Nektaria Dimopoulos

Regulatory T-Cell–Mediated Attenuation of T-Cell Responses to the NY-ESO-1 ISCOMATRIX Vaccine in Patients with Advanced Malignant Melanoma

Purpose: NY-ESO-1 is a highly immunogenic antigen expressed in a variety of malignancies, making it an excellent target for cancer vaccination. We recently developed a vaccine consisting of full-length recombinant NY-ESO-1 protein formulated with ISCOMATRIX adjuvant, which generated strong humoral and T-cell–mediated immune responses and seemed to reduce the risk of disease relapse in patients with fully resected melanoma. This study examines the clinical and immunologic efficacy of the same vaccine in patients with advanced metastatic melanoma. Experimental Design: Delayed-type hypersensitivity responses, circulating NY-ESO-1–specific CD4+ and CD8+ T cells, and proportions of regulatory T cells (Treg) were assessed in patients. Results: In contrast to patients with minimal residual disease, advanced melanoma patients showed no clinical responses to vaccination. Although strong antibody responses were mounted, the generation of delayed-type hypersensitivity responses was significantly impaired. The proportion of patients with circulating NY-ESO-1–specific CD4+ T cells was also reduced, and although many patients had CD8+ T cells specific to a broad range of NY-ESO-1 epitopes, the majority of these responses were preexisting. Tregs were enumerated in the blood by flow cytometric detection of cells with a CD4+CD25+FoxP3+ and CD4+CD25+CD127− phenotype. Patients with advanced melanoma had a significantly higher proportion of circulating Treg compared with those with minimal residual disease. Conclusions: Our results point to a tumor-induced systemic immune suppression, showing a clear association between the stage of melanoma progression, the number of Treg in the blood, and the clinical and immunologic efficacy of the NY-ESO-1 ISCOMATRIX cancer vaccine.

Reversal in the Immunodominance Hierarchy in Secondary CD8+ T Cell Responses to Influenza A Virus: Roles for Cross-Presentation and Lysis-Independent Immunodomination

Immunodominance is a central feature of CD8+ T cell (TCD8+) responses to pathogens, transplants, and tumors. Determinants occupy a stable position in an immunodominance hierarchy (α-, β-, etc.) defined by the frequencies of responding TCD8+. In this paper, we study the mechanistic basis for place-swapping between α- (acid polymerase (PA)224–233) and β-determinants (nuclear protein 366–374) in primary vs secondary anti-influenza A virus (IAV) responses in mice. This phenomena was recently correlated with the inability of IAV-infected nondendritic cells (DCs) to generate PA224–233, and it was proposed that secondary TCD8+ are principally activated by IAV-infected epithelial cells, while primary TCD8+ are activated by IAV-infected DCs. In this study, we show that the inability of non-DCs to generate PA224–232 is relative rather than absolute, and that the preferential use of cross-priming in secondary anti-IAV responses can also account for the revised hierarchy. We further show that immunodomination of PA224–233-specific TCD8+ by nucleoprotein 366–374-specific TCD8+ plays a critical role in the phenomena, and that this is unlikely to be mediated by TCD8+ lysis of APCs or other cells.

Melan-A–specific Cytotoxic T Cells Are Associated with Tumor Regression and Autoimmunity Following Treatment with Anti-CTLA-4

Purpose: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. Experimental Design: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. Results: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A–specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A–specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A–expressing tumor cells. Conclusions: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

MEK Inhibition, Alone or in Combination with BRAF Inhibition, Affects Multiple Functions of Isolated Normal Human Lymphocytes and Dendritic Cells

Vella and colleagues show that inhibition of BRAF (dabrafenib) had no effect on healthy donor T cells and monocyte-derived dendritic cells (MoDC), but that MEK inhibition (trametinib) suppressed T-cell proliferation, cytokine production, antigen-specific expansion, and MoDC cross-presentation. Combination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF-mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently, BRAF inhibition has been proposed as proimmunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and modulate dendritic cell function. In this study, we investigate the effects on isolated T lymphocytes and monocyte-derived dendritic cells (moDC) of a MEK (trametinib) and BRAF (dabrafenib) inhibitor combination currently being evaluated in a randomized controlled clinical trial. The effects of dabrafenib and trametinib, alone and in combination, were studied on isolated normal T lymphocytes and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production, and antigen-specific expansion, were assessed. MoDC phenotype in response to lipopolysaccharide stimulation was evaluated by flow cytometry, as were effects on antigen cross-presentation. Dabrafenib did not have an impact on T lymphocytes or moDCs, whereas trametinib alone or in combination with dabrafenib suppressed T-lymphocyte proliferation, cytokine production, and antigen-specific expansion. However, no significant decrease in CD4+ or CD8+ T-lymphocyte viability was observed following kinase inhibition. MoDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. The results of this study demonstrate that MEK inhibition, alone or in combination with BRAF inhibition, can modulate immune cell function, and further studies in vivo will be required to evaluate the potential clinical impact of these findings. Cancer Immunol Res; 2(4); 351–60. ©2014 AACR.

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10–specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.

A Long, Naturally Presented Immunodominant Epitope from NY-ESO-1 Tumor Antigen: Implications for Cancer Vaccine Design

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.

Striking Immunodominance Hierarchy of Naturally Occurring CD8+ and CD4+ T Cell Responses to Tumor Antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient’s immune system, especially the “cancer-testis” tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Combining MHC tetramer and intracellular cytokine staining for CD8+ T cells to reveal antigenic epitopes naturally presented on tumor cells

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.