Cell-Derived Factor 1α) and CXCR4-Dependent Migration of CTLs toward Melanoma Cells in Organotypic Culture

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, 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 melanoma patient’s CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1α) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.

Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture

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.

A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma.

CD4+ Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4+ Th cell clone (Th35‐1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV‐transformed B cells pulsed with melanoma tumor cell lysates. Th35‐1A produces IFN‐γ (a Th1‐type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II–restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II–matched and unmatched EBV‐B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA‐DR7 isolated from autologous EBV‐B cells rendered the cell line stimulatory for Th35‐1A cells. Specificity studies using autologous EBV‐B cells (EBV‐B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.

Shared MHC Class II–Dependent Melanoma Ribosomal Protein L8 Identified by Phage Display

Antigens recognized by T helper (Th) cells in the context of MHC class II molecules have vaccine potential against cancer and infectious agents. We have described previously a melanoma patients HLA-DR7-restricted Th cell clone recognizing an antigen, which is shared among melanoma and glioma cells derived from various patients. Here, this antigen was cloned using a novel antigen phage display approach. The antigen was identified as the ribosomal protein L8 (RPL8). A peptide of RPL8 significantly stimulated proliferation and/or cytokine expression of the Th cell clone and lymphocytes in four of nine HLA-DR7(+) melanoma patients but not in healthy volunteers. The RPL8 antigen may represent a relevant vaccine target for patients with melanoma, glioma, and breast carcinoma whose tumors express this protein.

Preferential involvement of CX chemokine receptor 4 and CX chemokine ligand 12 in T-cell migration toward melanoma cells

Our previous analysis of the role of chemokines in T lymphocyte trafficking toward human tumor cells revealed the migration of a melanoma patients cytotoxic T lymphocytes (CTL) toward autologous tumor cells, resulting in tumor cell apoptosis, in an organotypic melanoma culture. CTL migration was mediated by CX chemokine receptor (CXCR) 4 expressed by the CTL and CX chemokine ligand (CXCL) 12 secreted by the tumor cells, as evidenced by blockage of CTL migration by antibodies to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. Here, we present the results of T cell migration in one additional melanoma patient and T cell and tumor cell analyses for CXCR4 and CXCL12 expression, respectively, in 12 additional melanoma patients, indicating the preferential role of CXCR4 and CXCL12 in CTL migration toward melanoma cells. These studies add to the increasing body of evidence suggesting that CXCL12 is a potent chemoattractant for T cells.

Detection of HLA class II-dependent T helper antigen using antigen phage display.

Major histocompatibility complex (MHC) class II‐dependent antigens not only activate CD4+ T helper (Th) cells, but also cytolytic T lymphocytes and effector cells of the innate immune system. These antigens therefore are candidate vaccines against cancer and infectious agents. We have developed a novel approach using a model antigen, tetanus toxoid (TT), which provides the basis for the establishment of a novel strategy of cloning Th antigens. In the TT model system, a cDNA library encoding part of the TT light chain which contained a TT‐associated Th epitope recognized by TT‐specific Th clones was displayed on a phage vector (TT‐phage) and presented to TT‐specific Th cells by autologous Epstein‐Barr virus‐transformed B cells (APC). These TT‐phages were able to specifically activate two different TT‐specific CD4+ Th cell lines as demonstrated both in [3H]thymidine incorporation and cytokine release assays. Th cell stimulation by TT‐phages was significant at a ratio of one TT‐phage in 50 irrelevant phages. The described approach provides the basis for the development of a novel strategy of cloning MHC class II‐dependent Th antigens, using available Th cells. This strategy has several potential advantages over existing antigen cloning methods or biochemical peptide isolation.

Nucleophosmin is recognized by a cytotoxic T cell line derived from a rectal carcinoma patient

Immunotherapy of colorectal carcinoma (CRC) has great promise as the presence of T lymphocytes in CRC tissues in situ is correlated with reduced recurrence and increased survival. Thus, identification of the antigens recognized by T cells of CRC patients may permit development of vaccines with potential benefit for these patients. Using expression cloning, we identified the antigen, nucleophosmin (Npm), recognized by an HLA‐A1 restricted cytotoxic T lymphocyte (CTL) line derived from the peripheral blood mononuclear cells (PBMC) of a rectal cancer patient. A decamer peptide derived from the Npm sequence sensitized peptide‐pulsed HLA‐A1 positive cells to lysis by the CTL line. The peptide also induced proliferative and cytotoxic T lymphocytes in the PBMC of 4 of 6 CRC patients, which lysed HLA‐A1 positive peptide‐pulsed target cells and CRC cells endogenously expressing Npm. Overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.

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.