Annette Paschen

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Vemurafenib reverses immunosuppression by myeloid derived suppressor cells

Myeloid derived suppressor cells (MDSCs) suppress innate and adaptive immunity, thereby limiting anti‐tumor immune responses in cancer patients. In patients with advanced melanoma, the phenotype and function of MDSCs remains controversial. In our study, we further explored two distinct subpopulations of MDSCs and investigated the impact of Vemurafenib on these cells. Flow cytometry analysis revealed that in comparison to healthy donors and patients with localized disease, PBMCs from patients with metastatic melanoma showed an increased frequency of CD14+HLA‐DR−/low monocytic MDSCs (moMDSCs) and of a previously unrecognized population of CD14−CD66b+Arginase1+ granulocytic MDSCs (grMDSCs). In vitro, both populations suppressed autologous T‐cell proliferation, which was tested in CFSE‐based proliferation assays. Vemurafenib treatment of melanoma patients reduced the frequency of both moMDSCs and grMDSCs. According to our in vivo finding, conditioned medium (CM) from Vemurafenib treated melanoma cells was less active in inducing moMDSCs in vitro than CM from untreated melanoma cells. In conclusion, patients with advanced melanoma show increased levels of moMDSCs, and of a population of CD14−CD66b+Arginase1+ grMDSCs. Both MDSCs are distinct populations capable of suppressing autologous T‐cell responses independently of each other. In vitro as well as in vivo, Vemurafenib inhibits the generation of human moMDSCs. Thus, Vemurafenib decreases immunosuppression in patients with advanced melanoma, indicating its potential as part of future immunotherapies.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Interferon-γ down-regulates NKG2D ligand expression and impairs the NKG2D-mediated cytolysis of MHC class I-deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of αβ CD8+ T cells. Ligands of NKG2D, the MHC class I chain‐related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon‐γ (IFN‐γ) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I‐negative and ‐positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN‐γ treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down‐regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT‐PCR revealed a decrease of MICA‐specific mRNA levels upon IFN‐γ treatment and siRNA experiments pointed to an involvement of STAT‐1 in this process. Importantly, IFN‐γ‐treated MHC class I‐negative melanoma cells were less susceptible to NKG2D‐mediated NK cell cytotoxicity. Our study suggests that IFN‐γ, by down‐regulating ligand expression, might facilitate escape of MHC class I‐negative melanoma cells from NKG2D‐mediated killing by NK cells.

Implication of the β2-microglobulin gene in the generation of tumor escape phenotypes

Classical MHC molecules present processed peptides from endogenous protein antigens on the cell surface, which allows CD8+ cytotoxic T lymphocytes (CTLs) to recognize and respond to the abnormal antigen repertoire of hazardous cells, including tumor cells. The light chain, β2-microglobulin (β2m), is an essential constant component of all trimeric MHC class I molecules. There is convincing evidence that β2m deficiency generates immune escape phenotypes in different tumor entities, with an exceptionally high frequency in colorectal carcinoma (CRC) and melanoma. Damage of a single β2m gene by LOH on chromosome 15 may be sufficient to generate a tumor cell precommitted to escape. In addition, this genetic lesion is followed in some tumors by a mutation of the second gene (point mutation or insertion/deletion), which produces a tumor cell unable to express any HLA class I molecule. The pattern of mutations found in microsatellite unstable colorectal carcinoma (MSI-H CRC) and melanoma showed a striking similarity, namely the predominance of frameshift mutations in repetitive CT elements. This review emphasizes common but also distinct molecular mechanisms of β2m loss in both tumor types. It also summarizes recent studies that point to an acquired β2m deficiency in response to cancer immunotherapy, a barrier to successful vaccination or adoptive cellular therapy.

Identification of a new HLA-A(*)0201-restricted T-cell epitope from the tyrosinase-related protein 2 (TRP2) melanoma antigen.

For the development of peptide‐based immunotherapies, the identification of additional tumor antigens and T‐cell epitopes is required. Because HLA‐A*0201 is the most common allele in Caucasians, who represent the majority of patients with melanomas, 6 peptides carrying an HLA‐A*0201 motif were synthesized from tyrosinase‐related protein‐2 (TRP2) melanoma antigen and tested for binding affinity to the HLA allele using processing‐defective T2 cells. These peptides were then pulsed onto autologous dendritic cells and used to stimulate in vitro CD8+‐enriched T cells isolated from peripheral blood of HLA‐A*02+ healthy donors or melanoma patients for the induction of specific cytotoxic T lymphocytes (CTLs). One peptide, TRP2288–296 (SLDDYNHLV), the best HLA‐A*0201 binder, elicited specific CTLs from 1 of 4 patients and 3 of 4 healthy donors. The induced CTLs from the patient and from 1 donor efficiently recognized HLA‐A*02+ TRP2+ melanomas as well as COS‐7 cells expressing HLA‐A*0201 and TRP2 in an HLA class I–restricted manner, as assessed by cytokine production and direct cytolysis. The remaining 2 CTL lines derived from 2 donors displayed low T‐cell receptor avidity, which could lyse melanoma cells in the presence of exogenous peptide. Since TRP2 is an antigen expressed in most melanomas, identification of the TRP2/HLA‐A*0201 peptide SLDDYNHLV may facilitate the design of present peptide‐based immunotherapies for the treatment of a large fraction of melanoma patients. Int. J. Cancer 87:399–404, 2000.

