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Circulating T cells that specifically target normal self-proteins expressed by regulatory immune cells were first described in patients with cancer, but can also be detected in healthy individuals. The adaptive immune system is distinguished for its ability to differentiate between self-antigens and foreign antigens. Thus, it was remarkable to discover T cells that apparently lacked tolerance to important self-proteins, eg, IDO, PD-L1, and FoxP3, expressed in regulatory immune cells. The ability of self-reactive T cells to react to and eliminate regulatory immune cells can influence general immune reactions. This suggests that they may be involved in immune homeostasis. It is here proposed that these T cells should be termed antiregulatory T cells (anti-Tregs). The role of anti-Tregs in immune-regulatory networks may be diverse. For example, pro-inflammatory self-reactive T cells that react to regulatory immune cells may enhance local inflammation and inhibit local immune suppression. Further exploration is warranted to investigate their potential role under different malignant conditions and the therapeutic possibilities they possess. Utilizing anti-Tregs for anticancer immunotherapy implies the direct targeting of cancer cells in addition to regulatory immune cells. Anti-Tregs provide the immune system with yet another level of immune regulation and contradict the notion that immune cells involved in the adjustment of immune responses only act as suppressor cells., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The era of immunotherapies was unleashed in 2010 with the Food and Drug Administration (FDA) approval of the first therapeutic vaccine sipuleucel-T as a standard treatment for metastatic prostate cancer. Next, the first immune-activating anticytotoxic lymphocyte antigen-4 (CTLA-4) antibody ipilimumab exhibiting 'immune checkpoint blockade' was approved by FDA and European Medical Agency (EMA) for the treatment of patients with metastatic melanoma. New generations of immune checkpoint blockading antibodies targeting programmed cell death 1 (PD-1) and its ligand (PD-L1) are now under intense investigation in metastatic melanoma (MM) and non-small-cell lung cancer (NSCLC), and impressive clinical results are anticipated. Despite these successes, only a fraction of patients become clinical responders to therapy. Thus, to improve the selection of patients likely to respond, scrutinizing different immune parameters during treatment is essential. In the summary of this PhD thesis, we investigated changes in immune parameters and their possible correlation with clinical efficacy in patients with MM during treatments with the standard chemo- and immunotherapies, temozolomide (TMZ) and interferon-α2b/interleukin-2 (IFN-α/IL-2) immunotherapy. The overall aim was to assess changes in frequency and absolute counts of different immune cell subsets before and after treatment and correlate to clinical benefit. Furthermore, the thesis covers a finalized, clinical phase 1 study in patients with NSCLC testing a peptide vaccination with a HLA-A2-restricted epitope derived from indoleamine 2,3 dioxygenase (IDO). The overall aim in this trial was to evaluate safety and tolerability of IDO as an anticancer vaccine target in patients with NSCLC and to assess whether immunity correlated to clinical response., (© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The immune system is a complex arrangement of cells and molecules that preserve the integrity of the organism by eliminating all elements judged to be dangerous. Several regulatory mechanisms function to terminate immune responses to antigens, return the immune system to a basal state after the antigen has been cleared, and maintain unresponsiveness, or tolerance, to self-antigens. In recent years, reports have described T cell responses to several proteins involved in regulating the immune system, particularly under malignant conditions. The present review highlights specific T cells that recognize proteins involved in three, well-defined immunosuppressive mechanisms: (1) inhibitory T cell pathways (i.e., PD-L1), (2) regulatory T cells (i.e., Foxp3(+)), and (3) metabolic enzymes, like indoleamine-2,3-dioxygenase. Cytotoxic T cells can eliminate regulatory cells, thereby suppressing and/or delaying local immune suppression; conversely, regulatory CD4(+) and non-cytotoxic CD8(+) T cells enhance target-mediated immune suppression. The apparent lack of tolerance against endogenous proteins expressed by regulatory cells is intriguing, because it suggests that self-reactive T cells play a general role of fine-tuning the immune system. Thus, T cell responses may be generally used to maintain the homeostasis of the immune system. Further exploration is warranted to investigate the potential role of auto-reactive T cells under different physiological and/or pathological conditions.

