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The therapeutic response to immune checkpoint blockade (ICB) is highly variable, not only between different cancers but also between patients with the same cancer type. The biological mechanisms underlying these differences in response are incompletely understood. Identifying correlates in patient tumor samples is challenging because of genetic and environmental variability. Murine studies usually compare different tumor models or treatments, introducing potential confounding variables. This protocol describes bilateral murine tumor models, derived from syngeneic cancer cell lines, that display a symmetrical yet dichotomous response to ICB. These models enable detailed analysis of whole tumors in a highly homogeneous background, combined with knowledge of the therapeutic outcome within a few weeks, and could potentially be used for mechanistic studies using other (immuno-)therapies. We discuss key considerations and describe how to use two cell lines as fully optimized models. We discuss experimental details, including proper inoculation technique to achieve symmetry and one-sided surgical tumor removal, which takes only 5 min per mouse. Furthermore, we outline the preparation of bulk tissue or single-cell suspensions for downstream analyses such as bulk RNA-seq, immunohistochemistry, single-cell RNA-seq and flow cytometry.

Recently, there has been a coordinated effort from academic institutions and the pharmaceutical industry to identify biomarkers that can predict responses to immune checkpoint blockade in cancer. Several biomarkers have been identified; however, none has reliably predicted response in a sufficiently rigorous manner for routine use. Here, we argue that the therapeutic response to immune checkpoint blockade is a critical state transition of a complex system. Such systems are highly sensitive to initial conditions, and critical transitions are notoriously difficult to predict far in advance. Nevertheless, warning signals can be detected closer to the tipping point. Advances in mathematics and network biology are starting to make it possible to identify such warning signals. We propose that these dynamic biomarkers could prove to be useful in distinguishing responding from non-responding patients, as well as facilitate the identification of new therapeutic targets for combination therapy.

Malignant pleural mesothelioma (MPM) is a rare cancer with a heterogeneous prognosis. Prognostic models are not widely utilized clinically. Classification and regression tree (CART) analysis examines the interaction of multiple variables with a given outcome.

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

Malignant pleural mesothelioma is an aggressive, treatment-resistant tumor, which continues to increase in frequency throughout the world because the causative agent, asbestos, has high economic importance, particularly in developing countries. Patients typically present with breathlessness and chest pain with pleural effusions. Median survival is around 12 months from diagnosis. Palliative chemotherapy is beneficial for mesothelioma patients with high performance status. The role of aggressive surgery remains controversial. This review will outline some of the recent advances in the development of novel therapies for malignant pleural mesothelioma, with a focus on immunological approaches.

SUMMARY Given the impressive efficacy of immune checkpoint blockade in thoracic malignancies, and the recently discovered immune-stimulating properties of many cytotoxic drugs, a logical next step would be to combine these treatments. However, the rational design of clinical trials investigating these combinations is hampered by a lack of knowledge regarding the overall immunogenic effects of the different chemotherapeutics. Here, we give an overview of the field with regard to checkpoint blockade and the immunological effects of cytotoxic chemotherapeutics, with particular focus on preclinical and clinical studies investigating the combination of these two treatment modalities. We discuss the hurdles that need to be overcome in order to optimally exploit chemotherapy and immune checkpoint blockade combinations in thoracic cancers.

SUMMARY Mouse models of cancer are invaluable for obtaining detailed knowledge about tumor development and for screening therapeutic and preventive approaches. Mesothelioma is an unusual cancer because the same carcinogen, asbestos, causes a similar disease in both humans and animals. Unlike most other cancers, murine mesothelioma can therefore be regarded as a disease homolog, rather than a model as such. However, because asbestos-induced cancer has low penetrance and a long lag time, most translational studies have utilized more efficient models such as tumor transplantation. In consequence, many promising results have not translated into positive findings in patients. Here, we describe the widely used murine mesothelioma models and critically discuss their relative advantages and disadvantages. We emphasize the use of the appropriate model for the specific research question and the need to use multiple models in order to obtain robust and translatable data.

An incomplete understanding on the effect of surgery on tumor-specific immunity continues to hamper efforts to combine surgery with immunotherapy in the clinic. Herein, we describe the impact of tumor resection on the tumor-specific T-cell response, showing that complete tumor resection is associated with (1) a decline in the amount of cross-presented tumor antigens, (2) a decline of cytolytic tumor-specific CD8+T cell activity, and (3) the development of systemic CD8+T cell-mediated protective immunity. Our findings are consistent with a model whereby tumor resection releases antitumor CD8+T cells from chronic antigen exposure, allowing a gradual differentiation toward functional antitumor memory T cells. This process depends on sentinel lymph nodes, as their removal at the time of surgery was associated with a strong negative effect on survival. We conclude that complete tumor resection provides a unique environment that boosts protective immunological memory and might provide a powerful platform for immunotherapy. Our findings also carry important implications for the design and timing of post-surgery immunotherapeutic regimens.

