Tumor-specific Lymphocytes Research Articles

Longitudinal immune monitoring of patients receiving intratumoral injection of a MART-1 T-cell receptor-transduced cell line (C-Cure 709)

Adoptive transfer of tumor-specific lymphocytes is a promising strategy in the treatment of cancer. We conducted intratumoral administration of an allogeneic irradiated continuous T-cell line (C-Cure 709) expressing an HLA-A2-restricted MART-1-specific T-cell receptor (TCR) into HLA-A2(+) melanoma patients. The C-Cure 709 cell line is cytotoxic against MART-1(+) HLA-A2(+) melanoma cell lines and secretes several immune stimulatory cytokines upon stimulation. Anti-tumor immune responses against the commonly expressed tumor antigen (Ag) MART-1 were longitudinally analyzed in peripheral blood by fluorescence-activated cell sorting (FACS) before and after intratumoral injection of C-Cure 709. No treatment-induced increase in Ag-specific T-cell frequencies was observed in peripheral blood, and the phenotype of MART-1-specific T cells was very stable during the treatment. Interestingly, despite a very stable frequency of MART-1-specific T cells over the course of treatment, clonotype mapping revealed that the response was in fact highly diverse and dynamic, with new clonotypes emerging during treatment. Only a few clonotypes were recurrently detected in consecutive samples. One MART-1-specific T-cell clone disappearing from peripheral blood was later detected in a metastatic lesion. Sequence analyzes of the CDR3 region revealed conserved structural characteristics in the MART-1-specific TCR used by T-cell clones.

Adoptive transfer of allogeneic tumor-specific T cells mediates effective regression of large tumors across major histocompatibility barriers

AbstractGraft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8+ pmel-1 or CD4+ TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma–bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persisted at detectable levels for several weeks and mediated significant regression of large, vascularized tumors. We found that the risk of GVHD was low when tumor-specific T cells were transferred and significant toxicity was observed only when substantial numbers of open repertoire allogeneic naive T cells were mixed with the tumor-specific lymphocytes. Taken together, these data indicate that the use of tumor-specific allogeneic CD8+ T cells or CD4+ can result in significant antitumor effects in the absence of measurable GVHD.

Abstract LB-238: Immunotransplant filters out regulatory T cells and cures large lymphoma tumors.

Abstract Background: The potency of transferring ex vivo activated tumor-infiltrating lymphocytes into lymphodepleted recipients has been demonstrated in clinical trials, though the use of in vivo vaccine-primed anti-tumor lymphocytes could make this approach sufficiently feasible for broad clinical application. We previously described a vaccination maneuver against murine B-cell lymphoma combining chemotherapy (CTX) and intra-tumoral injection of an immunostimulant CpG, which induced T-cell immunity and eradicated established tumors. An ongoing clinical trial of this maneuver has demonstrated complete and partial responses in lymphoma patients, though immune-regulatory factors such as regulatory T cells (Tregs) and limiting amounts of “homeostatic” cytokines might block even more powerful antitumor immunity. Transfer of tumor-specific lymphocytes into lymphodepleted recipients may eliminate these immune-regulatory obstacles. Methods: Splenocytes and bone marrow from CpG/CTX vaccinated mice were transplanted into lethally irradiated recipients, i.e., “immunotransplant.” Recipients received high dose tumor challenge with A20 murine B-cell lymphoma and followed clinically for tumor growth and for tumor-specific CD8 T cell IFNγ responses by multi-color flow cytometry. Results: We observed selective filtering against Tregs, doubling of the transferred Teffector:Treg ratio, and preferential expansion of transferred tumor-specific CD8 T cells in immunotransplant recipients. Immunotransplant cures large subcutaneous (>100 mm2) and metastatic tumors in 100% of recipients and enhances tumor-specific T cell memory. The addition of a vaccine “boost” to the immunotransplant maneuver results in an additional 3-fold increase in tumor-specific immunity and the use of cured recipients as subsequent donors in serial immunotransplant experiments demonstrated even greater tumor protection. Conclusions: As hematopoietic stem cell transplantation is a standard therapy for lymphoma patients, pre-transplant vaccination followed by post-transplant transfer of tumor-specific T cells could be tested in clinical trials. Our model suggests that this immunotransplant approach would allow the benefit of cancer vaccines to be revealed and might lead to cure of otherwise resistant malignancies.

