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An understanding of the interactions between immune cells and trophoblast cells, as well as choriocarcinoma cells, are of extreme importance in reproductive immunology and cancer immunology. In this study, we found that the human HLA-G-positive choriocarcinoma cell line JEG-3 upregulates CD4 + CD25 hi CD127 lo T cells, increases the expression of HLA-G + CD4 + and CD8 + T cells, and decreases the expression of ILT2 + on CD4 + T cells in resting PBMCs after six days of co-culture. Expression of HLA-G on JEG-3 cells did not affect regulatory T cell phenotypes, but promoted modulation of pro-inflammatory cytokines IFN-γ, TNF-α and IL-17A. When JEG-3 cells were stimulated with rhIFN-γ prior to co-culture, CD4 + HLA-G + T cells were significantly increased, and IFN-γ and TNF-α elevated. Taken together, the results indicate that JEG-3 cells upregulate regulatory T cell phenotypes and modulate the level of pro-inflammatory cytokines, which might be important mechanisms in the tumor microenvironment and at the feto-maternal interface during pregnancy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background Aims: Maturation of dendritic cells (DCs) induces their homing from peripheral to lymphatic tissues guided by CCL21. However, in vitro matured human monocyte-derived DC cancer vaccines injected intradermally migrate poorly to lymph nodes (LNs). In vitro maturation protocols generate DCs with high (type 1 DCs) or low (prostaglandin E2 [PGE2]-DCs) autocrine CCL19 levels, which may potentially interfere with LN homing of DCs., Methods: Employing a three-dimensional (3D) chemotaxis assay, chemokine competition/desensitization studies and short interfering RNA (siRNA) against CCL19, we analyzed the effect of autocrine CCL19 on in vitro migration of human DCs toward CCL21., Results: Using human monocyte-derived DCs in a 3D chemotaxis assay, we are the first to demonstrate that CCL19 more potently induces directed migration of human DCs compared with CCL21. When comparing migration of type 1 DCs and PGE2-DCs, migration of type 1 DCs was strikingly impaired compared with PGE2-DCs, but only toward low concentrations of CCL21. When type 1 DCs were cultured overnight in fresh culture medium (reducing autocrine CCL19 levels), a rescuing effect was observed on migration toward low concentrations of CCL21 in a 3D chemotaxis assay. Finally pre-incubation with CCL19 negatively affected PGE2-DC migration, whereas silencing of CCL19 by siRNA improved type 1 DC migration. Importantly, in both cases, the effect was observed only at low concentrations of CCL21., Conclusions: Our results demonstrate that autocrine CCL19 negatively affects DC migratory potential toward CCL21, the potency difference between CCL19 and CCL21 being the underlying cause. CCL19 secretion level of in vitro matured DCs is an important indicator of DC vaccine homing potential., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aim: Indoleamine 2,3-dioxygenase (IDO) is an emerging new target in cancer therapy that can be targeted with active immunotherapy (e.g. through peptide vaccination). Furthermore, IDO has been identified as a key mechanism underlying resistance to treatment with the checkpoint blocking antibody ipilimumab (ipi)., Methods: Ten patients with metastatic melanoma participated in a phase I first-in-human clinical study assessing safety of combining ipi with a 21-mer synthetic peptide vaccine from IDO denoted IDOlong. Secondary and tertiary end points included vaccine and clinical response., Results: Treatment was generally safe and well tolerated. Vaccine related adverse reactions included grade I and II erythema, oedema and pruritus at the vaccination site, which were manageable with mild topical corticosteroids. One patient developed presumed ipi-induced colitis. It initially responded to high-dose parenteral corticosteroids but later relapsed while the patient was admitted to a local hospital, where he died after receiving suboptimal therapy. Vaccine-specific T-cell responses were detectable ex vivo in three patients. At first evaluation, five of the 10 treated patients were in stable disease, one of whom had an unconfirmed partial response., Conclusions: Treatment with IDOlong synthetic peptide vaccine in combination with ipi was generally safe and without augmented toxicity. The vaccine induced readily detectable T-cell responses in a subset of patients. Treatment showed signs of clinical activity, although not exceeding efficacy of ipi alone. Results should be confirmed in a larger study., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFNγ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating T-cell clones and functionality persists through out expansion among an oligoclonal composition of T-cells. Our findings mirror prior results on the oligoclonal composition of TIL cultures, further indicating a potential for a broader repertoire of specific effector cells recognizing the heterogeneous tumors upon adoptive transfer; increasing the probability of tumor control by minimizing immune evasion by tumor cell escape variants., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background Aims: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies., Methods: In a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr(51) release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion., Results: TIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8(+) and CD4(+) subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells., Conclusion: The procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO(+) suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-α while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate-guanosine, soluble cytotoxic T lymphocyte-associated antigen 4, or interferon γ) induced IDO-specific T cells among peripheral blood mononuclear cells from patients with cancer as well as healthy donors. In the clinical setting, IDO may serve as an important and widely applicable target for immunotherapeutic strategies in which IDO plays a significant regulatory role. We describe for the first time effector T cells with a general regulatory function that may play a vital role for the mounting or maintaining of an effective adaptive immune response. We suggest terming such effector T cells "supporter T cells."

