While T cell-based immunotherapies are steadily improving, there are still many patients who progress, despite T cell-infiltrated tumors. Emerging evidence suggests that T cells themselves may provoke immune escape of cancer cells. Here, we describe a well-controlled co-culture system for studying the dynamic T cell - cancer cell interplay, using human melanoma as a model. We explain starting material, controls, and culture parameters to establish reproducible and comparable cultures with highly heterogeneous tumor cells. Low passage melanoma cell lines and melanoma-specific CD8+ T cell clones generated from patient blood were cultured together for up to 3 days. Living melanoma cells were isolated from the co-culture system by fluorescence-activated cell sorting. We demonstrate that the characterization of isolated melanoma cells is feasible using flow cytometry for protein expression analysis as well as an Agilent whole human genome microarray and the NanoString technology for differential gene expression analysis. In addition, we identify five genes (ALG12, GUSB, RPLP0, KRBA2, and ADAT2) that are stably expressed in melanoma cells independent of the presence of T cells or the T cell-derived cytokines IFNγ and TNFα. These genes are essential for correct normalization of gene expression data by NanoString. Further to the characterization of melanoma cells after exposure to CTLs, this experimental system might be suitable to answer a series of questions, including how the affinity of CTLs for their target antigen influences the melanoma cell response and whether CTL-induced gene expression changes in melanoma cells are reversible. Taken together, our human T cell - melanoma cell culture system is well suited to characterize immune-related mechanisms in cancer cells.
<p>Supplementary Materials and Methods. Supplementary Figures: Fig. S1. Sorting strategy and purity. Fig. S2. Comparison of the two batches of NanoString experiments shown in Fig. 1 and Fig. 4. Fig. S3. Melanoma cells respond to CTL attack with gene expression changes. Fig. S4. CTL-induced gene expression changes are driven by IFNgamma and TNFalpha. Fig. S5. Representative histograms of protein expression regulated by cytokines. Fig. S6. Characterization of untreated and CTL-exposed melanocytes. Supplementary Tables: Table S1. Source of the 17 melanoma cell lines used in this study. Table S2. Antibodies (including isotype-matched controls) used to assess protein expression by flow cytometry. Table S3. Differential expression of genes that are part of the enriched gene sets in Supplementary Figs. S3C and S3E. Table S4. Selection of genes for 181-gene panel. Table S5. Differential expression of genes in melanoma cell lines after 24h treatment. Table S6. Protein expression of melanoma cells after 48h culture in presence or absence of IFNgamma and TNFalpha. Table S7. Differential expression of genes in melanocyte cell lines after 24h treatment. Table S8. Genes that have statistically different fold-changes (MelanA-specific CTLs/untreated) between melanoma cell lines and melanocyte lines. Supplementary References.</p>
Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.
Inhibitory receptors (iRs) are frequently associated with "T cell exhaustion". However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as "checkpoint blockade", is showing unprecendented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with "T cell exhaustion" and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are "downtuned" in order to limit tissue damage. Furthermore, we review the novel "checkpoint blockade" treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells.
Le melanome est le type de cancer de la peau le plus agressif. En Europe, pres de 22'000 deces des suites de ce cancer ont ete repertories en 2012 (statistiques les plus recentes). La Suisse etait alors le pays presentant la plus forte incidence (26.1 nouveaux diagnostiques par annee pour lOO'OOO habitants). Le melanome est egalement l'un des cancers provoquant les plus fortes reactions immunitaires. Un type de globules blancs, les lymphocytes T cytotoxiques (CTLs), joue un role cle dans la lutte contre le cancer. En effet, les CTLs peuvent reconnaitre, tuer, ou encore modifier le comportement des cellules cancereuses. Leur presence dans les tumeurs humaines a ete associee a un meilleur pronostic. Les CTLs sont donc au coeur des progres spectaculaires des nouveaux traitements contre le cancer par immunotherapie.