It is widely accepted that generation of tumor specific CD8+T-cell responses occur via cross-priming; however the source of tumor antigen for this event is unknown. We examined the source and form of tumor antigen required for cross-presentation in the local lymph node (LN) using a syngeneic mouse tumor model expressing a marker antigen. We found that cross-presentation of this model tumor antigen in the LN is dependent on continuous traffic of antigen from the tumor site, but without any detectable migration of tumor resident dendritic cells (DCs). Instead, small numbers of tumor cells metastasize to local LNs where they are exposed to a localized CTL attack, resulting in delivery of tumor antigen into the cross-presentation pathway.

Abstract Analysis of the crystal structure of human class II (HLA-DR1) molecules suggests that the aß heterodimer may be further ordered as a dimer of heterodimers (superdimer), leading to the hypothesis that T cell receptor dimerisation is a mechanism for initiating signaling events preceding T cell activation. The interface between pairs of molecules is stabilised by both salt bridges, polar and hydrophobic interactions. The residues that form the superdimer interface occur in three areas distinct from the antigen-binding groove. They can be defined as follows: region 1, ß-ß contacts in the helix of the ß1 domain; region 2, a-a contacts near the a1/a2 domain junction and region 3; a-ß contacts in the a2/ß2 domains adjacent to the plasma membrane. To determine whether salt bridges and polar interactions formed within these regions are involved in the immune function of the murine MHC class II molecule, I-Ab, appropriate residues in both the a and ß chain were identified and mutated to uncharged alanine. Cell lines transfected with different combinations of mutated a and ß chains were generated and tested for MHC class II expression, peptide binding capabilities, and ability to present antigenic peptide to an OVA-specific T cell hybridoma. With the exception of two residues in region 2, the substitutions tested did not modulate MHC class II expression, or peptide binding function. When tested for ability to present peptide to an antigen-specific T cell hybridoma, with the exception of mutations in region 2, the substitutions did not appear to abrogate the ability of I-Ab to stimulate the T cells. These results suggest that mutation of residues in region 2 of the putative superdimer interface have a gross effect on the ability of I-Ab to be expressed on the cell surface. However, abrogation of salt bridges in region 1 and 3 do not influence I-Ab cell surface expression, peptide binding or ability to stimulate antigen-specific T cells.

Mesothelin is a cell-surface antigen of unknown function that is expressed in a highly tissue-specific manner. Expression of the protein is limited to mesothelium, the tissue forming the pleural, pericardial, and peritoneal membranes. The purpose of this study was to characterize the gene and identify elements responsible for its transcription. The gene contains an 1884-bp open reading frame encoded by 15 exons occupying 8 kb of human chromosome 16. An 1850-bp region of genomic DNA at the 5′ end of the gene encompassing the proposed transcriptional start site was cloned. This region lacks a TATA box and other regulatory elements such as SP1 sites, which are commonly found in promoters. Transient transfection analyses demonstrated that mesothelium-specific control elements are present within the 1.85-kb region. Minimal constitutive promoter elements were localized to a 317-bp region. Tissue-specific enhancer elements upstream of the minimal promoter were found to activate transcription from the homologous and a heterologous promoter in a position- and orientation-independent manner.

This review describes several in vitromodels of anergy in both murine and human CD4+T cells, relating the role of lymphokine production to the induction and maintenance of tolerance. The division of CD4+T cells into Th1 and Th2 sub-populations and the capacity of Th2 lymphokines to abrogate the response of T cells belonging to the Th1 subset are discussed. The model of nonresponsiveness is described in terms of phenotypic modulation, responses to IL2, requirement for co-stimulatory molecules, and lymphokine profiles. The various methods for measuring lymphokines using bioassays, immunoassays, or molecular techniques are compared and critiqued.

A mixed antiglobulin reaction (MAR test), using antibody-coated red cells and spermatozoa, has been used to detect the presence of either lgA or IgG on the surface of the spermatozoa. Group O Rh-positive red cells were sensitized with serum containing anti-D, which was partly IgA, though mainly IgG. Spermatozoa were added to the sensitized red cells and tested either with anti-IgA or anti-IgG. MAR (IgA) test results were negative or doubtful in 42 (44%) of 94 samples with positive or strongly positive MAR (IgG) tests. Positive MAR (IgA) tests showed a highly significant correlation with the presence of antisperm antibodies in seminal plasma. Significantly impaired sperm penetration of cervical mucus was demonstrated for 15 patients, 12 of whom had positive MAR (IgA) tests, whereas good sperm penetration was observed for 5 patients with negative or dubious MAR (IgA) tests; all 20 patients had strongly positive MAR (IgG) tests and positive serum antisperm antibody tests.

This paper presents an analytical and graphical methodology, developed as part of a detailed feasibility study of railway transport of two products (crude oil and liquid natural gas) from two Arctic locations (Prudhoe Bay, Alaska, and the Mackenzie Delta, Northwest Territories) to a single southern destination, for determining the range of product and origin tariff levels under conditions of joint and common costs. Sample tariff ranges, computed to preclude cross-subsidization and to permit a realistic comparison of the railway with other transport modes are presented. The applicability of the methodology to more general circumstances is discussed.