The mechanisms of immune response to cancer have been studied extensively and great effort has been invested into harnessing the therapeutic potential of the immune system. Immunotherapies have seen significant advances in the past 20 years, but the full potential of protective and therapeutic cancer immunotherapies has yet to be fulfilled. The insufficient efficacy of existing treatments can be attributed to a number of biological and technical issues. In this review, we detail the current limitations of immunotherapy target selection and design, and review computational methods to streamline therapy target discovery in a bioinformatics analysis pipeline. We describe specialized bioinformatics tools and databases for three main bottlenecks in immunotherapy target discovery: the cataloging of potentially antigenic proteins, the identification of potential HLA binders, and the selection epitopes and co-targets for single-epitope and multi-epitope strategies. We provide examples of application to the well-known tumor antigen HER2 and suggest bioinformatics methods to ameliorate therapy resistance and ensure efficient and lasting control of tumors.

The immune system is a tightly regulated and complex system. An important part of this immune regulation is the assurance of tolerance toward self-antigens to maintain immune homeostasis. However, in recent years, antigen-specific cellular immune responses toward several normal self-proteins expressed in regulatory immune cells have been reported, especially in patients with cancer. The seemingly lack of tolerance toward such proteins is interesting, as it suggests a regulatory function of self-reactive T (srT) cells, which may be important for the fine tuning of the immune system. In particular, surprising has been the description of cytotoxic srT cells that are able to eliminate normal regulatory immune cells. Such srT cells may be important as effector cells that suppress regulatory suppressor cells. The current knowledge of the nature and function of srT cells is still limited. Still, the therapeutic targeting of srT cells offers a novel approach to harness immune-regulatory networks in cancer.

Haem oxygenase-1 (HO-1) catalyses the rate-limiting step in haem degradation. All three metabolites resulting from haem degradation (carbon monoxide (CO), biliverdin and free iron) have anti-inflammatory and anti-apoptotic properties. HO-1 is a stress-inducible enzyme found extensively expressed in a vast variety of both human and murine cancers, where it serves as an essential survival molecule by modulating expression of molecules regulating apoptosis and stimulating angiogenesis. In addition, HO-1 contributes in a critical manner to inhibition or termination of inflammation. Consequently, several anticancer strategies aim at targeting HO-1. The inhibition of HO-1 may cause tumour cells to become more sensitive to chemotherapy and radiation therapy. The water-soluble forms of the HO-1 inhibitor Zinc protoporphyrin (ZnPP) have seemed promising in different in-vivo models, in which it has induced growth arrest in tumour cells with few, if any, side effects. Studies have suggested that HO-1 may also function to disrupt the tumour metastasising process, since the expression of the metalloprotease MMP9 is inversely correlated with HO-1 expression. Additionally, HO-1 has anti-inflammatory functions which play a very important role in the negative regulation of the immune system. Immunological targeting of HO-1 might represent an interesting approach, as epitopes derived from HO- 1 have been found exclusively on tumour tissue. Natural HO-1-specific T-cell responses have been identified in cancer patients. Hence, recently HO-1-specific, CD8⁺ regulatory T cells were described in cancer patients, which in concert with HO-1 expression might be responsible for a highly immunosuppressive tumour microenvironment. Here, we summarise current knowledge of the role of HO-1 in cancer, report the different results of the targeting of HO-1 in preclinical and clinical settings, and discuss future opportunities.

Solid tumors are more than an accumulation of cancer cells. Indeed, cancerous cells create a permissive microenvironment by exploiting non-transformed host cells. Thus, solid tumors rather resemble abnormal organs composed of the cancerous cells itself and the stroma providing the supportive framework. The stroma can be divided into the extracellular matrix consisting of proteoglycans, hyaluronic acid, and fibrous proteins, as well as stromal cells including mesenchymal and immune cells; moreover, it contains various peptide factors and metabolites. Here, we will focus on immune-modulating capacities of the tumor microenvironment.

