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Over the last few years, cancer immunotherapy experienced tremendous developments and it is nowadays considered a promising strategy against many types of cancer. However, the exclusion of lymphocytes from the tumor nest is a common phenomenon that limits the efficiency of immunotherapy in solid tumors. Despite several mechanisms proposed during the years to explain the immune excluded phenotype, at present, there is no integrated understanding about the role played by different models of immune exclusion in human cancers. Hypoxia is a hallmark of most solid tumors and, being a multifaceted and complex condition, shapes in a unique way the tumor microenvironment, affecting gene transcription and chromatin remodeling. In this review, we speculate about an upstream role for hypoxia as a common biological determinant of immune exclusion in solid tumors. We also discuss the current state of ex vivo and in vivo imaging of hypoxic determinants in relation to T cell distribution that could mechanisms of immune exclusion and discover functional-morphological tumor features that could support clinical monitoring. [ABSTRACT FROM AUTHOR]

The dichotomy of immunology into innate and adaptive immunity has created conceptual barriers in appreciating the intrinsic two-way interaction between immune cells. An emerging body of evidence in various models of immune rejection, including cancer, indicates an indispensable regulation of innate effector functions by adaptive immune cells. This bidirectional cooperativity in innate and adaptive immune functions has broad implications for immune responses in general and for regulating the tumor-associated inflammation that overrides the protective antitumor immunity. Mechanistic understanding of this two-way immune cross-talk could provide insights into novel strategies for designing better immunotherapy approaches against cancer and other diseases that normally defy immune control. [ABSTRACT FROM AUTHOR]

The understanding of tumor–host interactions remains elusive despite significant progress in the identification of tumor antigens (TAs) recognized by autologous T cells. In particular, most human tumors do not regress and continue to grow in spite of spontaneous or immunization-induced immune responses demonstrated in circulating lymphocytes. Indeed, systemic immune responses might insufficiently address the complexity of tumor–host interactions because of factors, such as (1) the lack of productive T-cell receptor (TCR) engagement with epitope owing to qualitative and/or quantitative defects in the generation and maintenance of the immune response, (2) insufficient costimulation provided by the host, (3) the lack of localization of the immune response in target tissues and (4) the complexity of tumor–host interactions within the tumor microenvironment caused by temporal changes in tumor phenotypes and an array of immune mediators expressed in the tumor microenvironment. Here, we will review current knowledge of the different ‘levels’ of immune response that might be necessary for immunotherapy to be effective in the treatment of cancer. Furthermore, we will discuss the information still required in order to understand the mechanism(s) governing tumor rejection by the immune system in response to TA-specific immunization. [Copyright &y& Elsevier]

Background: Recent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity. Methods: MET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo. Results: MET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells. Conclusion: We generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo. [ABSTRACT FROM AUTHOR]

A recent commentary stirred intense controversy over the status of anti-cancer immunotherapy. The commentary suggested moving beyond current anti-cancer vaccines since active-specific immunization failed to match expectations toward a more aggressive approach involving the adoptive transfer of in vitro expanded tumor antigen-specific T cells. Although the same authors clarified their position in response to others' rebuttal more discussion needs to be devoted to the current status of T cell-based anti-cancer therapy. The accompanying publications review the status of adoptive transfer of cancer vaccines on one hand and active-specific immunization on the other. Hopefully, reading these articles will offer a balanced view of the current status of antigen-specific ant-cancer therapies and suggest future strategies to foster unified efforts to complement either approach with the other according to specific biological principles. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. Methods: We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). Results: The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. Conclusions: RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity. [ABSTRACT FROM AUTHOR]

