Anti-melanoma Cytotoxic T Lymphocytes Research Articles

Direct Toll-Like Receptor 8 signaling increases the functional avidity of human CD8+ T lymphocytes generated for adoptive T cell therapy strategies.

Adoptive transfer of in vitro activated and expanded antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic strategy for infectious diseases and cancers. Obtaining in vitro a sufficient amount of highly specific cytotoxic cells and capable of retaining cytotoxic activity in vivo remains problematic. We studied the role of Toll-Like Receptor-8 (TLR8) engagement on peripheral CTLs activated with melanoma antigen MART-1-expressing artificial antigen-presenting cells (AAPCs). After a 3-week co-culture, 3–27% of specific CTLs were consistently obtained. CTLs expressed TLR8 in the intracellular compartment and at the cell surface. Specific CTLs activated with a TLR8 agonist (CL075) 24 h before the end of the culture displayed neither any change in their production levels of molecules involved in cytotoxicity (IFN-γ, Granzyme B, and TNF-α) nor major significant change in their cell surface phenotype. However, these TLR8-stimulated lymphocytes displayed increased cytotoxic activity against specific peptide-pulsed target cells related to an increase in specific anti-melanoma CTL functional avidity. TLR8 engagement on CTLs could, therefore, be useful in different immunotherapy strategies.

Antimelanoma CTL recognizes peptides derived from an ORF transcribed from the antisense strand of the 3′ untranslated region of TRIT1

Noncoding regions of the genome play an important role in tumorigenesis of cancer. Using expression cloning, we have identified a cytotoxic T lymphocyte (CTL)–defined antigen that recognizes a protein sequence derived from an open reading frame transcribed from the reverse strand in the 3′ untranslated region of tRNA isopentenyltransferase 1 (TRIT1). A peptide derived from this open reading frame (ORF) sequence and predicted to bind to HLA-B57, sensitized HLA-B57+ tumor cells to lysis by CTL793. The peptide also induced a CTL response in peripheral blood mononuclear cells (PBMC) of patient 793 and in two other melanoma patients. The CTL lysed peptide-pulsed HLA-B57+ target cells and melanoma cells with endogenous antigen expression. The recognition of this antigen is not limited to HLA-B57-restricted CTLs. An HLA-A2 peptide derived from the ORF was able to induce CTLs in PBMC of 2 HLA-A2+ patients. This study describes for the first time a CTL-defined melanoma antigen that is derived from an ORF on the reverse strand of the putative tumor suppressor gene TRIT1. This antigen has potential use as a vaccine or its ability to induce CTLs in vitro could be used as a predictive biomarker.

Activin receptor‐like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.

Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes.

The adenylate cyclase (CyaA) of Bordetella pertussis is able to deliver CD8(+) T cell epitopes into the cytosol of CD11b(+) dendritic cells (DC) following its specific interaction with the alpha(M)beta(2) integrin (CD11b/CD18). This delivery results in intracellular processing and presentation by MHC class I molecules of the CD8(+) T cell epitopes inserted into CyaA. Indeed, we previously showed that CyaA toxins carrying a single cytotoxic T lymphocyte (CTL) epitope can induce efficient protective and therapeutic antitumor immunity in mice. With a view to elaborating cancer immunotherapy in humans using CyaA, we constructed two recombinant CyaA carrying HLA*0201-restricted melanoma epitopes. Here we show that these recombinant CyaA induce strong anti-melanoma CTL responses in HLA*0201 transgenic mice, even after a single i.v. immunization without adjuvant. These responses are long lasting, since they were also detected 5 months after the last injection. Finally, human DC treated with the recombinant CyaA were shown to process and present efficiently the melanoma epitopes to human CTL clones. Altogether, our results demonstrate that tumoral epitopes inserted into CyaA are efficiently processed and presented in association with human MHC molecules. These observations suggest that CyaA is capable of activating antitumoral CTL in humans and highlight the potential of CyaA for use in cancer immunotherapy.