Dendritic cells (DC) are the most potent antigen presenting cells and have proven effective in stimulation of specific immune responses in vivo. Competing immune inhibition could limit the clinical efficacy of DC vaccination. In this phase II trial, metronomic Cyclophosphamide and a Cox-2 inhibitor have been added to a DC vaccine with the intend to dampen immunosuppressive mechanisms. Twenty-eight patients with progressive metastatic melanoma were treated with autologous DCs pulsed with survivin, hTERT, and p53-derived peptides (HLA-A2(+)) or tumor lysate (HLA-A2(-)). Concomitantly the patients were treated with IL-2, Cyclophosphamide, and Celecoxib. The treatment was safe and tolerable. Sixteen patients (57 %) achieved stable disease (SD) at 1st evaluation and 8 patients had prolonged SD (7-13.7 months). The median OS was 9.4 months. Patients with SD had an OS of 10.5 months while patients with progressive disease (PD) had an OS of 6.0 months (p = 0.048) even though there were no differences in prognostic factors between the two groups. Despite the use of metronomic Cyclophosphamide, regulatory T cells did not decrease during treatment. Indirect IFN-γ ELISPOT assays showed a general increase in immune responses from baseline to the time of 4th vaccination. Induction of antigen-specific immune responses was seen in 9 out of 15 screened HLA-A2(+) patients. In conclusion, the number of patients obtaining SD more than doubled and 6-month survival significantly increased compared to a previous trial without Cyclophosphamide and Celecoxib. A general increase in immune responses against the tested peptides was observed.

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.

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.

S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.

During the past years numerous clinical trials have been carried out to assess the ability of dendritic cell (DC) based immunotherapy to induce clinically relevant immune responses in patients with malignant diseases. A broad range of cancer types have been targeted including malignant melanoma which in the disseminated stage have a very poor prognosis and only limited treatment options with moderate effectiveness. Herein we describe the results of a focused search of recently published clinical studies on dendritic cell vaccination in melanoma and review different vaccine parameters which are frequently claimed to have a possible influence on clinical response. These parameters include performance status, type of antigen, DC maturation status, route of vaccine administration, use of adjuvant, and vaccine induced immune response. In total, 38 articles found through Medline search, have been included for analysis covering a total of 626 patients with malignant melanoma treated with DC based therapy. Clinical response (CR, PR and SD) were found to be significantly correlated with the use of peptide antigens (p = 0.03), the use of any helper antigen/adjuvant (p = 0.002), and induction of antigen specific T cells (p = 0.0004). No significant correlations between objective response (CR and PR) and the tested parameters were found. However, a few non-significant trends were demonstrated; these included an association between objective response and use of immature DCs (p = 0.08), use of adjuvant (p = 0.09), and use of autologous antigen preparation (p = 0.12). The categorisation of SD in the response group is debatable. Nevertheless, when the SD group were analysed separately we found that SD was significantly associated with use of peptide antigens (p = 0.0004), use of adjuvant (p = 0.01), and induction of antigen specific T cells (p = 0.0003). No specific route of vaccine administration showed superiority. Important lessons can be learned from previous studies, interpretation of these findings should, however, be done with reservation for the many minor deviations in the different treatment schedules among the published studies, which were not considered in order to be able to process and group the data.

Harnessing of the immune system by the development of 'therapeutic' vaccines, for the battle against cancer has been the focus of tremendous research efforts over the past two decades. As an illustration of the impressive amounts of data gathered over the past years, numerous antigens expressed on the surface of cancer cells, have been characterized. To this end, recent years research has focussed on characterization of antigens that play an important role for the growth and survival of cancer cells. Anti-apoptotic molecules like survivin that enhance the survival of cancer cells and facilitate their escape from cytotoxic therapies represent prime vaccination candidates. The characterization of a high number of tumor antigens allow the concurrent or serial immunological targeting of different proteins associated with such cancer traits. Moreover, while vaccination in itself is a promising new approach to fight cancer, the combination with additional therapy could create a number of synergistic effects. Herein we discuss the possibilities and prospects of vaccination when combined with other treatments. In this regard, cell death upon drug exposure may be immunogenic or non-immunogenic depending on the specific chemotherapeutics. Also, chemotherapy represents one of several options available for clearance of CD4+ Foxp3+ regulatory T cells. Moreover, therapies based on monoclonal antibodies may have synergistic potential in combination with vaccination, both when used for targeting of tumor cells and endothelial cells. The efficacy of therapeutic vaccination against cancer will over the next few years be studied in settings taking advantage of strategies in which vaccination is combined with other treatment modalities. These combinations should be based on current knowledge not only regarding the biology of the cancer cell per se, but also considering how treatment may influence the malignant cell population as well as the immune system.