Immune-mediated rejection of human cancer is a relatively rare but well-documented phenomenon. Its rate of occurrence progressively increases from the occasional observation of spontaneous regressions to the high rate of complete remissions observed in response to effective treatments. For two decades, our group has focused its interest in understanding this phenomenon by studying humans following an inductive approach. Sticking to a sequential logic, we dissected the phenomenon by studying to the best of our capability both peripheral and tumor samples and reached the conclusion that immune-mediated cancer rejection is a facet of autoimmunity where the target tissue is the cancer itself. As we are currently defining the strategy to effectively identify the mechanisms leading in individual patients to rejection of their own tumors, we considered useful to summarize the thought process that guided us to our own interpretation of the mechanisms of immune responsiveness. [ABSTRACT FROM AUTHOR]

Whether the efforts of the last decade aimed at the development of vaccines against tumor-specific antigens encountered success or failure is a matter of expectations. On the bright side, we could optimistically observe that anti-cancer-vaccines stand as an outstanding example of the successful implementation of modern biotechnology tools for the development of biologically sound therapeutics. In particular, vaccines against melanoma (the prototype model of tumor immunology in humans) can reproducibly induce cytotoxic T cell (CTL) responses exquisitely specific for cancer cells. This achievement trespasses the specificity of any other anti-cancer therapy. The skeptics, on the other end, might point out that immunization only rarely leads to cancer regression, labeling, therefore, this approach is ineffective. In our opinion this judgment stems from the naïve expectation that CTL induction is sufficient for an effective immune response. Here we propose that more needs to be understood about the mechanisms required for the induction of a therapeutically relevant immune response in humans. In particular, we will discuss the variables related to cancer heterogeneity, the weight of individual patients’ polymorphism(s), the role of the T cell activation and differentiation and, finally, the complex relationship between immune and cancer cells within the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: The inefficacy of standard therapeutic strategies for ovarian cancer is reflected by the enduring poor prognosis of this malignancy. Due to the potential for exquisite specificity, sensitivity and long-term memory, immunotherapy offers an alternative modality for durable control of the disease, provided appropriate antigens can be identified and presented in the right context.Methods: We tested a novel dendritic cell vaccine formulation to reprogram autologous antigen-specific T-cells in vitro, in vivo in a murine model of ovarian cancer, and ex vivo using human cells from patients.Results: We show that dendritic cells (DCs) treated with a p38 MAPK inhibitor and transduced with a recombinant adenovirus associated vector (AAV) expressing Sperm protein (Sp) 17 are highly effective in generating antigen-specific T-cell cytotoxic response against ovarian cancer cells. Additionally, these DCs enhanced the differentiation of effector T-cells while reducing the frequency of Foxp3+ T-reg cells in vitro.Conclusions: This work provides a rationale for translation of pharmacologically reprogrammed DCs into clinical trials for prevention of tumor recurrence and progression in high-risk ovarian cancer patients. [ABSTRACT FROM AUTHOR]

The predictive accuracy of the traditional staging system is based on disease progression as a tumour cell-autonomous process, but it fails to incorporate the effects of the host immune response. A precise analysis of the immune component of the tumour microenvironment by computer-based analysis may be essential to managing patients better, opening the road to an expertise in this new emerging field. The Immunoscore as a new possible approach in the classification of cancer, designated TNM-Immune, studied in colon cancer patients with predictive and prognostic value. This new scoring system is derived from the immune contexture, and is based on the numeration of lymphocyte populations, both in the core of the tumour and in the invasive margin of tumours. The Immunoscore demonstrated to be quantitative, reproducible and robust. The usefulness of Immunoscore in advanced melanoma cancer patients has been as well demonstrated; the correlation of marker expression profile with clinical outcome is ongoing. More recently, the Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. A multivariable Cumulative "Suppression Index" scoring system has been also studied in Oral Squamous Cell Carcinoma patients: it evaluates both the tumor and stromal microcompartments at the invasive margin and summarizes them into the score, providing an accurate stratification, independent of stage, tumour classification. The introduction of Immunoscore requires a redefinition of the Laboratory system according to the LEAN Management process, which has been already implemented in referral research labs. The definition and test of hundreds of biomarkers, in the tumour contexture represents a definitive scientific progression. However, there is still a need of substantial body of work to reach the end of the tunnel to assure a personalize treatment. [ABSTRACT FROM AUTHOR]