IL-12 stimulation but not B7 expression increases melanoma killing by patient cytotoxic T lymphocytes (CTL).

Recent studies have demonstrated that rodent tumour cells engineered to secrete a variety of cytokines, or to express foreign antigens, MHC molecules or co-stimulatory molecules, are rejected by syngeneic animals. These observations have led to the initiation of a number of clinical trials using genetically modified tumour cells, to attempt to stimulate a patient anti-tumour immune response. In this study, a protocol has been developed to test in vitro the specific cytotoxic anti-tumour response generated from melanoma patient lymphocytes. The results showed that IL-12 in combination with IL-2 enhanced the autologous anti-melanoma CTL response, whereas B7.1 antigen expression on tumour cells did not increase anti-melanoma CTL generation. This method could be used to design more appropriate genetically modified tumour vaccines.

Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells.

In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.

Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy.

A central triple-stranded DNA structure created during HIV-1 reverse transcription, the central flap, acts as a cis-active nuclear import determinant of the HIV-1 DNA genome. Insertion of the sequences responsible for formation of the central DNA flap into an HIV-1-derived vector strongly enhances its transduction efficiency. HIV-1 vectors with or without inclusion of the DNA flap and encoding the same melanoma polyepitope were constructed to transduce dendritic cells (DCs) and to evaluate their capacity for induction of melanoma-specific cytotoxic T-lymphocyte (CTL) responses ex vivo and in vivo. HIV-1 vectors including the DNA flap transduced up to 100% of immature mouse and human DCs. Inoculation of HLA-A2.1 transgenic mice with this flap vector elicited vigorous and multi-specific long-term anti-melanoma CTL responses, whereas the parental vector lacking the flap sequence was less efficient. Furthermore, human DCs transduced ex vivo with the recombinant DNA flap vector displayed efficient multi-specific primary human CTL responses against melanoma. Lentiviral vectors including the DNA flap should be powerful tools both for active immunization and for the ex vivo priming of CTL for tumor immunotherapy.

Interferon-alpha (IFN-alpha) stimulates anti-melanoma cytotoxic T lymphocyte (CTL) generation in mixed lymphocyte tumour cultures (MLTC).

IFN-alpha administration after primary tumour resection improves the survival of melanoma patients at high risk of relapse. To investigate whether this response might be due to stimulation of anti-tumour immunity, the effect of IFN-alpha on anti-melanoma CTL generation in MLTC was measured. IFN-alpha increased both allogeneic and autologous anti-melanoma CTL generation from peripheral blood lymphocytes stimulated with irradiated primary melanoma cultures. IFN-alpha up-regulated MHC class I expression on primary melanoma cultures, whereas IFN-gamma up-regulated both MHC class I and II expression. However, the effect of IFN-alpha on anti-melanoma CTL generation was often more potent than that of IFN-gamma, equalling the effect of the optimal combination of IL-2 and IL-12. Pre-treatment of primary melanoma cultures with IFN-gamma was sufficient for CTL generation in MLTC, whereas IFN-alpha needed to be present during the MLTC. While direct anti-proliferative effects of IFN-alpha on some tumour cells have been described, IFN-alpha did not inhibit proliferation of primary melanoma cultures. These results suggest that the clinical effects of IFN-alpha in melanoma patients may be immune-mediated.

A Structure-based Approach to Designing Non-natural Peptides That Can Activate Anti-melanoma Cytotoxic T Cells

Tumor antigens presented by major histocompatibility complex (MHC) class I molecules and recognized by CD8(+) cytotoxic T lymphocytes (CTLs) may generate an efficient antitumor immune response after appropriate immunization. Antigenic peptides can be used in vivo to induce antitumor or antiviral immunity. The efficiency of naked peptides may be greatly limited by their degradation in the biological fluids. We present a rational, structure-based approach to design structurally modified, peptidase-resistant and biologically active analogues of human tumor antigen MAGE-1.A1. This approach is based on our understanding of the peptide interaction with the MHC and the T cell receptor and its precise degradation pathway. Knowledge of these mechanisms led to the design of a non-natural, minimally modified analogue of MAGE-1.A1, [Aib2, NMe-Ser8]MAGE-1.A1, which was highly peptidase-resistant and bound to MHC and activated MAGE-1.A1-specific anti-melanoma CTLs. Thus, we showed that it is possible to structurally modify peptide epitopes to obtain analogues that are still specifically recognized by CTLs. Such analogues may represent interesting leads for antitumor synthetic vaccines.