A strong T cell-specific enhancer is located 3′ to the human CD2gene. Six sequences within this enhancer are bound by proteins present in T cell nuclear extracts. These sequences share homology with sequences bound by several transcription factors involved in T cell- and lymphoid-specific transcription. The results presented here demonstrate that the human T cell-specific transcription factor, SOX4, is able to bind to one of these regions; further, SOX4 transactivates transcription of a reporter gene via three tandem copies of this sequence. The binding of SOX4 to this site is not via a canonical HMG protein binding sequence, identifying a novel class of binding site for this protein. A second sequence within the CD2enhancer closely resembles the IL-2NF-AT site. We show that it is bound by the ets-related factor, Elf1. However, unlike the IL-2NF-AT sequence, the CD2NF-AT-like sequence is unable to confer transcriptional inducibility on a reporter gene. Consistent with this result, we show that the observed increase in expression of CD2 protein on the cell surface following T cell activation is a post-transcriptional event.

MATTERS GERMANE TO TESTINGBefore I deal with the true substance matter of this paper, there are a few points to which I would direct attention, and foremost is that most interesting phenomenon of nature—sound. Not only is every human voice different; as an external configuration, family characteristics occur, so often in voice the same applies. One can tell by sound what variety of musical instrument is being played, and frequently distinguish the up from the down stroke note on a violin. In this instance there is a variation in the timbre of a note, due to a physical alteration in the string from which the sound is produced. The string is of twisted gut, the up stroke going in the direction of the twist tends to tighten the string, the down stroke on the other hand, tends to loosen or undo the twist. But the difference in the

ABSTRACTDexamethasone is a synthetic glucocorticoid commonly used for the prevention and management of side effects in cancer patients undergoing chemotherapy. While it is effective as an anti-emetic and in preventing hypersensitivity reactions, dexamethasone depletes peripheral blood lymphocytes and impacts immune responses. The effect of dexamethasone on the number and quality of tumour-infiltrating leukocytes has not been reported. To address this, we calibrated the dose in two different strains of mice to achieve the same extent of peripheral blood lymphocyte depletion observed in patients with cancer. Doses that caused analogous depletion of T and B lymphocytes and NK cells from the peripheral blood, elicited no change in these populations within the tumour. The expression of immune checkpoint molecules PD-1, OX40, GITR and TIM3 on tumour-infiltrating lymphocytes was not altered. We found that dexamethasone had a small but significant deleterious impact on weakly efficacious chemoimmunotherapy but had no effect when the protocol was highly efficacious. Based on these results, we predict that dexamethasone will have a modest negative influence on the overall effectiveness of chemoimmunotherapy treatment.

A STAT1-driven inflammatory phenotype associated with response to checkpoint blocking antibodies sensitizes cancers to immunotherapy.

ABSTRACTMesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated. Here, we used our well characterised mesothelioma tumour model to investigate the efficacy of different ICBP treatments to generate effective therapy for mesothelioma. We show that tumour resident regulatory T cell co-express high levels of CTLA-4, OX40 and GITR relative to T effector subsets and that these receptors are co-expressed on a large proportion of cells. Targeting any of CTLA-4, OX40 or GITR individually generated effective responses against mesothelioma. Furthermore, the combination of αCTLA-4 and αOX40 was synergistic, with an increase in complete tumour regressions from 20% to 80%. Other combinations did not synergise to enhance treatment outcomes. Finally, an early pattern in T cell response was predictive of response, with activation status and ICP receptor expression profile of T effector cells harvested from tumour and dLN correlating with response to immunotherapy. Taken together, these data demonstrate that combination ICPB can work synergistically to induce strong, durable immunity against mesothelioma in an animal model.

ABSTRACTThe glucocorticoid (GC) steroid dexamethasone (Dex) is used as a supportive care co-medication for cancer patients undergoing standard care pemetrexed/platinum doublet chemotherapy. As trials for new cancer immunotherapy treatments increase in prevalence, it is important to track the immunological changes induced by co-medications commonly used in the clinic, but not specifically included in trial design or in pre-clinical models. Here, we document a number of Dex -induced immunological effects, including a large-scale lymphodepletive effect particularly affecting CD4+T cells but also CD8+T cells. The proportion of regulatory T cells within the CD4+compartment did not change after Dex was administered, however a significant increase in proliferation and activation of regulatory T cells was observed. We also noted Dex -induced proportional changes in dendritic cell (DC) subtypes. We discuss these immunological effects in the context of chemoimmunotherapy strategies, and suggest a number of considerations to be taken into account when designing future studies where Dex and other GCs may be in use.

A key to improving cancer immunotherapy will be the identification of tumor-specific “neoantigens” that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

Tumor antigen cross-presentation by dendritic cells (DCs) to specific CD8+T cells is central to antitumor immunity. Although highly efficient in draining lymph nodes, it is defective within the tumor site itself. Importantly, an immunogenic chemotherapy, gemcitabine, reverses this defect, allowing the potential re-stimulation of cytotoxic T lymphocytes within tumor sites.

Molecular and cellular events that govern a disease resolution may pinpoint new druggable pathways.