Skin biopsies in DC vaccines for stage III–IV melanoma patients: Role of neutrophils?

Dendritic cell (DC) vaccines are used for the induction of anti-tumor T cell reaction in melanoma patients. DC are generated in vitro, pulsed with antigen and matured prior to injection. They are supposed to migrate to lymph nodes and to present the processed antigen to naive T cells allowing activation of tumor-specific lymphocytes. It has been suggested that intradermal injection allows a superior migration to the lymph node. Eight HLA-A2 positive patients with stage III or IV melanomas expressing NA 17 antigen were collected. They were included in a pilot trial of vaccination in which they received IL3/INFb DC presenting the NA17 A2 antigen. In each patient, a skin biopsy was performed at the injection site, 24 h after inoculation. The striking features of the biopsies were the presence of a perivascular CD3+/CD8+ T cell infiltrate with a slight population of CD4+ cells and the presence of a massive neutrophilic infiltrate associated with the injected DC still present, realizing a suppurative granuloma. The persistence of DC 24 h after the injection suggests that migration in the lymph node is not necessary for the induction of the immune response. The skin itself could be the location of a reaction starting with a massive recruitment of neutrophils.

Targeting death-inducing receptors in cancer therapy

Deregulated cell death pathways may lead to the development of cancer, and induction of tumor cell apoptosis is the basis of many cancer therapies. Knowledge accumulated concerning the molecular mechanisms of apoptotic cell death has aided the development of new therapeutic strategies to treat cancer. Signals through death receptors of the tumor necrosis factor (TNF) superfamily have been well elucidated, and death receptors are now one of the most attractive therapeutic targets in cancer. In particular, DR5 and DR4, death receptors of TNF-related apoptosis-inducing ligand (TRAIL or Apo2L), are interesting targets of antibody-based therapy, since TRAIL may also bind decoy receptors that may prevent TRAIL-mediated apoptosis, whereas TRAIL ligand itself selectively induces apoptosis in cancer cells. Here, we review the potential therapeutic utility of agonistic antibodies against DR5 and DR4 and discuss the possible extension of this single-antibody-based strategy when combined with additional modalities that either synergizes to cause enhanced apoptosis or further engage the cellular immune response. Rational design of antibody-based therapies combining the induction of tumor cell apoptosis and activation of tumor-specific adaptive immunity enables promotion of distinct steps of the antitumor immune response, thereby enhancing tumor-specific lymphocytes that can eradicate TRAIL/DR5-resistant mutating, large established and heterogeneous tumors in a manner that does not require the definition of individual tumor-specific antigens.

Partly MHC Matched Allogeneic Tumor Specific T Cells Mediate Tumor Regression without Inducing GVHD in Immunosuppressed Host.

Adoptive cell transfer (ACT) immunotherapy following lymphodepleting conditioning is a promising strategy for the treatment of metastatic solid tumors, however the difficulties in generating autologous tumor specific lymphocytes for every patient has significantly limited its applications. Allogeneic partly matched tumor specific T cells could be used for patients in whom autologous cells are not available, however their rapid rejection by the host restricts this approach. When CD8+ pmel-1 T cells from B6-BALB-C F1 (b/d) were transferred into B16 tumor-bearing B6 mice (b/b) or into a different F1 (b/k), these tumor-specific T cells were rapidly rejected, and had no impact on the tumor regression. Here we show that following myeloablative conditioning, the adoptive transfer of allogeneic, major histocompatibility mismatched tumor-specific T lymphocytes resulted in the regression of large vascularized tumors. The ability of adoptively transferred allogeneic T cells to mediate tumor regression was directly proportional to the dose of irradiation given prior T cell transfer which also correlated with the in vivo survival of the transferred cells. At the highest irradiation dose used (i.e. 11 Gy) allogeneic T cells could survive for as long as 4 weeks and their efficacy was comparable to syngeneic tumor-reactive T cells. In addition we found that the risk of inducing a graft versus host (GVH) reaction was minor when the specificity of transferred TCR is confined. In fact co-transfer of transgenic cells and small amounts of open repertoire T cells (2*10e4) able to react with the host did not result in any measurable toxicity whereas co transfer of greater quantities (2*10e5 or more) could cause fatal GVHD effect. Interestingly GVH reaction was associated with an improved tumor treatment, though this effect was transient as most of the animals succumbed to GVHD. Here we demonstrate that allogeneic T cells might represent an important tool in cancer immunotherapy allowing treatment of patients for whom it is not possible to obtain autologous cells. Furthermore the possible synergy between tumor specific allogeneic T cells and GVH effect could be exploited in bone marrow transplant protocols.