Identification of gp100-derived, melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 supertype molecules by primary in vitro immunization with peptide-pulsed dendritic cells.

The human melanocyte lineage-specific antigen gp100 contains several epitopes recognized by cytotoxic T lymphocytes (CTL). However, most of the epitopes reported to date are HLA-A2.1-restricted. Despite the high frequency of HLA-A2.1 in melanoma patients, effective population coverage requires the identification of epitopes restricted by other frequent HLA alleles. Herein, HLA-A3 binding, gp100-derived synthetic peptides were tested for their capacity to elicit anti-melanoma CTL in vitro using CD8+ T cells from healthy donors as responders and peptide-pulsed autologous dendritic cells as antigen-presenting cells. Of 7 peptides tested, 2 (gp100[9(87)] and gp100[10(86)]) induced CTLs that killed melanoma cell lines expressing HLA-A3 and gp100. Additional MHC-binding studies to various HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301 and -A*6801) were performed to determine whether these CTL epitopes could further increase potential population coverage. Further experiments indicated that the peptide gp100[9(87)], which bound to HLA-A11 with high affinity, was capable of inducing specific CTLs that killed melanoma cells expressing gp100 and HLA-A11 molecules. Our results indicate that the gp100[9(87)] peptide corresponds to a CTL epitope which may be restricted by either the HLA-A3 or HLA-A11 allele, emphasizing its utility for the design and development of epitope-based therapies for melanoma.

Human Melanoma Cells Transfected with the B7-2 Co-Stimulatory Molecule Induce Tumor-Specific CD8+Cytotoxic T LymphocytesIn Vitro

Neoplastic cells express tumor-associated antigens, but tumor rejection seldom occurs in vivo. The absence of an effective immune response may be explained by the inability of tumor cells to deliver co-stimulatory signals. Indeed, transfection of either B7-1 or B7-2 co-stimulatory molecules into mouse tumor cells enhances antitumor immune responses. In this study, we stably transfected human melanoma cells with the cDNA encoding the B7-2 molecule to evaluate in vitro: (i) the induction of anti-melanoma cytotoxic T lymphocytes (CTL) by stimulation of CD8+ T cells, purified from healthy donors and a melanoma patient, with B7-2 transfected allogeneic HLA-matched melanoma cells; (ii) the tumor specificity and the HLA restriction of the induced CTL; and (iii) the feasibility to propagate long-term antimelanoma CTL lines. We found that B7-2 transfected, but not untransfected or mock-transfected, melanoma cells activated MHC-class I-restricted, melanoma-specific CD8+ CTL from healthy donors. More importantly, CD8+ tumor-associated lymphocytes, purified from a tumor-invaded lymph node of a melanoma patient and stimulated with B7-2-transfected melanoma cells, acquired a strong reactivity toward the autologous tumor. CTL lines with specific cytolytic activity could be propagated in long-term culture. These results indicate that: (i) the expression of the B7-2 molecule into human melanoma cells makes them immunogenic and able to act as antigen-presenting cells and (ii) purified CD8+ cells, stimulated with B7-2+ allogeneic HLA-matched melanoma cells, preferentially recognize melanoma-specific rather than allogeneic antigens. This study may have clinical implications for passive and/or active immunotherapy in melanoma patients.