A large proportion of melanoma patients host a spontaneous T-cell response specifically against ML-IAP-derived peptides. In this study, we describe that some ML-IAP-specific cytotoxic T cells isolated from melanoma patients cross react with an epitope from the auto-antigen SS56. SS56 is a recently described target of autoantibody responses in Sjögren's syndrome (SS) as well as systemic lupus erythematosus (SLE). Here, we describe that SS56 is also an auto-antigen for T cells in SS and SLE. Hence, SS56-specific T cells could readily be detected in circulation and among the infiltrating cells of SLE skin lesions. SS56-specific T cells were able to lyse target cells presenting the peptide epitope on the surface. Notably, SS56-specific CD8 T cells isolated from an SS patient cross reacted with the ML-IAP epitope. This early evidence of a target for auto-reactive CTL in SS and SLE patients; it is to our knowledge previously unreported and underscores the important role of CD8 T cells in autoimmune disorders. Furthermore, the cross-reactivity against the auto-antigen SS56 and the tumor-antigen ML-IAP confirms the link between autoimmunity and anti-cancer cellular immune responses.

Background Aims: 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., Methods: 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., Results: 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., Conclusions: Sequence analyzes of the CDR3 region revealed conserved structural characteristics in the MART-1-specific TCR used by T-cell clones.

The immune system is a complex arrangement of cells and molecules that preserve the integrity of the organism by elimination of all elements judged dangerous. Within the immune system, a humoral and a cellular as well as an innate and an adaptive arm can be differentiated. The key players of adaptive cellular immune responses are T lymphocytes in general and, for the effector function, cytotoxic T lymphocytes (CTLs) in particular. T lymphocytes arise in the bone marrow and migrate to the thymus for maturation. During this process, T cells somatically rearrange gene segments, eventually leading to the expression of a unique antigen-binding molecule, the T-cell receptor (TCR). This receptor allows them to monitor all cells of the body, ready to destroy any cell posing a threat to the organism. Cytotoxicity is exerted directly through the Fas or perforin pathway and/or indirectly by the release of cytokines. Obviously, the activity of such a potent cell is tightly regulated. Indeed, a predominance of stimulatory over inhibitory signals is required for effective immune responses to pathogens, and a predominance of inhibitory over stimulatory signals is required for maintenance of self-tolerance. Still, several situations occur in which an inappropriate CTL response leads to either autoimmune disease or persistence of pathogens.

B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from patients suffering from unrelated tumor types (ie, pancreatic cancer, breast cancer, acute myeloid leukemia [AML], and chronic lymphocytic leukemia [CLL]). Additionally, we show that these Bcl-2-reactive T cells are indeed peptide-specific, cytotoxic effector cells. Thus, Bcl-2 may serve as an important and widely applicable target for anticancer immunotherapeutic strategies (eg, in the combination with conventional radiotherapy and chemotherapy).

The identification of tumor antigens which expression is essential for the survival of tumor cells is a new avenue to prevent antigen loss variants emerging due to immunoselection, particularly during immune therapy. The melanoma inhibitor of apoptosis protein, ML-IAP (also named livin) counteracts apoptosis induced by death receptors, hypooxgenic conditions, or chemotherapeutic agents. Thus, elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the oncogenic phenotype. Here, we demonstrate that T cells in a large proportion of melanoma patients infiltrating the tumor or circulating in the peripheral blood specifically recognize ML-IAP-derived peptides. Interestingly, the responses against the peptide epitope ML-IAP280-289 were not restricted to melanoma patients but present among peripheral blood T cells in a few healthy controls. In situ peptide/HLA-A2 multimer staining, however, confirmed the infiltration of ML-IAP-reactive cells into the tumor microenvironment. Moreover, ML-IAP-reactive T cells isolated by magnetic beads coated with peptide/HLA-A2 complexes were cytotoxic against HLA-matched melanoma cells. In conclusion, out data strongly indicate ML-IAP as a suitable target for immunologic intervention.

Cellular immune responses are initiated by direct interaction of naive T cells with professional antigen-presenting cells, i.e., dendritic cells. In general, this interaction takes place in secondary lymphoid organs to which both naive T cells and mature dendritic cells preferentially home. This physiologic scenario differs substantially, however, from therapeutic dendritic-cell-based vaccinations used to treat human cancer. In fact, only a small fraction of intradermally injected dendritic cells migrate to the draining lymph node and the majority of cells remain at the site of inoculation. These sites are characterized by a distinct oligoclonal T cell infiltrate comprising both L-Selectin+/CD45RA+ and L-Selectin+/CD45RA- cells. Blood vessels expressing peripheral lymph node addressin represent possible entry channels for such naive and central memory T cells, the former probably attracted by dendritic cell-CK1 produced by the injected dendritic cells. In situ staining with multimeric peptide/major histocompatibility complex class I complexes revealed that infiltrating T cells specifically recognize peptide epitopes presented by the injected dendritic cells. Thus, the fraction of dendritic cells not migrating to secondary lymphatic tissue after therapeutic inoculation nevertheless seem to be involved in a specific immune modulation.

Selection and activation of T cells is tightly regulated by both antigen-specific receptors and co-receptors to ensure that responses to self antigens are largely avoided. By T cell receptor clonotypic mapping and staining with tetrameric HLA-peptide complexes, we demonstrate the presence of melanocyte differentiation antigen MART-1 specific T cells in the areas of destruction of both neoplastic and normal melanocytic cells in a case of a primary melanoma and its associated hypopigmentation. These self reactive T cells expressed CD94/NKG2 major histocompatibility complex class I specific C-type lectin-like receptors. This family of receptors includes both activating and inhibitory isoforms. Thus, we performed a detailed analysis that revealed the exclusive presence of inhibitory NKG2-A/B receptors in the vitiligo-like leukoderma, whereas both the inhibitory receptors and the activating NKG2-C/E isoforms were present within the tumor. Our data suggest the differential expression of killer inhibitory receptors as a possible mechanism to regulate T cell responses to self antigens.