Timothy J. Eberlein

SOLUBLE MUC1 SECRETED BY HUMAN EPITHELIAL CANCER CELLS MEDIATES IMMUNE SUPPRESSION BY BLOCKING T-CELL ACTIVATION

Solid tumors may secrete factors that mediate immune suppression in patients. We investigated the effect of supernatants from 25 human tumor cell lines on T‐lymphocytes from healthy donors. A profound inhibition of proliferation, cytokine secretion and cytotoxic activity was seen when T‐cells were cultured in concentrated tumor supernatants from 6 cell lines fractionated into high (>100 kDa) m.w. molecules. Interestingly, the inhibitory effects were reversed when the tumor supernatant was removed. Cell cycle studies of inhibited T‐cells showed most of them were growth arrested in the G0/G1 phase similar to naïve T‐cells. In addition, these T‐cells did not express IL2‐receptors and expression of CD54 (ICAM‐1) and CD58 (LFA‐3) resembled that of resting T‐cells. Protein gel electrophoresis of the tumor supernatants and western blot analysis demonstrated the presence of soluble MUC1 in the inhibitory tumor supernatants but not in control supernatant. Most importantly, depletion of soluble MUC1 by immunoprecipitation from the tumor supernatants neutralized the inhibitory effects on T‐lymphocytes. Therefore, our results show that MUC1 shed by cultured epithelial tumor cells mediates inhibition of T‐cell proliferation and function by inducing cell growth arrest. Int. J. Cancer, 82:721–726, 1999.

THE ROLE OF CD4+ TUMOR-INFILTRATING LYMPHOCYTES IN HUMAN SOLID TUMORS

Many, if not all, solid tumors are characterized by a T cell infiltrate, usually consisting of CD4+ and CD8+ T cells. Characterization of both subsets of tumor-infiltrating lymphocytes (TIL) have shown that each population can be divided into tumor-specific and tumor-nonspecific T cells. A small proportion of tumor-specific CD4+ TIL can directly lyse tumor cells in an HLA class I- or II-restricted fashion. The majority of tumor-specific CD4+ TIL, however, recognize tumor antigens presented on HLA class II molecules by antigen-presenting cells (APC). At the same time, APC in the tumor environment express elevated levels of heat shock antigen (Hsp) 70 (and perhaps other antigens) that can be specifically recognized by tumor-nonspecific CD4+ TIL when presented by HLA class II. Functionally, CD4+ TIL cells can be distinguished into Th0 (production of IL-2, IL-4, and IFN-γ), Th1 (IL-2 and IFN-γ), and Th2 (IL-4). In addition, stressed CD4+ TIL have the ability to produce the growth factors heparin binding epidermal growth factor and basic fibroblast growth factor that support tumor growth. Since the efficacy of an antitumor immune response is codetermined by the net effect of stimulatory and inhibitory cytokines, a detailed understanding of the developmental pathways of CD4+ TIL subsets and their interactions is critical for the design of clinical protocols.

Specific activation of retinoic acid receptors (RARs) and retinoid X receptors reveals a unique role for RARgamma in induction of differentiation and apoptosis of S91 melanoma cells.

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRα, RXRβ, RARα, RARγ, and RARβ in low levels. Among these, only RARβ gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARβ expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARβ gene expression, and blocks cellular proliferation. However, only the RARγ-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARβ, but suggest that RARγ has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.

Reactivation of murine tumour-infiltrating lymphocytes with solid-phase anti-CD3 antibody: in vitro cytokine production is associated with in vivo efficacy

Previously we described the use of solid-phase anti-CD3 monoclonal antibody (mAb) to stimulate murine tumour-infiltrating lymphocytes (TIL) and their subsequent expansion in recombinant interleukin 2 (rIL-2). In a pulmonary metastases model using the methylcholanthrene-induced sarcoma MCA-105 anti-CD3 activated TIL were capable of eradicating disease similar to TIL cultured in rIL-2 only. Here we extend these observations by characterizing the biological effects of sequential solid-phase anti-CD3 activation. TIL from MCA-105 tumour activated with solid-phase anti-CD3 on day 1 were reactivated on day 14, or day 26, or both and compared to TIL grown in rIL-2 only or TIL activated with anti-CD3 once on day 1. Reactivation enhanced in vitro proliferation 1.8- to 4-fold compared to TIL activated once with anti-CD3 (P < 0.05). In addition, the total lytic capacity of the cultures was enhanced after reactivation without changing the phenotype of the TIL cultures. Reactivation resulted in a greater in vivo efficacy when the TIL were administered within 72 h of reactivation. In contrast, TIL activated with anti-CD3 on day 1 and day 14 were least effective of all TIL cultures (P < 0.05). This correlated with in vitro cytokine production. The most effective TIL cultures in vivo produced 4- to 100-fold higher amounts of cytokines, especially interferon gamma (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF), than the other cultures. On the other hand, the least effective in vivo TIL culture, TIL activated with anti-CD3 on day 1 and 14, produced little or no cytokines. These data suggest that in vitro production of cytokines is indicative of in vivo efficacy of anti-CD3 activated TIL.