Immunodominance Across HLA Polymorphism: Implications for Cancer Immunotherapy

Recent advances in the understanding of the mechanisms leading to tumor recognition by the immune system have shown that, at least in the case of human melanoma, the majority of cytotoxic T lymphocytes (CTL) identified in association with in vivo tumor regression after interleukin-2 therapy recognize nonmutated molecules expressed by most melanoma cells. For this reason, peptide-based or whole protein vaccination protocols against melanoma-associated antigens (MAA) are ongoing in several institutions, with the goal of inducing tumor regression by enhancing in vivo specific antitumor CTL reactivity. The rationale for the use of such vaccines is supported by: (a) preclinical evidence that vaccination with major histocompatibility complex class I restricted epitopes can enhance effectively cellular immunity, (b) evidence that potent antimelanoma CTL reactivity can be generated by repetitive in vitro stimulation of peripheral blood monocytes with MAA, and (c) evidence that the systemic administration of the same MAA can elicit antitumor CTL reactivity in vivo. As strategies are being developed for the development of sound vaccines, two basic approaches are investigated: one vaccination strategy is based on the administration of the specific amino acid sequence recognized by the CTL in association with a particular human leukocyte antigen (HLA) restriction element, and the other is based on the administration of the whole antigenic molecule, which relies on the organism's antigen-processing capabilities to render suitable the antigen for induction of HLA class I restricted CTL reactivity in vivo. Among the various factors complicating T-cell-based vaccination approaches stands the polymorphism of the HLA molecules. HLA are the most polymorphic of human genes, and because such polymorphism is clustered in the functional peptide-binding region, the binding of antigenic peptides is necessarily restricted to specific HLA alleles. This limits the interactions between CTL and antigen to specific sequences for each HLA allele. For this reason, the ability of an individual antigen to function as a T-cell immunogen in the context of different HLA allele restriction elements is an open question. It seems logical that whole-molecule vaccines have the potential advantage of broader use across patient populations. In particular, large antigenic molecules may contain multiple peptide sequences with putative binding properties for different HLA alleles, which in turn may elicit T-cell reactivity across the polymorphism of HLA. Such a concept, however, relies on the assumption that the same antigen may function with similar efficiency as an immunogen in association with different HLA alleles, independently from the epitopic sequence recognized in the various situations. This concept has been challenged recently by several practical observations and remains, in our opinion, an open question. This review will address the practical question of immunogenicity of molecules across the HLA polymorphism. We postulate that the complexity and success of the development of peptide-based vaccination strategies depend on the severity of this restriction, which is currently only incompletely studied and understood. Although no solutions are offered to the problem, emphasis is placed on the importance of this question, hopefully to stimulate the interest of other researchers, particularly in clinical settings, toward the investigation of this type of problem.

Anti-melanoma cytotoxic T lymphocytes (CTL) recognize numerous antigenic peptides having 'self' sequences: autoimmune nature of the anti-melanoma CTL response.

A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.

PMel17 is recognised by monoclonal antibodies NKI-beteb, HMB-45 and HMB-50 and by anti-melanoma CTL.

Recently, we cloned the cDNA encoding the melanocyte lineage-specific antigen gp100 and demonstrated that gp100 is recognised by three different monoclonal antibodies (MAbs) used to diagnose malignant melanoma. In addition, we showed that tumour-infiltrating lymphocytes (TIL 1200) from a melanoma patient reacted specifically with cells transfected with the gp100 cDNA. Molecular characterisation of the gp100 cDNA revealed that the gp100 antigen is highly homologous, but not identical, to another melanocyte-specific protein, pMel17. Here, we report that cells transfected with pMel17 cDNA also react with all three MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-45 and HMB-50. Moreover, pMel17 transfectants are specifically lysed by TIL1200. These data demonstrate that antigenic processing of both gp100 and pMel17 give rise to peptides seen by anti-melanoma cytotoxic T lymphocytes (CTL) and are therefore potential targets for immunotherapy of malignant melanoma.

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens.

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristics of B16 melanomas (H-2b) persisting in C57BL/6 mice (H-2b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the expression of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-antigen-positive immunogens was indicated by the effect of interferon gamma (IFN gamma) or N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN gamma or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.