The immune system's ability to detect and destroy tumor cells offers an attractive approach to broaden the spectrum of cancer therapies. Survivin, a member of the apoptosis inhibitor protein family, is a tumor antigen, overexpressed in human cancers giving rise to peptides eliciting spontaneous CD8+ and CD4+ responses. Due to its dual function, blockade of apoptosis and regulation of cell division, survivin is directly associated with tumor survival and therefore regarded as an ideal target structure for immunotherapeutic approaches. Strong evidence that survivin acts as a T-cell activating antigen has been collected in recent years and the first clinical trials using survivin-based vaccines aim to prove its therapeutic efficacy in the clinic. We focus on the role of survivin in hematological malignancies, including a list of survivin-derived peptides eliciting potent immune responses.

Monitoring and evaluation of biological responses induced by immunotherapy may provide important information with regards to efficacy, side effects, and potential improvements of treatment. Herein, we describe results from monitoring of T cell reactivity against survivin derived peptides, in a melanoma patient in complete remission following IL-2 based immunotherapy. The patient remains in complete remission five years after completion of therapy. Long-time persistence of anti-tumor responses is rarely monitored, however, in the present patient longitudinal examination of anti-survivin reactivity exceeded seven years. Throughout, survivin reactivity was monitored both by INFgamma-ELISPOT as well as by flow cytometry using HLA-multimers. Survivin specific T cell reactivity was found at all time points examined over the 7-year period. Moreover, using these two methods, similar precursor frequencies was found indicating that the majority of the survivin specific T cells posses functional capabilities. Our data demonstrate that anti-survivin T cells may persist in the periphery for extended periods in the absence of clinical manifestation of disease as well as autoimmunity.

Expression of the cancer-testis antigen Taxol resistance-associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01-restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3-derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01-binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3-specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2(+) donors, but no responses were detectable in 9 HLA-A2(-) healthy donors or 4 HLA-A2(-) melanoma patients. The identified HLA-A*02.01-restricted TRAG-3-derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.

Activating BRAF somatic missense mutations within the kinase domain are present in 60-66% of melanomas. The vast majority of these represent a single substitution of glutamate for valine (V599E). Here, we demonstrate spontaneous HLA-B*2705-restricted cytotoxic T-cell responses against an epitope derived from (V599E)BRaf. These T-cell responses were mutation specific as the corresponding epitope derived from wild-type BRaf was not recognized. The loss of the (V599E)BRAF genotype during progression from primary to metastatic melanoma in patients with (V599E)BRaf specific T-cell responses suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host.

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.

Cells of the immune system spontaneously recognize autologous tumor cells and T cells are believed to be the main effector cells for the immune surveillance of cancer. Recent advances in our understanding of basic and tumor immunology together with methodological developments implies that tumor specific T cells can now be studied functionally, phenotypically as well as molecularly. T cells recognize peptide antigens in the context of MHC molecules through the clonally distributed T-cell receptor (TCR), thus, the clonal distribution of the TCR offers the means to detect and track specific T cells based upon detection of the unique TCR. In this review, we present and discuss available data on TCR utilization of tumor specific T cells in murine models as well as spontaneous and treatment induced anti-tumor T-cell responses in humans.

Two HLA-A2 restricted epitopes have recently been identified from the broadly expressed tumor antigen survivin, and several vaccination trials in cancer patients based on these survivin-derived peptides have been initiated. Consequently, there is a crucial need for the identification of survivin epitopes restricted to other HLA-molecules in order to extend the proportion of patients that can enter these ongoing clinical trials. In the present study, we characterized 2 survivin-derived epitopes, which are restricted to HLA-B35. Specific T-cell reactivity against these survivin-derived epitopes was found in the peripheral blood from patients with different B-cell malignancies and melanoma. Substitution of the C-terminal anchor residue of the survivin-derived peptides improved the recognition by tumor-infiltrating lymphocytes from melanoma patients. Furthermore, we demonstrated spontaneous cytotoxic T-cell responses to survivin in a primary melanoma lesion. The characterization of these epitopes allows more patients can be included in the ongoing peptide-based survivin vaccination trials against cancer., (Copyright 2003 Wiley-Liss, Inc.)

