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Cancer vaccination drives the generation of anti-tumor T cell immunity and can be enhanced by the inclusion of effective immune adjuvants such as type I interferons (IFNs). Whilst type I IFNs have been shown to promote cross-priming of T cells, the role of individual subtypes remains unclear. Here we systematically compared the capacity of distinct type I IFN subtypes to enhance T cell responses to a whole-cell vaccination strategy in a pre-clinical murine model. We show that vaccination in combination with IFNβ induces significantly greater expansion of tumor-specific CD8+T cells than the other type I IFN subtypes tested. Optimal expansion was dependent on the presence of XCR1+dendritic cells, CD4+T cells, and CD40/CD40L signaling. Therapeutically, vaccination with IFNβ delayed tumor progression when compared to vaccination without IFN. When vaccinated in combination with anti-PD-L1 checkpoint blockade therapy (CPB), the inclusion of IFNβ associated with more mice experiencing complete regression and a trend in increased overall survival. This work demonstrates the potent adjuvant activity of IFNβ, highlighting its potential to enhance cancer vaccination strategies alone and in combination with CPB.

Advances in cancer immunology have increased the use of immune checkpoint inhibitors in clinical practice, however not all patients respond, and treatment can have severe side-effects. Blood-based immunological biomarkers are an attractive method for predicting which patients will respond to therapy, however, reliable biomarkers for immune checkpoint blockade are lacking. This study aimed to identify patients before or early in treatment who would best respond to PD-1 inhibitors. We hypothesised that higher baseline PD-L1 and/or PD-1 on peripheral blood T cells could predict radiological response to PD-1 inhibitors. This pilot prospective cohort study assessed 26 patients with melanoma or non-small cell lung cancer, treated with pembrolizumab, nivolumab, or nivolumab/ipilimumab combined. Response was assessed by RECIST 1.1. Peripheral blood lymphocytes collected at baseline, after one cycle, 10 weeks and at discontinuation of therapy were analysed by flow cytometry. Patients with a higher proportion of PD-L1+ T cells at baseline had improved objective response to PD-1 inhibitor therapy, and patients with a lower proportion of regulatory T cells at baseline experienced more immune-related adverse events. These findings may prove useful to assist in clinical decision making. Further studies with larger cohorts are required to validate these findings.

Background: Single-agent cyclophosphamide can deplete regulatory T-cells (Treg). We aimed to determine optimal dosing and scheduling of oral cyclophosphamide, alongside pemetrexed-based chemotherap...

Immunotherapy has revolutionised the treatment of cancers by harnessing the power of the immune system to eradicate malignant tissue. However, it is well recognised that some cancers are highly resistant to these therapies, which is in part attributed to the immunosuppressive landscape of the tumour microenvironment (TME). The contexture of the TME is highly heterogeneous and contains a complex architecture of immune, stromal, vascular and tumour cells in addition to acellular components such as the extracellular matrix. While understanding the dynamics of the TME has been instrumental in predicting durable responses to immunotherapy and developing new treatment strategies, recent evidence challenges the fundamental paradigms of how tumours can effectively subvert immunosurveillance. Here, we discuss the various immunosuppressive features of the TME and how fine-tuning these mechanisms, rather than ablating them completely, may result in a more comprehensive and balanced anti-tumour response.

The use of dendritic cells (DCs) to generate effective anti-tumor T cell immunity has garnered much attention over the last thirty-plus years. Despite this, limited clinical benefit has been demonstrated thus far. There has been a revival of interest in DC-based treatment strategies following the remarkable patient responses observed with novel checkpoint blockade therapies, due to the potential for synergistic treatment. Cross-presenting DCs are recognized for their ability to prime CD8+ T cell responses to directly induce tumor death. Consequently, they are an attractive target for next-generation DC-based strategies. In this review, we define the universal classification system for cross-presenting DCs, and the vital role of this subset in mediating anti-tumor immunity. Furthermore, we will detail methods of targeting these DCs both ex vivo and in vivo to boost their function and drive effective anti-tumor responses.

The use of dendritic cells (DCs) to generate effective anti-tumor T cell immunity has garnered much attention over the last thirty-plus years. Despite this, limited clinical benefit has been demonstrated thus far. There has been a revival of interest in DC-based treatment strategies following the remarkable patient responses observed with novel checkpoint blockade therapies, due to the potential for synergistic treatment. Cross-presenting DCs are recognized for their ability to prime CD8+ T cell responses to directly induce tumor death. Consequently, they are an attractive target for next-generation DC-based strategies. In this review, we define the universal classification system for cross-presenting DCs, and the vital role of this subset in mediating anti-tumor immunity. Furthermore, we will detail methods of targeting these DCs both ex vivo and in vivo to boost their function and drive effective anti-tumor responses.