Systematic Comparison of Dendritic Cell-based Immunotherapeutic Strategies for Malignant Gliomas: In Vitro Induction of Cytolytic and Natural Killer-like T Cells

To compare the efficacy of various immunotherapeutic strategies of loading dendritic cells (DCs) with whole-glioma cell antigens and characterize the effector responses induced. DCs were either fused with major histocompatibility complex (MHC)-matched glioma cells (Fusion) or pulsed with apoptotic tumor cells (DC/Apo), total tumor ribonucleic acid (RNA) (DC/RNA), or tumor lysate (DC/Lys). These tumor-DC preparations were then assessed for their phenotype, cytokine profile, and capacity to stimulate autologous peripheral blood mononuclear cells (PBMCs) in vitro. Phenotype and tumor-specific cytolytic activities of various effector cell populations were characterized and compared. The various tumor-DC preparations exhibited similar phenotype and cytokine profiles irrespective of the method of loading tumor-cell antigens. However, the fusion, DC/Apo, and DC/RNA induced superior tumor cytolytic activities in PBMCs compared with DC/Lys or DC and tumor controls. DC/Apo induced the greatest expansion of tumor-specific lymphocytes, as detected by trypan blue exclusion and thymidine incorporation assays. Flow cytometric analyses also revealed the highest relative percentages of T helper cells (CD3+CD4+), cytotoxic T lymphocytes (CTLs) (CD3+CD8+), and natural killer (NK)-like T cells (CD3+CD56+) in the DC/Apo group among all the groups studied, indicating that DC/Apo induced expansion of PBMCs bearing multiple T and NK cell markers. Interestingly, isolated NK-like T cells demonstrated significantly higher tumor cytotoxicity compared with CTLs isolated from the same groups and was also non-MHC-restricted. Apoptotic tumor cells may be an optimal source of whole-tumor-cell antigen for immunotherapy of gliomas. The study also demonstrates for the first time that both CTLs and NK-like T cells are expanded and stimulated by mature, tumor-pulsed DCs.

Vaccination with dendritic cells pulsed in vitro with tumor antigen conjugated to cholera toxin efficiently induces specific tumoricidal CD8 + cytotoxic lymphocytes dependent on cyclic AMP activation of dendritic cells

We investigated the development of CD8 + tumor-specific cytotoxic lymphocytes (CTL) and protection against tumor growth after vaccination with bone marrow-derived dendritic cells (DC) pulsed with a model protein ovalbumin conjugated to cholera toxin (OVA-CT) in B6 mice using E.G7 tumor cells expressing OVA 257–264 peptide (SIINFEKL) as target cells in vitro and in vivo. Vaccination with OVA-CT-pulsed DC concurrently induced strong CTL in vitro activity and anti-E.G7 tumor protection in vivo in WT, NK-depleted and CD4-deficient mice as well as in IL-12−/− and IFN-γ−/− mice but not in CD8-deficient mice. Importantly, activation of CTL by OVA-CT-pulsed DC was dependent on CT-induced activation of adenylate cyclase and increased cAMP production by DC associated with increased expression of MHC class I and co-stimulatory molecules (CD80, CD86 and CD40). These results show that vaccination with DC pulsed with antigens (Ag) conjugated to CT induces a strong CTL response and suggest that conjugation of tumor Ag to CT for DC vaccination represents a promising approach for tumor vaccination and immunotherapy.

IL-2 production by virus- and tumor-specific human CD8 T cells is determined by their fine specificity.

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

Growth and functional reactivity of lymphocytes obtained from three anatomic compartments in patients with non-small-cell lung cancer (NSCLC).

