Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment

Experimental cancer immunology and therap

Benchmarked performance charts using principal components analysis to improve the effectiveness of feedback for audit data in HIV care

Abstract Background Feedback tools for clinical audit data that compare site-specific results to average performance over all sites can be useful for quality improvement. Proposed tools should be simple and clearly benchmark the site’s performance, so that a relevant action plan can be directly implemented to improve patient care services. We aimed to develop such a tool in order to feedback data to UK HIV clinics participating in the 2015 British HIV Association (BHIVA) audit assessing compliance with the 2011 guidelines for routine investigation and monitoring of adult HIV-1- infected individuals. Methods HIV clinic sites were asked to provide data on a random sample of 50–100 adult patients attending for HIV care during 2014 and/or 2015 by completing a self-audit spreadsheet. Outcomes audited included the proportion of patients with recorded resistance testing, viral load monitoring, adherence assessment, medications, hepatitis testing, vaccination management, risk assessments, and sexual health screening. For each outcome we benchmarked the proportion for a specific site against the average performance. We produced performance charts for each site using boxplots for the outcomes. We also used the mean and differences from the mean performance to produce a dashboard for each site. We used principal components analysis to group correlated outcomes and simplify the dashboard. Results The 106 sites included in the study provided information on a total of 7768 patients. Outcomes capturing monitoring of treatment of HIV-infection showed high performance across the sites, whereas testing for hepatitis, and risk assessment for cardiovascular disease and smoking, management of flu vaccination, sexual health screening, and cervical cytology for women were very variable across sites. The principal components analysis reduced the original 12 outcomes to four factors that represented HIV care, hepatitis testing, other screening tests, and resistance testing. These provided simplified measures of adherence to guidelines which were presented as a 4 bar dashboard of performance. Conclusion Our dashboard performance charts provide easily digestible visual summaries of locally relevant audit data that are benchmarked against the overall mean and can be used to improve feedback to HIV services. Feedback from clinicians indicated that they found these charts acceptable and useful

A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens

Deficiencies in MHC class I antigen presentation are a common feature of tumors and allows escape from cytotoxic T lymphocyte (CTL)-mediated killing. It is crucial to take this capacity of tumors into account for the development of T-cell-based immunotherapy, as it may strongly impair their effectiveness. A variety of escape mechanisms has been described thus far, but progress in counteracting them is poor. Here we review a novel strategy to target malignancies with defects in the antigenic processing machinery (APM). The concept is based on a unique category of CD8+ T-cell epitopes that is associated with impaired peptide processing, which we named TEIPP. We characterized this alternative peptide repertoire emerging in MHC-I on tumors lacking classical antigen processing due to defects in the peptide transporter TAP (transporter associated with peptide processing). These TEIPPs exemplify interesting parallels with the folktale figure Cinderella: they are oppressed and neglected by a stepmother (like functional TAP prevents TEIPP presentation), until the suppression is released and Cinderella/TEIPP achieves unexpected recognition. TEIPP-specific CTLs and their cognate peptide-epitopes provide a new strategy to counteract immune evasion by APM defects and bear potential to targeting escape variants observed in a wide range of cancers

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials

Abstract B111: Vaccination against TAP downregulation-induced neoantigens to prevent future tumor development in the setting of recurrence or premalignancy