Combination chemotherapy is currently standard care for advanced mesothelioma. Checkpoint blockade is a promising new treatment.This review covers clinical use and biomarkers of checkpoint blockade. Medline search used keywords 'mesothelioma' combined with 'checkpoint blockade' OR 'PD-L1' OR 'PD1' OR 'anti-CTLA4'; the search terms AND 'clinical trial' or AND 'biomarker*' were added. Handsearching covered abstracts from relevant meetings from 2016 to 2018 and reference lists. Data informed a narrative review.Single agent anti-CTLA4 blockade is inactive in mesothelioma. Single agent PD-1 blockade as second or subsequent treatment gives 20-29% partial responses; no randomized comparisons against placebo or chemotherapy are available. Biomarkers of response have been difficult to identify. There is no consensus as to whether tumor PD-L1 expression predicts outcomes. Combination checkpoint inhibitors (CTLA4 and PD1 blockade) provide a small incremental increase in response rates and progression-free survival. Chemoimmunotherapy is the next frontier.

Objective Pleural effusion (PE) is a common feature of malignant pleural mesothelioma. These effusions typically contain lymphocytes and malignant cells. We postulated that the PE would be a source of lymphocytes for analysis of tumor immune milieu. The aim of this study was to compare the phenotype and T cell receptor usage of pleural effusion T cells with paired concurrently drawn peripheral blood lymphocytes. We used multi-parameter flow cytometry and high-throughput T cell receptor sequencing to analyse peripheral blood and pleural effusion mononuclear cells. Results Both CD8+ and CD4+ T cells from effusion showed increased expression of T cell inhibitory receptors PD-1, LAG-3 and Tim-3 compared to blood. Comprehensive T cell receptor sequencing on one of the patients showed a discordant distribution of clonotypes in the antigen-experienced (PD-1+) compartment between effusion and blood, suggesting an enrichment of antigen specific clonotypes in the effusion, with potential as an immunological response biomarker.

BACKGROUND Data from murine models suggest that CD40 activation may synergize with cytotoxic chemotherapy. We aimed to determine the maximum tolerated dose (MTD) and toxicity profile and to explore immunological biomarkers of the CD40-activating antibody CP-870,893 with cisplatin and pemetrexed in patients with malignant pleural mesothelioma (MPM). PATIENTS AND METHODS Eligible patients had confirmed MPM, ECOG performance status 0-1, and measurable disease. Patients received cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 and CP-870,893 on day 8 of a 21-day cycle for maximum 6 cycles with up to 6 subsequent cycles single-agent CP-870,893. Immune cell subset changes were examined weekly by flow cytometry. RESULTS Fifteen patients were treated at three dose levels. The MTD of CP-870,893 was 0.15 mg/kg, and was exceeded at 0.2 mg/kg with one grade 4 splenic infarction and one grade 3 confusion and hyponatraemia. Cytokine release syndrome (CRS) occurred in most patients (80%) following CP-870,893. Haematological toxicities were consistent with cisplatin and pemetrexed chemotherapy. Six partial responses (40%) and 9 stable disease (53%) as best response were observed. The median overall survival was 16.5 months; the median progression-free survival was 6.3 months. Three patients survived beyond 30 months. CD19+ B cells decreased over 6 cycles of chemoimmunotherapy (P < 0.001) with a concomitant increase in the proportion of CD27+ memory B cells (P < 0.001) and activated CD86+CD27+ memory B cells (P < 0.001), as an immunopharmacodynamic marker of CD40 activation. CONCLUSIONS CP-870,893 with cisplatin and pemetrexed is safe and tolerable at 0.15 mg/kg, although most patients experience CRS. While objective response rates are similar to chemotherapy alone, three patients achieved long-term survival. AUSTRALIA NEW ZEALAND CLINICAL TRIALS REGISTRY NUMBER ACTRN12609000294257.