Non-small-cell lung cancer (NSCLC) claims the lives of both men and women worldwide. In this study, we describe the ability to expand lymphocytes from solid tumor biopsies, pleural fluid, and peripheral blood of patients with NSCLC. While lymphocytes readily expanded from both solid biopsies and pleural fluids, the incidence of obtaining tumor-specific lymphocytes was low. Specific cytolytic and/or cytokine-releasing activity was observed in tumor-infiltrating lymphocytes (TIL) from 3/15 solid tumor biopsies (20%) and 2 of 4 pleural fluid samples (50%). Two of the CTL derived from solid tumor were cytolytic, one being HLA-A2 restricted while the other was either restricted at HLA-A30 or - B18. One of the pleural fluid cytotoxic T lymphocyte (CTL) was also HLA-A2 restricted. Peripheral blood from NSCLC patients was studied using mixed lymphocyte tumor cell cultures (MLTC) as reported previously. Peripheral blood lymphocytes were cultured with allogeneic NSCLC-TC line 29.7, which expressed by gene transfer the lymphocyte costimulatory molecule CD80. Of 9 HLA-A2 patient samples tested, 8 gave rise to lymphocytes, which lysed NSCLC tumor cells expressing HLA-A2 in an major histocompatibility complex (MHC)-restricted fashion. While specific CTL can be generated from all anatomic sites, for clinical trials peripheral blood appears to be the site of choice for obtaining specific precursors.

Immunosensitization of resistant human tumor cells to cytotoxicity by tumor infiltrating lymphocytes

Most anti-cancer therapies induce apoptotic cell death, but a major barrier to long-term cancer treatments is the generation of apoptosis-resistant tumor cells. Tumor cells that become resistant to one therapy are usually cross-resistant to subsequent therapies, including those with different cellular/molecular targets, suggesting that resistant tumor cells acquire modifications of the general apoptotic pathway. Most solid tumors are characterized by infiltration of lymphocytes (tumor infiltrating lymphocytes, TIL), which may serve as a basis for new strategies to generate tumor specific lymphocytes. However, TIL frequently are unable to kill autologous tumor cells suggesting that they are anergic/tolerant. It is possible that the TIL are functional but the tumor cells are resistant to TIL-mediated apoptotic pathways. Previous findings revealed that resistant tumor cells can be sensitized with cytokines or subtoxic concentrations of chemotherapeutic drugs and restore killing by cytotoxic lymphocytes. In this study, we examined whether TIL can kill autologous and allogeneic tumor cells following sensitization with chemotherapeutic drugs. Renal and prostate cancer-derived TIL were cytotoxic to chemosensitized resistant tumor cells. Killing by TIL was found to be perforin-dependent and perforin-independent. These findings demonstrate that combination drug and immunotherapy may be able to overcome tumor cell resistance to killing by TIL. Further, in vivo sensitization of drug-resistant tumor cells by subtoxic doses of sensitizing chemotherapeutic drugs may result in tumor regression by the host immune system.

The Fas/Fas ligand system and cancer: immune privilege and apoptosis.

The Fas/FasL system has been suggested to play an important role in the establishment of immune privilege status for tumors by inducing Fas-mediated apoptosis in tumor-specific lymphocytes. However, the role of cell-surface expressed FasL in tumor cell protection has recently become controversial. Our laboratory has focused on the study of the role of the Fas/FasL system in the normal tissue remodeling of the female reproductive tract and in immune-privileged organs. Our studies have demonstrated a connection between sex hormones and the regulation of the Fas/FasL pathway in immune and reproductive cells. More recently, we have investigated the resistance of tumor cells to Fas-mediated apoptosis. We have also characterized a new form of FasL, different from the classical membranal form, which is secreted by ovarian cancer cells. In this review we describe the main techniques used in these studies.

Crosstalk between Tumor T Lymphocytes and Reactive T Lymphocytes in Cutaneous T Cell Lymphomas

