The functions of autophagy at the tumour-immune interface

Autophagy is frequently induced in the hypoxic tumour microenvironment. Accumulating evidence reveals important functions of autophagy at the tumour-immune interface. Herein, we propose an update on the roles of autophagy in modulating tumour immunity. Autophagy promotes adaptive resistance of established tumours to the cytotoxic effects of natural killer cells (NKs), macrophages and effector T cells. Increased autophagic flux in tumours dampen their immunogenicity and inhibits the expansion of cytotoxic T lymphocytes (CTLs) by suppressing the activation of STING type I interferon signalling (IFN-I) innate immune sensing pathway. Autophagy in suppressive tumour-infiltrating immune subsets maintains their survival through metabolic remodelling. On the other hand, autophagy is involved in the antigen processing and presentation process, which is essential for anti-tumour immune responses. Genetic deletion of autophagy induces spontaneous tumours in some models. Thus, the role of autophagy is context-dependent. In summary, our review has revealed the dichotomous roles of autophagy in modulating tumour immunity. Broad targeting of autophagy may not yield maximal benefits. The characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into certain tumours are among the most urgent tasks to sensitize cold cancers to immunotherapy.     

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a “push and pull” manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.     

Immunotherapy and immunoselection -- tumour escape as the final hurdle

Tumours are immunogenic and are commonly infiltrated by anti-cancer effector cells. Why, then, are they not completely rejected by the host? Unfortunately, tumours are Darwinian paragons, winning the battle against the forces of natural immune selection. Some of the latter can even act as double-edged swords, actually being subverted to become pro-tumorigenic. Prevention or reversal of tumour escape from the immune response therefore offers the possibility of reconstituting effective anti-tumour immunity and remains the major challenge for 21st century tumour immunology.     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Immune surveillance and natural resistance: an evaluation

Summary Concepts in tumour immunology are changing fundamentally.Around 1970 tumour immunology contained the following related concepts: 1. Thousands of tumour cells arise de novo each day. 2. Tumour cells are antigenic in their host. 3. All these antigenic tumour cells are killed by a strong immune surveillance system.A more likely set of concepts looks as follows: 1. Tumour cells do not arise frequently. 2. Tumour cells may be antigenic or not. 3. There is no need to postulate a very strong immune surveillance or natural resistance system.In this paper I am reviewing our present knowledge of immune surveillance and natural resistance. Only scanty information appears to be available. This information suggests that virally induced tumours are usually killed by cytotoxic T lymphocytes, and natural killer cells, whereas immune surveillance and natural resistance against other tumours may be quite weak.     

OP9-DL1 cell co-culture enhances anti-tumour immunity of mouse bone marrow-derived dendritic cells

DCs (dendritic cells) are the strongest professional APCs (antigen-presenting cells) to initiate immune responses against pathogens, but they are usually incompetent in initiating efficient immune responses in the progress of solid tumours. We have shown that Notch signalling plays a pivotal role in DC-dependent anti-tumour immunity. Compared with the control DCs, OP9-DL1 (Delta-like1) cell co-cultured DCs gained increased tumour suppression activity when inoculated together with tumour cells. This was probably due to the activation of Notch signalling in DCs enhancing their ability to evoke anti-tumour immune responses in solid tumours. Indeed, the OP9-DL1 cell co-cultured DCs expressed higher levels of MHC I, MHC II, CXCR4 (CXC chemokine receptor 4), CCR7 (CC chemokine receptor 7), IL-6 (interleukin 6), IL-12 and TNFα (tumour necrosis factor α), and a lower level of IL-10 than control DCs, resulting in more efficient DC migration and T-cell activation in vivo and in vitro. T-cells stimulated by OP9-DL1 cells co-cultured DCs more efficiently; and were cytotoxic against tumour cells, in contrast with control DCs. These results indicated that up-regulation of Notch signalling in DCs by co-culturing with OP9-DL1 cells enhances DC-dependent anti-tumour immune reactions, making the Notch signalling pathway a target for the establishment of the DC-based anti-tumour immunotherapies.     