Malgre l'importance fondamentale de l'infiltration de CTLs sur le pronostic du melanome, la reaction immediate des cellules du melanome au contact des CTLs est encore peu connue. En effet, beaucoup de cancers progressent en depit de l'infiltration de CTLs. Le systeme immunitaire peut contrecarrer, mais egalement parfois favoriser le developpement d'un cancer. Nous avons emis l'hypothese que les CTLs pourraient provoquer chez certaines cellules des reactions en lien avec la progression du cancer autres que la mort cellulaire.
Afin d'etudier les interactions entre CTLs et cellules de melanome, nous avons developpe un systeme de co-culture de CTLs avec des lignees cellulaires de melanome provenant de patients differents. Les proteines des cellules du melanome ont ete quantifiees par cytometrie en flux. Les variations de l'expression des genes des cellules tumorales ont egalement ete evaluees. Nous avons pu observer que la reponse des cellules tumorales au contact des CTLs est tres rapide, et qu'elle est similaire entre les differentes lignees de melanome. Cette reponse est en grande partie provoquee par les cytokines IFNy and TNFa. Ces cytokines sont secretees par les CTLs lors de la reconnaissance d'antigenes specifiques aux cellules tumorales. Les lignees cellulaires de melanocytes benignes reagissent de manieres similaires aux lignees non-benignes, ce qui suggere que ces reactions ne sont pas specifiques a la tumeur. Cette reponse comprend l'augmentation de la secretion de facteurs solubles, et induit de nombreux mecanismes qui peuvent a la fois bloquer et stimuler le systeme immunitaire. Au contact des CTLs, les cellules du melanome modifient la transcription de nombreux genes, produisant des facteurs qui vont influencer leur environnement immunitaire. L'homogeneite de la reponse contraste avec 1 heterogeneite genetique des lignees de melanome. Ces resultats contribuent a la meilleure compensions des mecanismes immunitaires induits en presence de CTLs, et pourraient contribuer a l'amelioration des traitements par immunotherapie.
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Melanoma is the most aggressive type of skin cancer. In 2012 (latest statistics), 22'000 deaths from melanoma were reported in Europe with the highest incidence in Switzerland (26.1 new diagnosis per year per IOO'OOO inhabitants). Melanoma is also one of the most immunogenic cancer types (meaning it can provoke an immune response). In human tumors, the presence of cytotoxic T lymphocytes (CTLs), a specialized type of white blood cell, has been associated with better clinical outcome. CTLs can recognize and kill cancer cells or modify their behavior, and are being exploited as a central player of immunotherapy with recent spectacular clinical outcome.
Despite the high importance of CTL infiltration for good prognosis, the reactions of cancer cells to CTL attack are poorly characterized. In fact, many cancers progress in spite of tumor-infiltrating CTLs. Conversely, immune system components can counteract but also support cancers, and we speculated that CTLs may provoke responses in targeted cancer cells other than cell death that may be relevant to cancer progression.
To investigate the interplay between CTLs and melanoma cells, we have established a co-culture system using CTLs and human melanoma cell Unes derived from different patients. We determined protein expression using multi-parameter flow cytometry, and gene expression using multiplexed gene expression analysis. We found rapid and predominantly homogeneous changes in our melanoma cells in response to CTL attack. These responses were mostly mediated by the cytokines IFNy and TNFa secreted by the CTLs upon antigen-specific recognition of the melanoma cells. Interestingly, benign melanocyte lines reacted to CTL attack in a similar way as melanoma cell lines, indicating that the reactions were conserved upon acquisition of malignancy. The melanoma response included increased secretion of soluble factors that can attract different immune cells to the tumor microenvironment and drive multiple immune activatory and inhibitory functions. In response to CTLs, melanoma cells modify their transcriptional program to produce immune-related factors and shape their immune microenvironment. The surprising homogeneity of the observed melanoma response contrasts to the genetic heterogeneity of melanoma cells. These results contribute to a better understanding of immune-related mechanisms in cancer cells challenged by CTLs, and may therefore help improving cancer immunotherapy.