We have established several tumor T cell lines, both from the skin and from the blood of a patient with an MHC class II-/class I+, CD4+ cutaneous T cell lymphoma (CTCL). These cell lines, like the initial tumor cells, had a CD3+CD4+CD8- phenotype. We also isolated two cytotoxic T lymphocyte clones from the tumor site of this CTCL patient. These clones displayed a CD4+CD8dim+ (TC5) and CD4+CD8- (TC7) phenotype and mediated a specific MHC class I-restricted cytotoxic activity toward noncultured tumor cells and autologous tumor cell lines. Despite surface expression of Fas on tumor cells and Fas-L induction on TC5 and TC7 cell membrane after coculture with autologous tumor cells, the CD4+ CTL clones did not use this cytotoxic mechanism to lyse their specific target. TC7 used a granzyme/perforin-dependent pathway, whereas TC5 used a TRAIL-dependent mechanism. Quantitative analysis of cytokine mRNA expression indicated that while the tumor cells displayed a Th2-type profile, the CTL clones expressed Th1-type cytokines. Preincubation of TIL clones with autologous tumor cells in a short-term culture induced their activation and subsequent amplification of the Th1-type response, which indicates a direct contribution of the malignant cells in the Th1/Th2 imbalance. However, we found that tumor cells produced high amounts of TGF-beta, which could explain the inhibition of a specific antitumor immune response. Another mechanism to avoid the host immune response was the expression of CD158a, CD158b, p70, and CD94/NKG2A inhibitory receptors by tumor-specific lymphocytes. Finally, we present recent data on new antigen structures expressed both by long-term CTCL lines and uncultured tumor cells.

Loss of MHC class II inducibility in hyperplastic tissue in Rb-defective mice

Retinoblastoma gene (Rb) defects occur frequently in human tumors. Studies of Rb-defective human tumor cell lines and Rb−/− murine embryonic fibroblasts demonstrate that Rb is required for interferon-gamma (IFN-γ) induced major histocompatibility complex (MHC) class II expression. MHC class II expressing tumors generate anti-tumor immune responses associated with tumor-specific infiltrating lymphocytes. The role of Rb in IFN-γ induced MHC class II expression on an endogenous tumor was examined by immunohistochemical staining for IAβ and Rb on tissues from Rb+/− mice. MHC class II IAβ is not induced by IFN-γ in Rb-deficient neoplastic cells, but remains inducible in related normal tissue.

Cyclophosphamide induces the development of early myeloid cells suppressing tumor cell growth by a nitric oxide-dependent mechanism.

Adoptive immunotherapy with cyclophosphamide (Cy) increases the host resistance against tumor growth. The precise mechanism(s) by which this therapy enhances tumor suppression is unclear. Cy induces the development of early myeloid cells that may be strongly antiproliferative through NO production. These cells are similar to the natural suppressor cells found in normal bone marrow with a potential antitumor effect. Here we have addressed whether the development of NO-producing cells may be involved in this tumor resistance in Cy-treated mice. The results show a synergism between Cy treatment and tumor-specific lymphocytes transferred systemically (i.v.) or locally (Winn's assay) that results in a strong tumor suppression. Inhibition of NO production by N(G)-monomethyl-L-arginine at the site of tumor inoculation results in a loss of the protection achieved by the combined therapy. Cy-treated mice develop splenic early myeloid (CD11b, Gr-1, CD31 (ER-MP12), ER-MP20, ER-MP54) cells producing large amounts of NO upon T cell-derived signals (IFN-gamma plus CD40 ligation) able to inhibit tumor cell growth in vitro. Early myeloid cells (ER-MP54(+)) and cells expressing inducible NO synthase are increased at the site of tumor challenge in mice treated with the combined therapy, but not in those treated with Cy or immune cell transfer alone. Thus, Cy induces the expansion of early myeloid cells, inhibiting tumor cell growth by a mechanism involving NO. Both the recruitment and the activation of these myeloid cells at the site of tumor challenge appear to be dependent on the presence of tumor-specific lymphocytes.

Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis

Mature and activated dendritic cells (CD83-positive DCs) are essential for the recruitment and survival of activated tumor-specific lymphocytes during carcinogenesis. The frequencies of CD83 positive DCs were almost same in peripheral blood from patients with hepatocellular carcinoma (HCC) and cirrhosis of liver (LC). However, the numbers of CD83 positive DCs in liver tissues were significant lower in HCC compared with LC (6.6±10.9 vs. 33.3±24 DCs/specimen, P<0.05). Most importantly, there were no CD83-positive DCs at cancer nodules in HCC. A role of infiltration of activated DCs in liver during hepatocarcinogenesis is shown.