The identification of tumor antigens, which are essential for the survival of tumor cells is a new avenue to prevent antigen loss variants emerging due to immunoselection, particularly during immune therapy. In the search for such immunogenic tumor antigens, we recently identified spontaneous cytotoxic lymphocyte (CTL) responses against the inhibitor of apoptosis protein survivin. Thus, we identified two HLA-A2-restricted, survivin-derived CTL epitopes, which both were targets for spontaneous CTL responses in melanoma, breast cancer, and CLL. Here, we extend these data and describe the characterization of novel HLA-A1-, HLA-A2-, HLA-A3-, and HLA-A11-restricted survivin epitopes on the basis of spontaneous CTL responses in cancer patients. These epitopes significantly increase the number of patients eligible for immunotherapy based on survivin derived peptides. Additionally, the collective targeting of several restriction elements is likely to decrease the risk of immune escape by HLA-allele loss.

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.

Interleukin 2 (IL-2) is a promising immunotherapeutic agent for the treatment of metastatic melanoma and renal cell carcinoma. Systemic administration of high dose IL-2 produces objective responses in up to 25% of melanoma patients, and a low but significant proportion of these patients experience durable responses. Nevertheless, the cells and molecules responsible for induction of tumor regression over the course of IL-2 treatment remain unknown. New strategies in tumor immunotherapy have evolved over the past decade as a consequence of significant progress in the field, in particular with respect to the characterization of peptide epitopes derived from tumor associated antigens, and the role of antigen presenting cells in the initiation of cellular immune responses. Alongside with these factual as well as conceptual advances, new methods have been developed to monitor and characterize anti-tumor T cell responses in cancer patients. Application of these tools to dissect anti-tumor responses has demonstrated that various immune therapeutic approaches can induce powerful systemic anti-tumor cytotoxic T lymphocyte (CTL) responses. However, only limited efforts have been made to use present days tool to analyze anti-tumor immune responses in patients treated with IL-2 based immunotherapy. We have examined CTL responses against known tumor antigens in melanoma patients over the course of IL-2 based immunotherapy (electrochemotherapy). Surprisingly, anti-tumor CTL responses significantly declined upon initiation of therapy, but reappeared when IL-2 administration was paused. Molecular analyses of the clonotypic composition of responding T cells demonstrated that new clones emerged over the course of treatment, and that tumor-specific T cells that had left the peripheral blood could subsequently be detected at the tumor site. These data provide new insight into the biological actions of IL-2 and highlight the difficulties associated with the monitoring of anti-tumor immune responses. This underlines the importance of frequent sampling of blood and tumor biopsies to be analyzed with a combination of state of the art technologies in order to gain detailed information on the interactions between cancer cells and cells of the immune system.

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.

Significant advances in the understanding of the cells and molecules involved in tumour/host interactions in humans have been made over the last decades. Thus, through studies of the interactions between cells of the immune system and cancer cells, several tumour antigens and their epitopes recognised by human leucocyte antigen class I-restricted cytotoxic T lymphocytes have been identified. Likewise, specialised antigen presenting cells have been characterised and methods developed that allow the use of such cells in clinical immunology. In the clinic, systemic administration of immune stimulatory cytokines, in particular IL-2, has strengthened the notion that the immune system is capable of mediating rejection of cancer. More recently, more sophisticated methods of inducing anti-cancer immune responses have been pursued, for instance the use of antigen presenting cells in combination with various sources of antigen and cytokines for therapeutic vaccinations against cancer. The present paper summarizes current knowledge in the field of tumour immunology, and reviews various approaches undertaken with the aim of inducing strong anti-tumour responses. To this end, we review data from clinical trials, and discuss the potential of these treatment modalities as an adjunct to conventional modes of cancer treatment. Further, novel methodologies for monitoring and analysing cancer patients over the course of immunotherapeutic treatments are described, and future directions put forward.

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.