Background CD40 signalling can synergise with chemotherapy in preclinical cancer models, and early clinical studies are promising. We set out to define the immunological changes associated with this therapeutic combination to identify biomarkers for a response to the therapy. Here, we present serial immunomonitoring examining dendritic cell and T cell subpopulations over sequential courses of chemoimmunotherapy. Methods Fifteen patients with mesothelioma received up to six 21-day cycles of pemetrexed plus cisplatin chemotherapy and anti-CD40 (CP-870,893). Peripheral blood was collected weekly, and analysed by flow cytometry. Longitudinal immunophenotyping data was analysed by linear mixed modelling, allowing for variation between patients. Exploratory analyses testing for any correlation between overall survival and immunophenotyping data were undertaken up to the third cycle of treatment. Results Large statistically significant cyclical variations in the proportions of BDCA-1+, BDCA-2+ and BDCA-3+ dendritic cells were observed, although all subsets returned to baseline levels after each cycle and no significant changes were observed between start and end of treatment. Expression levels of CD40 and HLA-DR on dendritic cells were also cyclically modulated, again without significant change between start and end of treatment. CD8 and CD4 T cell populations, along with regulatory T cells, effector T cells, and markers of proliferation and activation, showed similar patterns of statistically significant cyclical modulation in response to therapy without changes between start and end of treatment. Exploratory analysis of endpoints revealed that patients with a higher than average proportion of BDCA-2+ dendritic cells (p = 0.010) or a higher than average proportion of activated (ICOS+) CD8 T cells (0.022) in pretreatment blood samples had better overall survival. A higher than average proportion of BDCA-3+ dendritic cells was associated with poorer overall survival at both the second (p = 0.008) and third (p = 0.014) dose of anti-CD40. Conclusions Substantial cyclical variations in DC and T cell populations during sequential cycles of chemoimmunotherapy highlight the critical importance of timing of immunological biomarker assessments in interpretation of results and the value of linear mixed modelling in interpretation of longitudinal change over a full treatment course. Trial registration Australia New Zealand Clinical Trials Registry number ACTRN12609000294257 (18th May 2009).

Cross-presentation defines the unique capacity of an APC to present exogenous Ag via MHC class I molecules to CD8(+) T cells. DCs are specialized cross-presenting cells and as such have a critical role in antitumor immunity. DCs are routinely found within the tumor microenvironment, but their capacity for endogenous or therapeutically enhanced cross-presentation is not well characterized. In this study, we examined the tumor and lymph node DC cross-presentation of a nominal marker tumor Ag, HA, expressed by the murine mesothelioma tumor AB1-HA. We found that tumors were infiltrated by predominantly CD11b(+) DCs with a semimature phenotype that could not cross-present tumor Ag, and therefore, were unable to induce tumor-specific T-cell activation or proliferation. Although tumor-infiltrating DCs were able to take up, process, and cross-present exogenous cell-bound and soluble Ags, this was significantly impaired relative to lymph node DCs. Importantly, however, systemic chemotherapy using gemcitabine reversed the defect in Ag cross-presentation of tumor DCs. These data demonstrate that DC cross-presentation within the tumor microenvironment is defective, but can be reversed by chemotherapy. These results have important implications for anticancer therapy, particularly regarding the use of immunotherapy in conjunction with cytotoxic chemotherapy.

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.

One of the clear paradoxes in tumor immunology is the fact that cross-presentation of cell-associated tumor antigens to CD8(+) T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross-presenting DC using a solid tumor model expressing a membrane bound neo-antigen (hemagglutinin, HA). Tumor antigen was constitutively cross-presented in the tumor-draining LN throughout tumor progression by CD11c(+) DC. Further analysis revealed that both CD8alpha(+) and CD8alpha(-) DC subsets, but not plasmacytoid DC, were effective at cross-presenting HA tumor antigen. The proportions of DC subsets in the tumor-draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7-DC, was noted and appeared to be restricted to the CD8alpha(-) DC subset. Therefore LN resident CD8alpha(+) DC are not the sole DC subset capable of cross-presenting cell-associated tumor antigens. Migratory tumor DC subsets with altered co-stimulatory receptor expression may contribute to induction and regulation of tumor-specific responses.

Tumors have evolved multiple mechanisms to evade immune destruction. One of these is expression of T cell inhibitory ligands such as programmed death-ligand 1 (PD-L1; B7-H1). In this study, we show that PD-L1 is highly expressed on mesothelioma tumor cells and within the tumor stroma. However, PD-L1 blockade only marginally affected tumor growth and was associated with the emergence of activated programmed death-1+ ICOS+ CD4 T cells in tumor-draining lymph nodes, whereas few activated CD8 T cells were present. Full activation of antitumor CD8 T cells, characterized as programmed death-1+ ICOS+ Ki-67+ and displaying CTL activity, was only observed when CD4 T cells were depleted, suggesting that a population of suppressive CD4 T cells exists. ICOS+ foxp3+ regulatory T cells were found to be regulated through PD-L1, identifying one potentially suppressive CD4 T cell population. Thus, PD-L1 blockade activates antitumor CD8 T cell most potently in the absence of CD4 T cells. These findings have implications for the development of PD-L1-based therapies.