Regulatory T cell: a protection for tumour cells

Characterized by immunosuppression regulatory T cells (Tregs) play a key role in maintaining immune tolerance. A growing number of tumours have been found with Tregs accumulating in microenvironment and patients with high density of Tregs in tumour stroma get a worse prognosis, which suggests that Tregs may inhibit anti-tumour immunity in stroma, resulting in a poor prognosis. In this paper, we demonstrate the accumulation of Tregs in tumour stroma and the possible suppressive mechanisms. We also state the immunotherapy that has being used in animal and clinical trials.     

Adjuvants and the promotion of Th1-type cytokines in tumour immunotherapy

Immunotherapy includes both active and passive mechanisms that have the potential to treat many tumour types. Whereas monoclonal antibodies may kill cells by merely binding to them, 'cancer vaccines' involve the induction of an active immune response. The activation of tumour antigen-specific T-helper and cytotoxic T lymphocytes or non-specific macrophages and natural killer (NK) cells using immunotherapeutic approaches may lead to the subsequent destruction of tumour tissue. Administration of a tumour antigen alone is often not sufficient to stimulate an appropriate immune response. However, incorporating an immunological adjuvant into a vaccine regime often improves anti-tumour immunity. There are various types of adjuvants used in immunotherapy, ranging from microbial, chemical, and cellular components to proteins and cytokines. Previous reports have demonstrated that the induction of Th1-promoting cytokines, using specific adjuvants, can enhance anti-tumour immunity and can reduce or even prevent tumour growth. There is also increasing evidence that many adjuvants induce Th1-type cytokines, which correlates with the induction anti-tumour immunity. Th1-type responses which comprise cell-mediated immunity are characterised by the secretion of interferon-gamma by T cells, which is induced by antigen-presenting cell (APC)-derived IL-12. This review describes immunoadjuvants that are currently undergoing preclinical investigation, and emerging clinical data revealing that adjuvants which induce Th1-type responses can improve the efficacy of cancer vaccines. Therefore, the use of Th1-inducing adjuvants may provide an essential strategy for the future success of immunotherapy.     

Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer

Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade.     

Toxoplasma gondii and MHC-restricted antigen presentation: on degradation, transport and modulation

Resistance against Toxoplasma gondii, an obligate intracellular protozoan parasite surrounded by a parasitophorous vacuolar membrane, is mediated by the cellular arm of the immune system, namely CD8+ and CD4+ T cells. Thus, priming and activation of these cells by presentation of antigenic peptides in the context of major histocompatibility complex class I and class II molecules have to take place. This is despite the fact that the vacuolar membrane avoids fusion with the endocytic compartment and acts like a molecular sieve, restricting passive diffusion of larger molecules. This raises several cell biological and immunological questions which will be discussed in this review in the context of our current knowledge about major histocompatibility complex-restricted antigen presentation in other systems: (1) By which pathways are parasite-derived antigens presented to T cells? (2) Has the parasite evolved mechanisms to interfere with major histocompatibility complex-restricted antigen presentation in order to avoid immune recognition? (3) To what extent and by which mechanism is antigenic material, originating from the parasite, able to pass through the vacuolar membrane into the cytosol of the infected cell and is it then accessible to the antigen presentation machinery of the infected cell? (4) What are the actual antigen-presenting cells which prime specific T cells in lymphoid organs? An understanding of these mechanisms will not only provide new insights into the pathogenesis of Toxoplasma gondii and possibly other intravacuolar parasites, but will also improve vaccination strategies.     

A novel tumour model system for the study of long-term protective immunity and immune T cell memory

We present a novel non-transgenic system to be used for studies on anti-tumour adoptive immunotherapy (ADI) and long-term T cell memory. Tumour-reactive donor immune cells against lacZ-transfected syngeneic tumour cells (ESbL-Gal) were generated from a naíve T cell repertoire in DBA/2 mice by a well-established priming/restimulation protocol, and transferred to tumour-inoculated athymic nu/nu mice. The donor immune cells efficiently mediated protective anti-tumour immunity involving both CD4(+) and CD8(+) T cells, and anti-metastatic effects were stronger in 4.5 Gy pre-irradiated than in non-irradiated tumour-inoculated hosts. Long-term persistence of beta-galactosidase (Gal)-specific T cells was shown ex vivo by tetramer staining of CD8(+) T cells specific for an immunodominant Gal epitope. Resistance of treated nu/nu mice against tumour rechallenge revealed the existence of long-term protective immune memory.     