Abstract T cell-based immunotherapies have brought great progress for cancer patients, but many questions still remain open with regard to mechanisms of action and immune regulation in the tumor microenvironment. Methods: We setup a well-controlled co-culture system to study the dynamic T cell - cancer cell interplay. Low passage MelanA-expressing melanoma cell lines were cultured with MelanA-specific CD8+ T cells and characterized using differential gene expression analysis and flow cytometry. Results: As expected, significant fractions of melanoma cells died in presence of melanoma-specific CD8+ T cells. However, still some melanoma cells persisted during up to three days of co-culture. Characterization of the surviving melanoma cells revealed increased mRNA levels of genes associated with antigen processing and presentation: HLA Class I-encoding genes (HLA-A and HLA-B), HLA Class 2-encoding genes (HLA-DRA and HLA-DRB) and CD74, the invariant chain that stabilizes the complex of HLA Class II α and β chains until it is loaded with a peptide, were strongly increased after co-culture, as well as TAP1 and TAP2 (encoding transporter associated with antigen presentation), responsible for peptide transfer from the cytosol to the endoplasmic reticulum (Neefjes et al., 2011). HLA-Class I and HLA-DR expression was also strongly increased at the protein level as assessed at 48h of co-culture. These gene and protein expression changes were dependent on antigen-specific interaction of CTLs with melanoma cells and were mediated by IFNγ and TNFα, two cytokines secreted by CTLs upon antigen recognition. Discussion: So far, mainly immunosuppressive mechanisms of cancer cells upon exposure to CTLs were described, such as increased expression of IDO1 and the inhibitory receptor ligand PDL1 (Spranger et al., 2013). Here we show that further to these immunosuppressive mechanisms, melanoma cells respond to melanoma-specific CTLs with an IFNγ-driven upregulation of genes involved in antigen presentation. Increased antigen-presentation likely increases target cell recognition of CTLs. These findings may play a role in successful immunotherapy, and might explain why IFNγ blockade in situations of anti-tumor immune attack not only blocks the immunosuppressive response of melanoma cells but also the anti-tumor T cell response (McGray et al., 2013). References: McGray, A.J.R., Hallett, R., Bernard, D., Swift, S.L., Zhu, Z., Teoderascu, F., VanSeggelen, H., Hassell, J.A., Hurwitz, A.A., Wan, Y., et al. (2013). Immunotherapy-induced CD8+ T Cells Instigate Immune Suppression in the Tumor. Mol Ther 22, 206-218. Neefjes, J., Jongsma, M.L.M., Paul, P., and Bakke, O. (2011). Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11, 823-836. Spranger, S., Spaapen, R.M., Zha, Y., Williams, J., Meng, Y., Ha, T.T., and Gajewski, T.F. (2013). Up-Regulation of PD-L1, IDO, and Tregs in the Melanoma Tumor Microenvironment Is Driven by CD8+ T Cells. Sci Transl Med 5, 200ra116-200ra116. Citation Format: Natalie J. Neubert, Laure Tillé, David Barras, Charlotte Soneson, Petra Baumgaertner, Donata Rimoldi, Mauro Delorenzi, Silvia A. Fuertes Marraco, Daniel E. Speiser. Melanoma cells respond to CD8+ T cell attack by upregulation of genes associated with antigen processing and presentation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B34.
The immune system defends us and combats many microbes, like the viruses that cause the common cold or bacteria that enter wounds. Our defenses can also learn to protect us from more difficult situations—and they can do so with the lessons learned from vaccines. For example, the poliovirus vaccine teaches the immune system to recognize and eliminate the poliovirus, in case it ever enters the body. In recent years, scientists have found that the immune system can also be taught to attack another type of disease: cancer. Cancer is a big mistake in our body. It happens when cells lose control and start to grow without limits. The good news is that we can educate the immune system in cancer patients—this is called immunotherapy. In this article, we will explain how our defenses can learn to attack and eliminate cancer.