Benign schwannoma of the esophagus presenting as a giant fibrovascular polyp

The case of a 62-year-old man with benign schwannoma associated with a giant polyp of the esophagus is presented. His initial symptom was dysphagia. The polyp was removed through cervical esophagotomy. He had no recurrence of symptoms 5 years after this procedure. Pathologic examination showed a rare histology.

Human pancreatic cancer cells (MPANC‐96) recognized by autologous tumor‐infiltrating lymphocytes after in vitro as well as in vivo tumor expansion

A human tumor line designated MPanc‐96 has been established from a poorly differentiated primary pancreatic adenocarcinoma. MPanc‐96 has a doubling time of 27 hr and grows as a confluent monolayer in various culture media. Cytogenetic analysis of in vitro–cultured tumor cells revealed a large number of clonal chromosomal aberrations, confirming their neoplastic origin. MPanc‐96 grows in SCID mice when injected s.c. Xenografts established from the tumor line had a similar histology as the primary tumor. Tumor‐infiltrating lymphocytes (TILs) were isolated from the primary tumor, and cytotoxic T lymphocytes (CTLs) were generated after activation on immobilized anti‐CD3 monoclonal antibody (MAb) for 48 hr, expansion in low‐dose IL‐2 and repeated stimulation with irradiated MPanc‐96 tumor cells. The generated CTLs lysed fresh autologous tumor cells as well as in vitro and in vivo expanded tumor cells from passages 9–53, suggesting that one or more tumor‐associated antigens (TAAs) are stably expressed. CTLs lysed tumor cells in an HLA‐class I–restricted fashion but showed no significant cytotoxicity against autologous fibroblasts, several allogeneic pancreatic cancer cell lines or K562. Our findings may be significant for the design of an animal model for studying the mechanisms of immunotherapy in human pancreatic cancer or for the identification of TAAs in pancreatic cancer. Int. J. Cancer 71: 993‐999, 1997.

Vaccine therapy for cancer.

AbstractBackground: Tumor-specific cytotoxic T-lymphocytes (CTLs) can be isolated from the solid tumors, draining lymph nodes, metastatic effusions, and peripheral blood of cancer patients. Despite this evidence for a cell-mediated immune response to cancer, attempts at active specific immunotherapy using cancer vaccines have met with little success in clinical trials.nMethods: We have reviewed the immunobiology of the cell-mediated immune response to cancer by focusing on what is known about the major histocompatibility complex (MHC)-restricted interaction between tumor cells and CD8+ or CD4+ T-cells. In addition, we review the recent advances in the identification of tumor-associated antigens (TAAs) that are recognized by tumor-specific CTLs in melanoma and other cancers. In discussing these antigens, we highlight the recent identification of several MHC-restricted antigenic peptides that are recognized by CTLs from patients with melanoma and those with ovarian and breast cancer. We examine the implications that the discovery of these TAAs and peptides will have on the development of new anticancer vaccines. We review the most recent vaccine trials in melanoma and other cancers and focus on current concepts aimed at improving the therapeutic efficacy of future vaccines, including genetically engineered tumor cell vaccines.nConclusions: With the recent identification of several TAAs and antigenic peptide epitopes in melanoma and other cancers, immunotherapy researchers are now focusing on new strategies for the development of anticancer vaccines. As the repertoire of known TAAs increases and our understanding of the immunobiology of cell-mediated immunity to cancer improves, immunotherapists remain cautiously optimistic in their quest for effective cancer vaccines.