The immune effects of EGFR tyrosine kinase inhibitors (EGFR-TKIs) are poorly understood. Identifying immune biomarkers could guide patient selection and optimisation of EGFR-TKI-immunotherapy combinations.33 patients with NSCLC treated with an EGFR-TKI were prospectively enrolled. Peripheral blood mononuclear cells were collected pre-treatment, and after 1, 3 and 8 weeks. Flow cytometry was used to identify immune cell subsets, including PD-1 and PD-L1 expressing T cells. Immune parameters were correlated with clinical outcomes.Compared to healthy donors (n=10), patients had higher pre-treatment proportions of proliferating and PD-L1(+)CD3(+) T cells (p0.001). Compared to patients with an EGFR mutation (n=12), patients without a known mutation (n=21) had higher proportions of proliferating CD4(+) and PD-L1(+)CD3(+) T cells (p=0.03). There was a significant increase in PD-L1(+) T cells after 1 week of EGFR-TKI in patients whose disease progressed compared to non-progressors. Patients with higher PD-L1(+)CD3(+) T cells at 1-week were more likely to progress (OR 30.3, p0.01) and had shorter PFS (1.6 vs. 8.8m; p0.01) and OS (3.8 vs 23.2m; p0.001) than those with fewer PD-L1(+)CD3(+) T cells. On multivariate analysis, high PD-L1(+)CD3(+) T cells was the only independent predictor for PFS (HR 3.7, p=0.01), while for OS independent predictors were high PD-L1(+)CD3(+) T cells (HR 6.5, p0.01) and EGFR-negative status (HR 3.3, p=0.04).There was a significant correlation between PD-L1 expression on peripheral T cells and clinical outcomes in EGFR-TKI-treated NSCLC. This warrants further validation as a blood-based biomarker that may identify candidates for PD-1 inhibitors or immunotherapy-EGFR-TKI combinations.

The 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.

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.

The immune system plays an important role in the surveillance of neoplastic cells by eliminating them before they manifest as full-blown cancer. Despite this, tumors do develop in the presence of a functioning immune system. Conventional chemotherapy and its ability to directly kill tumor cells is one of the most effective weapons in the fight against cancer, however, increasing evidence suggests that the therapeutic efficacy of some cytotoxic drugs relies on their capacity to interact with the immune system. Killing of tumor cells in a manner that favors their capture by immune cells or selective targeting of immunosuppressive pathways by specific chemotherapies promotes the generation of an effective anti-cancer response; however, this alone is rarely sufficient to cause elimination of advanced disease. An understanding of the immunological events occurring in both animal models and patients undergoing chemotherapy will guide decisions for the development of appropriate combinations and scheduling for the integration of chemotherapy with immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that are critical for the generation of effective cytotoxic T lymphocyte (CTL) responses; however, their function and phenotype are often defective or altered in tumor-bearing hosts, which may limit their capacity to mount an effective tumor-specific CTL response. In particular, the manner in which exogenous tumor antigens are acquired, processed, and cross-presented to CD8 T cells by DCs in tumor-bearing hosts is not well understood, but may have a profound effect on antitumor immunity. In this paper, we have examined the role of DCs in the cross-presentation of tumor antigen in terms of their subset, function, migration, and location with the intention of examining the early processes that contribute to the development of an ineffective anti-tumor immune response.

Cross presentation defines the unique capacity of an antigen presenting cell (APC) to present exogenous antigen via MHC class I molecules to CD8+ T cells. This process serves a critical role in host anti-tumor immunity. Dendritic cells (DC), specifically the CD8α+ subset, are specialised cross-presenting cells; however there is little understanding of how different DC subsets contribute to anti-tumour immunity. In this study we have examined the cross-presentation of a marker tumour antigen (transfected HA) expressed by the murine AB1-HA cell line. We have found that tumor antigen is constitutively cross-presented in the draining lymph node throughout disease progression and that both CD8α+ and CD8α - DC subsets are responsible for the observed cross-presentation of HA antigen to CD8+ T cells. Interestingly, tumor-infiltrating DCs fail to cross present, though they are not deficient in tumor antigen uptake or processing. Importantly, a tumor apoptosis-inducing false nucleotide agent, gemcitabine, which primes for anti-tumor immunity, also reverses the defect in antigen cross presentation of tumor DCs. These data suggest that lymph node resident CD8α+ DC are not the sole DC subset capable of cross-priming against tumor antigens and that local cross-presentation within tumors ie. TCR restimulation of tumor-infiltrating CD8 T-cells may be essential for effective anti-tumor immunity. These data have important implications for tumor immunotherapy particularly the use of immunotherapy in conjunction with tumor apoptosis-inducing therapy.