Abstract Development of therapeutic strategies to prevent recurrence in cancer patients, or tumor progression in individuals at high risk of developing cancer, has been challenging given the long and often unpredictable time to the emergence of the malignant tumors. Mutation-generated neoantigens represent the most potent antigens to induce antitumor immunity, yet the ability to predict which neoantigens will be expressed in future tumors is at present not an option.To overcome the limitations of targeting mutated neoantigens, we have developed a simple and broadly applicable approach to induce neoantigens in tumor cells in situ by reducing the expression of peptide transporter TAP, whereby a TAP-specific siRNA is targeted to tumor cells in mice by conjugation to a broad-range nucleolin-binding aptamer. Nucleolin, normally expressed in the cytoplasm and nucleolus of all somatic cells, is translocated to the cell surface of most murine and human tumors and hence could serve as an almost universal target to deliver therapeutics to tumors in vivo. Previous results have demonstrated that genetic ablation of TAP not only inhibits the canonical antigen processing pathway but also upregulates alternative pathways presenting new epitopes, essentially neoantigens, that can be recognized by the immune system to elicit effective CD8+ T-cell responses. Such epitopes are shared among all (tumor) cells in which TAP is downregulated, corresponding to the functional equivalent of clonal mutation-generated neoantigens. Our study shows that transiently increasing the neoantigen burden of tumor cells in situ by targeted downregulation of TAP represent a potent way of generating antitumor immunity in the absence of measurable toxicity.Exploiting the ability to induce neoantigens in situ, we are developing a novel vaccination strategy targeting potent neoantigens to control the growth of the future tumors—whereby mice in remission or with premalignant lesions are first vaccinated against TAP downregulation-induced neoantigens, and when or if tumor develops the same antigens are induced in the tumor, termed prorapeutic vaccination (prophylactic + therapeutic). To induce an immune response against TAP downregulation-induced neoantigens, mice were vaccinated with TAP siRNAs that are targeted to DC in situ by conjugation to a short CpG oligonucleotide leading to the downregulation of TAP, expression of neoantigens, as well as activation of the DC, and thereby priming of a potent T-cell response. To induce neoantigens in the future tumors, the TAP siRNA is targeted to the developing tumor lesion by conjugation to the nucleolin (Nucl) aptamer. Both Nucl and CpG targeted siRNA are administered systemically via intraperitoneal injection to reach the disseminated tumor lesions and resident DC present throughout the body. In murine models of recurrence and premalignant disease, prorapeutic vaccination elicited an adaptive and innate antitumor immune response and inhibited tumor growth. The prorapeutic vaccination approach was more effective than vaccinating against prototypic mutated neoantigens, and did not elicit measurable autoimmune toxicity, in stark contrast to a comparatively therapeutic dose of CTLA-4.The ability to vaccinate against experimentally induced neoantigens using a broadly applicable procedure with two chemically synthesized reagents, introduces a new paradigm in cancer immunotherapy of vaccinating against neoantigens induced in future tumors, to prevent recurrence in patients in remission or preventing tumor development in individuals at high risk of developing cancer. Citation Format: Greta Garrido Hidalgo, Brett Schrand, Ailem Rabasa, Agata Levay, Tal Gefen, Giri Bhuwan Bhuwan, Anthony R. Ferrantella, Vikas Dudeja, Koen Marijt, Thorbald T. van Hall, Eli Gilboa. Vaccination against TAP downregulation-induced neoantigens to prevent future tumor development in the setting of recurrence or premalignancy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B111

Correlation between <i>HLA-</i> gene expression and the expression of the genes encoding the HLA transcriptional regulators and peptide-loading machinery.

<p>Correlation between <i>HLA-</i> gene expression and the expression of the genes encoding the HLA transcriptional regulators and peptide-loading machinery.</p

Chromosomal aberrations and HLA expression.

<p>Comparison between chromosomal aberrations and the expression of HLA class I and II antigens in a set of 27 primary UM. Tumors are divided according to their chromosome 3 and 6 status (disomy or monosomy of chromosome 3, and disomy of chromosome 6 or gain of 6p). HLA gene expression was determined using an Illumina microarray (A) and protein expression by immunohistochemistry (B) in UM. Additionally, HLA gene expression was determined using qPCR, which served to validate the Illumina findings (C). Four data points of the qPCR that are outside the axis limits (> 11 and < 24) are not shown (HLA-A, D3D6p: 17; HLA-B, D3D6p: 24, and M3D6p: 12; B2M, D3D6p: 13). Only significant p-values are shown, all other comparisons between the groups were not significant (p-values not shown). Error-bars represent the interquartile range. Results were obtained using the Mann-Whitney U tests.</p