Immunoregulatory effects of CD4+ T helper subsets in human melanoma

BACKGROUNDnThe elucidation of CD4+ T helper (Th) cell traits is important for the understanding of immunoregulatory mechanisms in patients with cancer, in particular the Th-cell effect on cytotoxic CD8+ tumor-specific lymphocytes (CTL).nnnMETHODSnSixty-six T-cell receptor alpha beta+/CD4+ clones were generated from tumor-infiltrating lymphocytes of five patients with melanoma and classified into subsets by cytokine production. Transwell experiments were performed to test how the soluble factors of each Th-clone subset affected the cytotoxicity of the tumor-specific CTL against autologous tumor.nnnRESULTSnTh0 clones enhanced cytotoxicity of the CD8+ CTL compared with control CTL cultured in cytokine-free medium. Th1-clone supernatant also enhanced cytotoxicity by CD8+ CTL. In contrast, Th2 clones decreased killing compared with control CTL. Replacement of the Th clones by exogenous interleukin (IL)-2 in concentrations similar to that produced by Th0 and Th1 clones enhanced cytotoxicity. However, suppression of cytotoxicity was observed when similar concentrations of IL-4 were added instead. The helper effect of Th0-soluble factors could be inhibited by anti-IL-2 antibody, whereas anti-IL-4 antibody did not show a significant enhancement.nnnCONCLUSIONSnThe majority of the CD4+ tumor-infiltrating lymphocytes (Th0) in patients with melanoma enhance the CTL response to autologous tumor by their soluble factors, whereas Th2 cells suppress the CTL response.

Generation of peptide-specific cytotoxic T lymphocytes using allogeneic dendritic cells capable of lysing human pancreatic cancer cells☆☆☆

BACKGROUNDnDendritic cells (DCs) are potent antigen presenting cells (APCs), able to efficiently induce primary T cell-mediated responses to foreign antigens. In earlier studies we were able to identify a histocompatibility antigen (HLA)-A 2-restricted nine amino acid peptide (GP2, peptide 654-662) from the transmembrane portion of the protooncogene HER2/neu as a tumor-associated antigen (TAA) in human pancreatic cancer.nnnMETHODSnPeripheral blood mononuclear cells (PBMCs) of HLA-A2+ and HLA-A2 healthy volunteers were isolated. PBMCs were grown with initial anti-CD3, low-dose interleukin-2 (IL-2), and peptide-pulsed DC stimulation. T-cell lines were analyzed in functional studies.nnnRESULTSnAfter 4 weeks, T-cell cultures were more than 50% CD8+. All peptide-pulsed T cells significantly lysed APC pulsed with the immunizing antigen in an HLA-A2 restricted fashion. Furthermore, HLA-A2+,HER2/neu+ human pancreatic cancer cells were lysed significantly higher than HLA-A2 HER2/neu+ pancreatic cancer cells. Transfection of an HLA-A2 pancreatic cancer cell line with the HLA-A2 gene resulted in a significantly higher lysis of the transfected cell line compared to the wild type. In HLA-A2+ pancreatic cancer targets, specific lysis was HLA-A2 restricted.nnnCONCLUSIONnThe ability to use DCs for presentation of either tumor or peptide antigen in an HLA-restricted fashion to stimulate T-cell proliferation, as well as cytotoxicity, demonstrates the potential of this technology for future development of a pancreatic cancer vaccine.

Results of radical radiotherapy for inflammatory breast cancer

We performed a retrospective review of 65 patients with nonmetastatic clinical inflammatory breast carcinoma treated with radical radiotherapy as the sole local treatment between 1968 and 1986. Chemotherapy was given to 47 patients (72%). The median total radiation dose to the target volume was 6,984 cGy. With a median follow-up in survivors of 41 months, the 5-year actuarial probability of relapse-free survival was 17% and the overall survival was 28%. Thirty patients experienced failure in the treated breast, skin, or draining lymph nodes, for a crude, uncensored local recurrence rate of 46%. Of the factors analyzed, only the response to initial chemotherapy was predictive of local recurrence. Local recurrence was noted in 0 of 3 patients with a complete response (CR) to initial chemotherapy, 5 of 17 patients with a partial response (PR), and 12 of 17 patients with less than a partial response (CR/PR versus less than PR, p = 0.009). We conclude that conventional radical radiotherapy in unselected patients is insufficient to manage the local tumor burden presented by inflammatory breast cancer, even when high doses are employed.