Introduction: Infiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases. Patients and methods: Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression. Results and conclusions: Four eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma. [ABSTRACT FROM AUTHOR]

Introduction: Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9-11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases. Patients and methods: Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression. Results: Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures. Conclusion: The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases. [ABSTRACT FROM AUTHOR]

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer. [ABSTRACT FROM AUTHOR]

Background: Dendritic cells (DC) are currently implemented as immunotherapeutic strategy for the treatment of tumor patients based on their central role in the immune system. Despite good results were obtained in vitro and in animal models, their clinical use has provided limited success suggesting the requirement to optimise the protocol for their production.Methods: A cDNA array was performed on FastDC obtained from the differentiation of human peripheral blood monocytes stimulated with the clinical gold standard or with two alternative maturation cocktails combining interferon (IFN)γ and ligands for different toll like receptors (TLR).Results: A stronger modulation of the DC transcriptome with respect to immature DC was found in alternatively stimulated DC when compared to DC stimulated with the clinical gold standard. A major class of molecules differentially expressed using distinct DC stimulation protocols were chemokines. Validation of their differential expression pattern at the mRNA and protein level confirmed the secretion of inflammatory chemokines by the alternative DC. Functional analyses of the chemotactic properties of DC "wash out" supernatants highlighted the ability of alternative, but not of gold standard DC to efficiently recruit immune cells with a prevalence of monocytes. Effector cells belonging to the innate as well as adaptive immunity were also attracted and the interaction with alternative DC resulted in enhanced secretion of IFNγ and induction of cytotoxic activity. Using leukocytes from cancer patients, it was demonstrated that the monocyte-attracting activity targeted cells with an inflammatory phenotype characterised by high levels of HLA-DR expression.Conclusions: Despite other classes of immune modulatory genes differently expressed in the alternative DC require to be investigated and characterised regarding their functional consequences, the reduced maturation state and chemoattractive properties of the gold standard versus alternative DC clearly promote the necessity to change the clinically used maturation cocktail of DC in order to improve the outcome of patients treated with DC-based vaccines. [ABSTRACT FROM AUTHOR]

A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects of interferon- γ ( IFN- γ) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (mo DC). Here, we report that following differentiation of monocytes into mo DC with granulocyte-macrophage colony-stimulating factor and interleukin-4, IFN- γ induces mo DC maturation and up-regulates the co-stimulatory markers CD80/ CD86/ CD95 and MHC Class I, enabling mo DC to effectively generate antigen-specific CD4+ and CD8+ T-cell responses for multiple viral and tumour antigens. Early exposure of monocytes to high concentrations of IFN- γ during differentiation promotes the formation of macrophages. However, under low concentrations of IFN- γ, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN- γ or lipopolysaccharide and an inability to generate effective antigen-specific CD4+ and CD8+ T-cell responses. These findings demonstrate that IFN- γ imparts differential programmes on mo DC that shape the antigen-specific T-cell responses they induce. Timing and intensity of exposure to IFN- γ can therefore determine the functional capacity of mo DC. [ABSTRACT FROM AUTHOR]

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m2/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56bright NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA [105]. In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. [ABSTRACT FROM AUTHOR]

The transcriptional repressor Blimp-1 promotes the differentiation of CD8+ T cells into short-lived effector cells (SLECs) that express the lectin-like receptor KLRG-1, but how it operates remains poorly defined. Here we show that Blimp-1 bound to and repressed the promoter of the gene encoding the DNA-binding inhibitor Id3 in SLECs. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited the ability of SLECs to persist as memory cells. Enforced expression of Id3 was sufficient to restore SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of the transcriptional activity of E2A and induction of genes regulating genome stability. Our findings identify the Blimp-1-Id3-E2A axis as a key molecular switch that determines whether effector CD8+ T cells are programmed to die or enter the memory pool. [ABSTRACT FROM AUTHOR]

Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO?, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7R?+ T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2R?, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies. [ABSTRACT FROM AUTHOR]

The Society for Immunotherapy of Cancer, SITC (formerly the International Society for Biological Therapy of Cancer, iSBTc), aims to improve cancer patient outcomes by advancing the science, development and application of biological therapy and immunotherapy. The society and its educational programs have become premier destinations for interaction and innovation in the cancer biologics community. For over a decade, the society has offered the Primer on Tumor Immunology and Biological Therapy of Cancer™ in conjunction with its Annual Scientific Meeting. This report summarizes the 2010 Primer that took place October 1, 2010 in Washington, D.C. as part of the educational offerings associated with the society's 25th anniversary. The target audience was basic and clinical investigators from academia, industry and regulatory agencies, and included clinicians, post-doctoral fellows, students, and allied health professionals. Attendees were provided a review of basic immunology and educated on the current status and most recent advances in tumor immunology and clinical/translational caner immunology. Ten prominent investigators presented on the following topics: innate immunity and inflammation; an overview of adaptive immunity; dendritic cells; tumor microenvironment; regulatory immune cells; immune monitoring; cytokines in cancer immunotherapy; immune modulating antibodies; cancer vaccines; and adoptive T cell therapy. Presentation slides, a Primer webinar and additional program information are available online on the society's website. [ABSTRACT FROM AUTHOR]

Background: High-dose interferon-alpha 2b (IFN-α 2b) is the only approved systemic therapy in the United States for the adjuvant treatment of melanoma. The study objective was to explore the immunomodulatory mechanism of action for IFN-α 2b by measuring serum regulatory T cell (Treg), serum transforming growth factor-β (TGF-β), interleukin (IL)-10, and autoantibody levels in patients with melanoma treated with the induction phase of the high-dose IFN-α 2b regimen. Methods: Patients with melanoma received IFN-α 2b administered intravenously (20 MU/m² each day from day 1 to day 5 for 4 consecutive weeks). Serum Treg levels were measured as whole lymphocytes in CD4+ cells using flow cytometry while TGF-β, IL-10, and autoantibody levels were measured using enzyme-linked immunosorbent assays. Results: Twenty-two patients with melanoma received IFN-α 2b treatment and were evaluated for Treg levels. Before treatment, Treg levels were significantly higher in patients with melanoma when compared with data from 20 healthy subjects (P = 0.001; Mann-Whitney test). Although a trend for reduction of Treg levels following IFN-α 2b treatment was observed (average decrease 0.29% per week), statistical significance was not achieved. Subgroup analyses indicated higher baseline Treg levels for stage III versus IV disease (P = 0.082), early recurrence versus no recurrence (P = 0.017), deceased versus surviving patients (P = 0.021), and preoperative neoadjuvant versus postoperative adjuvant treatment groups (not significant). No significant effects were observed on the levels of TGF-β, IL-10, and autoantibodies in patients with melanoma treated with IFN-α 2b. Conclusions: Patients with melanoma in this study showed increased basal levels of Treg that may be relevant to their disease and its progression. Treg levels shifted in patients with melanoma treated with IFN-α 2b, although no firm conclusions regarding the role of Tregs as a marker of treatment response or outcome can be made at present. [ABSTRACT FROM AUTHOR]