Nitroreductase-based therapy of prostate cancer, enhanced by raising expression of heat shock protein 70, acts through increased anti-tumour immunity

Gene-directed enzyme-prodrug therapy (GDEPT) using nitroreductase (NTR), with efficient adenoviral delivery, and CB1954 (CB), is an effective means of directly killing tumours. However, an immune-mediated bystander effect remains an important product of GDEPT since it is often critical to the elimination of untransduced tumour cells both locally and at distal metastatic sites through generation of tumour-specific immunity without the need for tumour antigen identification or the generation of a personalised vaccine. The mode of induced tumour cell death is thought to contribute to the immunisation process, together with the induction and release of stress proteins. Here, RM-9 murine prostate tumour cells were efficiently killed by adenovirally delivered NTR/CB treatment both in vitro and in vivo, and bystander effects were observed. Cells appeared to die by pathways that suggest necrosis more than that of classical apoptosis. NTR/CB-induced expression of a range of stress proteins was determined by proteomic analysis, revealing chiefly heat shock protein (HSP)25 and HSP70 upregulation, whilst immune responses in vivo were weak. In an attempt to enhance the anti-tumour effect, an adenoviral vector was constructed that co-expressed NTR and HSP70, the latter being a known immune stimulator and chaperone of antigen. This combination elicited significantly enhanced protection over NTR alone for both the treated tumour and a subsequent re-challenge. Protection was CD4⁺ and CD8⁺ T cell-dependent and was associated with tumour-specific CTL, IFNγ and IL-5 responses. The use of such a cytotoxic and immunomodulatory gene combination in cancer therapy warrants further pursuit.     

Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX?

Background

NY-ESO-1 protein formulated in ISCOMATRIX? results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial.

Methods

Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252?C1,155?days (median?=?681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX? adjuvant at three protein doses 10???g, 30???g or 100???g (n?=?14); 100???g NY-ESO-1 protein (n?=?8) or placebo (n?=?6), together with 1???g of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2?days after DTH skin testing with NY-ESO-1 protein (1???g) or peptides (10???g). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17.

Results

Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX? adjuvant. In contrast, immunity only persisted in 3/14 who received 100???g un-adjuvanted NY-ESO-1 protein (3/8) or 2???g DTH protein (0/6) P?=?0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred.

Conclusion

Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX?. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.     

Oligoclonal T cells in human cancer

Many solid tumors are characterised by the infiltration of lymphocytes and their presence has been correlated with a more favourable prognosis. These tumor-infiltrating lymphocytes (TIL), have been shown to possess specific cytolytic reactivity towards autologous tumours, thus suggesting that tumour cells may express antigens capable of eliciting an immune response. Expression of such tumour-associated antigens (TAA) in combination with appropriate accessory signals would lead to thein vivo accumulation of T cells with anti-tumour specificity. Analysis of the composition of the specific T-cell receptor (TCR) of TIL could thus provide information on the nature of the antigen(s) recognised by TIL. In this review, different aspects of the presence of clonal T cells in patients with cancer are discussed.     

Modified peptides in anti-cancer vaccines: are we eventually improving anti-tumour immunity?