Tumor Infiltration by Adoptively Transferred T Cells Is Independent of Immunologic Specificity but Requires Down-Regulation of L-Selectin Expression

Adoptive immunotherapy with anti-CD3/IL-2 activated tumor-draining lymph node (LN) T cells is capable of eradicating tumor established at various histological sites. Tumor-specific effector lymphocytes have recently been identified to be LN T cells with down-regulated L-selectin (L-selectin-). Using fluorochrome labeling, the present study determined the early trafficking pattern of systemically transferred cells. In mice with 10-day established pulmonary 3-methylcholanthrene (MCA) 205 metastases, accumulation of cells in tumors was evident as early as 2 h after i.v. cell transfer, and, by 24 h, >50-fold higher numbers of cells were seen in metastases than in normal tissues. Similarly, transferred cells selectively infiltrated s.c. tumors, albeit at a lower rate. Analysis of the transferred cells isolated from recipient mice revealed that tumor-infiltrating cells were mostly L-selectin- (>95%). By contrast, only 24% and 58% L-selectin- cells were found in the LN and spleen, respectively. The ability of L-selectin- cells to accumulate at tumor sites was confirmed by the transfer of purified cell populations. Despite this selective tumor infiltration, the trafficking pattern did not reflect antigenetic specificity, and tumor regression occurred only after the transfer of tumor-specific effector cells. These results, thus, suggest that there are two distinct mechanisms operative in successful adoptive immunotherapy. Early infiltration of tumors by transferred cells is dictated by the physiological properties of cells and is independent on their immunologic specificity. Tumor regression, however, requires immunologically specific interactions at the site of tumor.

Adoptive immunotherapy after hematopoietic stem cell transplantation.

Adoptive immunotherapy may be performed with either autologous or allogeneic lymphocytes. Autologous cellular immunotherapy is hindered by the inability to break anergy or self-tolerance. Advances in this area have focused on the use of tumor-specific lymphocytes activated by antigen-pulsed or transgene modified dendritic cells. Allogeneic cellular immunotherapy is effective even with the use of non-specific and unactivated donor lymphocytes. Allogeneic cellular therapy is, however, complicated by graft-versus-host disease (GVHD). Advances in allogeneic immunotherapy will require better control of GVHD.

Isolation and Characterization of PAGE-1 andGAGE-7: NEW GENES EXPRESSED IN THE LNCaP PROSTATE CANCER PROGRESSION MODEL THAT SHARE HOMOLOGY WITH MELANOMA-ASSOCIATED ANTIGENS

The LNCaP progression model of human prostate cancer consists of lineage-related sublines that differ in their androgen sensitivity and metastatic potential. A differential display polymerase chain reaction was employed to evaluate mRNA expression differences between the LNCaP sublines in order to define the differences in gene expression between the androgen-sensitive, nontumorigenic LNCaP cell line and the androgen-insensitive, metastatic LNCaP sublines, C4-2 and C4-2B. An amplicon, BG16.21, was isolated that showed increased expression in the androgen-independent and metastatic LNCaP sublines, C4-2 and C4-2B. Hybridization screening of a lambda gt11 expression library with BG16.21 revealed two transcripts, both homologous to BG16.21 at the 3' end. A GenBankTM data base search using the GCG Wisconsin software package revealed the shorter approximately 600-bp transcript (designated GAGE-7) to be a new member of the GAGE family. The second approximately 700-bp transcript was a novel gene (designated PAGE-1, "prostate associated gene") with only 45% homology to GAGE gene family members. RNA blot analysis demonstrated that GAGE-7 mRNA was expressed at equal levels in all lineage related prostate cancer cell sublines, while PAGE-1 mRNA levels were elevated 5-fold in C4-2 and C4-2B as compared with LNCaP cells. Neither GAGE-7 nor PAGE-1 demonstrated any regulation by androgens in the prostate cancer cell lines used in this study. PAGE-1 and GAGE-7 expression was found to be restricted to testes (high) and placenta (low) on human multiple tissue Northern blots. As GAGE/MAGE antigens were reported previously to be targets for tumor-specific cytotoxic lymphocytes in melanoma, these results suggest that PAGE-1 and GAGE-7 may be related to prostate cancer progression and may serve as potential targets for novel therapies.

Immunoregulatory effects of CD4+ T helper subsets in human melanoma

The 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). Sixty-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. Th0 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. The 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.