Background: Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated. Methods: We performed miR microarray analyses on in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells. Results: Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR- 17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4+ T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-γ production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD). Conclusion: The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Over the last decade remarkable progress has been made in enhancing the efficacy of CAR T therapies. However, the clinical benefits are still limited, especially in solid tumors. Even in hematological settings, patients that respond to CAR T therapies remain at risk of relapsing due to several factors including poor T-cell expansion and lack of long-term persistence after adoptive transfer. This issue is even more evident in solid tumors, as the tumor microenvironment negatively influences the survival, infiltration, and activity of T-cells. Limited persistence remains a significant hindrance to the development of effective CAR T therapies due to several determinants, which are encountered from the cell manufacturing step and onwards. CAR design and ex vivo manipulation, including culture conditions, may play a pivotal role. Moreover, previous chemotherapy and lymphodepleting treatments may play a relevant role. In this review, the main causes for decreased persistence of CAR T-cells in patients will be discussed, focusing on the molecular mechanisms underlying T-cell exhaustion. The approaches taken so far to overcome these limitations and to create exhaustion-resistant T-cells will be described. We will also examine the knowledge gained from several key clinical trials and highlight the molecular mechanisms determining T-cell stemness, as promoting stemness may represent an attractive approach to improve T-cell therapies. [ABSTRACT FROM AUTHOR]

Background: Upon Ag-activation cytotoxic T cells (CTLs) produce IFN-γ GM-CSF and TNF-α, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown. Methods: Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression. Results: Ag-activated CTLs displayed higher levels of IFN-γ, GM-CSF (CSF2) and GM-CSF/IL-3/IL-5 receptor common β- chain (CD131) but lacked completely expression of IFN-γ receptor-II and IFN-stimulated genes (ISGs). This observation suggested that Ag-activated CTLs in preparation for the release of IFN-γ and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF. Conclusion: The selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation. [ABSTRACT FROM AUTHOR]

Latent membrane protein (LMP)-2 is one of the Epstein–Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient’s CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab. [ABSTRACT FROM AUTHOR]

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (109) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2+ MUC1+ MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were “consensus” agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LL PWTVLTV) and p15 ( VLL WTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-α, IFN-γ, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2. [ABSTRACT FROM AUTHOR]

The evaluation and characterization of epitope-specific human leukocyte antigen (HLA)-restricted memory T-cell reactivity is an important step for the development of preventive vaccines and peptide-based immunotherapies for viral and tumor diseases. The past decade has witnessed the use of HLA-restricted peptides as tools to activate strong immune responses of naïve or memory T cells specifically. This has fuelled an active search for methodological approaches focusing on HLA and peptide associations. Here, we outline new perspective on the emerging opportunity of evaluating HLA and peptide restriction by using novel approaches, such as quantitative real-time PCR, that can identify epitope specificities that are potentially useful in clinical settings. [Copyright &y& Elsevier]

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities. [ABSTRACT FROM AUTHOR]

Several HLA class I alleles have been associated with slow human immunodeficiency virus (HIV) disease progression, supporting the important role HLA class I-restricted cytotoxic T lymphocytes (CTL) play in controlling HIV infection. HLA-B63, the serological marker for the closely related HLA-B∗1516 and HLA-B∗1517 alleles, shares the epitope binding motif of HLA-B57 and HLA-B58, two alleles that have been associated with slow HIV disease progression. We investigated whether HIV-infected individuals who express HLA-B63 generate CTL responses that are comparable in breadth and specificity to those of HLA-B57/58-positive subjects and whether HLA-B63-positive individuals would also present with lower viral set points than the general population. The data show that HLA-B63-positive individuals indeed mounted responses to previously identified HLA-B57-restricted epitopes as well as towards novel, HLA-B63-restricted CTL targets that, in turn, can be presented by HLA-B57 and HLA-B58. HLA-B63-positive subjects generated these responses early in acute HIV infection and were able to control HIV replication in the absence of antiretroviral treatment with a median viral load of 3,280 RNA copies/ml. The data support an important role of the presented epitope in mediating relative control of HIV replication and help to better define immune correlates of controlled HIV infection. [ABSTRACT FROM AUTHOR]

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose-escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-gamma. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma. [ABSTRACT FROM AUTHOR]