The discovery of tumour antigens recognized by T cells and the features of immune responses directed against them has paved the way to a multitude of clinical studies aimed at boosting anti-tumour T cell immunity as a therapeutic tool for cancer patients. One of the different strategies explored to ameliorate the immunogenicity of tumour antigens in vaccine protocols is represented by the use of optimized peptides or altered peptide ligands, whose amino acid sequence has been modified for improving HLA binding or TCR interaction with respect to native epitopes. However, despite the promising results achieved with preclinical studies, the clinical efficacy of this approach has not yet met the expectations. Although multiple reasons could explain the relative failure of altered peptide ligands as more effective cancer vaccines, the possibility that T cells primed by modified tumour peptides might may be unable to effectively cross-recognize tumour cells has not been sufficiently addressed. Indeed, the introduction of conservative amino acid substitutions may still produce diverse and unpredictable changes in the HLA/peptide interface, with consequent modifications of the TCR repertoire that can interact with the complex. This could lead to the expansion of a broad array of T cells whose TCRs may not necessarily react with equivalent affinity with the original antigenic epitope. Considering the results presently achieved with this vaccine approach, and the emerging availability of alternative strategies for boosting anti-tumour immunity, the use of modified tumour peptides could be reconsidered. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications”, Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Manipulation of tumour-infiltrating B cells and tertiary lymphoid structures: a novel anti-cancer treatment avenue?

Combining different standard therapies with immunotherapy for the treatment of solid tumours has proven to yield a greater clinical benefit than when each is applied separately; however, the percentage of complete responses is still far from optimal, and there is an urgent need for improved treatment modalities. The latest literature data suggest that tertiary lymphoid structures (TLS), previously shown to correlate with the severity of autoimmune diseases or transplant rejection, are also formed in tumours, have a significant beneficial effect on survival and might reflect the generation of an effective immune response in close proximity to the tumour. Thus, the facilitation of TLS formation in tumour stroma could provide novel means to improve the efficiency of immunotherapy and other standard therapies. However, little is known about the mechanisms regulating the formation of tumour-associated TLS. Studies of chronic inflammatory diseases and transplant rejection have demonstrated that TLS formation and/or function requires the presence of B cells. Additionally, the infiltration of B cells into the tumour stroma has been demonstrated to be a significant prognostic factor for improved survival in different human tumours. This suggests that B cells could play a beneficial role in anti-tumour immune response not only in the context of antibody production, antigen presentation and Th1-promoting cytokine production, but also TLS formation. This review focuses on the latest discoveries in tumour-infiltrating B cell functions, their role in TLS formation and relevance in human tumour control, revealing novel opportunities to improve cancer therapies.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Cancer testis antigen PRAME: An anti-cancer target with immunomodulatory potential

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.     

Cooperation between chemotherapy and immune checkpoint blockade to enhance anti-tumour T cell immunity in oesophageal adenocarcinoma

Response rates to immune checkpoint blockade (ICB) remain low in oesophageal adenocarcinoma (OAC). Combining ICB with immunostimulatory chemotherapies to boost response rates is an attractive approach for converting ‘cold’ tumours into ‘hot’ tumours. This study profiled immune checkpoint (IC) expression on circulating and tumour-infiltrating T cells in OAC patients and correlated these findings with clinical characteristics. The effect of first-line chemotherapy regimens (FLOT and CROSS) on anti-tumour T cell immunity was assessed to help guide design of ICB and chemotherapy combinations in the first-line setting. The ability of ICB to enhance lymphocyte-mediated cytolysis of OAC cells in the absence and presence of post-FLOT and post-CROSS chemotherapy tumour cell secretome was assessed by a CCK-8 assay. Expression of ICs on T cells positively correlated with higher grade tumours and a subsequent poor response to neoadjuvant treatment. First-line chemotherapy regimens substantially altered IC expression profiles of T cells increasing PD-1, A2aR, KLRG-1, PD-L1, PD-L2 and CD160 and decreasing TIM-3 and LAG-3. In addition, pro-inflammatory T cell cytokine profiles were enhanced by first-line chemotherapy regimens. T cell activation status was significantly altered; both chemotherapy regimens upregulated co-stimulatory markers ICOS and CD69 yet downregulated co-stimulatory marker CD27. However, ICB attenuated chemotherapy-induced downregulation of CD27 on T cells and promoted differentiation of effector memory T cells into a terminally differentiated state. Importantly, dual nivolumab-ipilimumab treatment increased lymphocyte-mediated cytolysis of OAC cells, an effect further enhanced in the presence of post-FLOT tumour cell secretome. These findings justify a rationale to administer ICBs concurrently with first-line chemotherapies.