Human dendritic cells infected by Listeria monocytogenes: induction of maturation, requirements for phagolysosomal escape and antigen presentation capacity.

An important feature of microbial infections is the ability of the microorganisms to interfere with and modulate the induction of host immune reactions. However, little is known about the effects of broad host range pathogens such as Listeria monocytogenes on similar cell types in different hosts. Here we examine the effects of the human and animal pathogen L. monocytogenes on human dendritic cells (DC) since this type of cells is essential for the initiation of immune responses. Listeria are phagocytosed efficiently by immature human DC and the bacteria escape from the phagolysosome quickly. Lack of the pore‐forming activity of listeriolysin, which was found to be essential for the vacuolar escape of this bacterium in other cell types, retarded but did not prevent egress from the vacuole. Treatment of cultures of immature DC with L. monocytogenes resulted in rapid changes in morphology and cellular constitution followed by maturation of the DC. This could be judged by the appearance of maturation‐specific cell surface markers. Antigen presentation to CD4 T cells was apparently not impaired by the infection. These results are in clear contrast to results obtained previously in the mouse system (Guzman et al., Mol. Microbiol. 1996. 20: 119 – 126; Darji et al., Eur. J. Immunol. 1997. 27: 1696 – 1703.).

Cell-based vaccination against melanoma – background, preliminary results, and perspective

Melanoma is the prototype of a tumor to which many forms of immunotherapy have been applied extensively over the past two decades. Melanoma vaccines (active specific immunotherapy) are designed to modulate the immune system and have subsequent antitumor effects with minimal toxicity. Previous attempts to produce melanoma vaccines include immunization with whole tumor cells/cell lysates admixed with nonspecific adjuvants. While these vaccines generate enhanced antitumor immunity in a subset of patients, some of whom survive for longer than historical controls, no clinical benefit has so far been demonstrated in a properly controlled phase III study. Genetic modifications of tumor cells to make them express cytokines afford new-generation melanoma vaccines, and generate long-lasting systemic antitumor immunity in animal models. Translation of these preclinical results primarily into melanoma patients with advanced diseases shows the potential to induce systemic antitumor immune responses and in some instances tumor regression with acceptably low toxicity. The efficacy of this novel vaccine approach would be expected to be higher when used in a postsurgical adjuvant setting when the tumor load is small. Other novel vaccine approaches such as dendritic cell-based therapy also hold promise for the treatment of melanoma. The clinical value of all these new approaches will eventually have to be established in prospectively randomized clinical studies.

Genetic Evolution of T-cell Resistance in the Course of Melanoma Progression

Purpose: CD8+ T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell–stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor–T-cell interaction. Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell–stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I–negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of β2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second β2m-deficient melanoma model. Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell–based immunotherapy. Clin Cancer Res; 20(24); 6593–604. ©2014 AACR.

Identification of tumor antigens and T-cell epitopes, and its clinical application

The capability of antigen-specific CD8+ and CD4+ T lymphocytes to mediate antitumor immunity has generated remarkable interest in the identification of target antigens and their epitopes. The classical strategy to define tumor antigens is based on the employment of in vivo sensitized tumor-reactive T lymphocytes from cancer patients. These lymphocytes are used to screen an autologous tumor cDNA expression library for the target antigen. Alternatively, antibodies from the serum of cancer patients can be applied to screen a tumor-derived phage expression library for immunogenic cellular structures. In addition, potential target antigens have been selected by gene expression profiling searching for overexpressed gene products in neoplastic cells compared with normal tissues. B-cell target structures and overexpressed gene products have to be verified as T-cell antigens by the strategy of “reverse immunology.” Therefore, T cells are sensitized in vitro by autologous dendritic cells loaded with predicted antigenic peptide ligands for a given HLA allele or with the global antigen. These different approaches led to the identification of a still growing number of antigenic peptides providing the basis for the development of new active and passive immunotherapies and for the monitoring of spontaneous and vaccine-induced T-cell responses. Some of these antigens and/or their epitopes are now validated in different clinical regimens for their capability to mediate potent T-cell immunity in cancer patients.