We have recently developed a candidate HIV-1 vaccine model based on HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs). The HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing Antibodies as well as cytotoxic T lymphocytes, by intra-peritoneal as well as intra-nasal administration. Moreover, we have recently shown that the baculovirus-expressed HIV-VLPs induce maturation and activation of monocyte-derived dendritic cells (MDDCs) which, in turn, produce Th1- and Th2-specific cytokines and stimulate in vitro a primary and secondary response in autologous CD4+ T cells. In the present manuscript, the effects of the baculovirus-expressed HIV-VLPAs on the genomic transcriptional profile of MDDCs obtained from normal healthy donors have been evaluated. The HIV-VLPA stimulation, compared to both PBS and LPS treatment, modulate the expression of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. The results of gene profiling analysis here presented are highly informative on the global pattern of gene expression alteration underlying the activation of MDDCs by HIV-VLPAs at the early stages of the immune response and may be extremely helpful for the identification of exclusive activation markers. [ABSTRACT FROM AUTHOR]

Background: Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.Methods: To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.Results: We found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.Conclusions: These results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors. [ABSTRACT FROM AUTHOR]

Summary We examined how the immune microenvironment molds tumor evolution at different metastatic organs in a longitudinal dataset of colorectal cancer. Through multiplexed analyses, we showed that clonal evolution patterns during metastatic progression depend on the immune contexture at the metastatic site. Genetic evidence of neoantigen depletion was observed in the sites with high Immunoscore and spatial proximity between Ki67+ tumor cells and CD3+ cells. The immunoedited tumor clones were eliminated and did not recur, while progressing clones were immune privileged, despite the presence of tumor-infiltrating lymphocytes. Characterization of immune-privileged metastases revealed tumor-intrinsic and tumor-extrinsic mechanisms of escape. The lowest recurrence risk was associated with high Immunoscore, occurrence of immunoediting, and low tumor burden. We propose a parallel selection model of metastatic progression, where branched evolution could be traced back to immune-escaping clones. The findings could inform the understanding of cancer dissemination and the development of immunotherapeutics. Graphical Abstract Highlights • Different escape mechanisms delineated by lack of adaptive immunity or immunoediting • Non-recurrent clones are immunoedited; progressing clones are immune privileged • Immunoediting and Immunoscore are predictive factors of metastasis recurrence • Parallel selection model describes clonal immunoediting and tumor evolution A longitudinal analysis of clonal evolution of tumors across multiple tissues identifies a parallel selection model that explains the role of immune editing in controlling metastatic growth. [ABSTRACT FROM AUTHOR]

Metastatic melanoma represents a challenging clinical situation and, until relatively recently, there was an absence of effective treatment options. However, in 2011, the advanced melanoma treatment landscape was revolutionised with the approval of the anti-cytotoxic T-lymphocyte-associated protein-4 checkpoint inhibitor ipilimumab and the selective BRAF kinase inhibitor vemurafenib, both of which significantly improved overall survival. Since then, availability of new immunotherapies, especially the anti-programmed death-1 checkpoint inhibitors, as well as other targeted therapies, have further improved outcomes for patients with advanced melanoma. Seven years on from the first approval of these novel therapies, evidence for the use of various immune-based and targeted approaches is continuing to increase at a rapid rate. Improved understanding of the tumour microenvironment and tumour immuno-evasion strategies has resulted in different approaches to target and harness the immune response. These new immune-based approaches offer the opportunity for various approaches with distinct modes of action being used in combination with one another, as well as combined with other treatment modalities such as targeted therapy, electrochemotherapy and surgery. The increasing number of treatment options that are now available has resulted in a growing need to identify which patients will derive most benefit from which treatments. Much research is now focused on the identification of biomarkers that can be utilised to help select patients for treatment. These and other recent advances in the management of melanoma were the focus of discussions at the third Melanoma Bridge meeting (30 November-2 December, 2017, Naples, Italy), which is summarised in this report. [ABSTRACT FROM AUTHOR]

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+TIL subset and their CD8+BTLA−counterparts. We found that the CD8+BTLA+TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longerin vivoafter infusion. In contrast, CD8+BTLA−TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+T cells. These attributes may explain our previous observation that BTLA expression on CD8+TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma. [ABSTRACT FROM PUBLISHER]