MHC Antigen Presentation Research Articles

Abstract 1217: Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma

Abstract Introduction: Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in ~25% of cases. Targeted therapy strategies are currently lacking for NRAS-mutant melanoma. Here, we characterized the anti-tumor activity and immune effects of RMC-7977, a RASMULTI(ON) preclinical tool compound, representative of the investigational agent RMC-6236, in preclinical mouse and human models of NRAS-mutant melanoma. Methods: The in vitro potency of RMC-7977 was determined using mouse and human NRAS-mutant melanoma cell lines. Recovery of signaling was quantified by Western Blot and receptor tyrosine kinase (RTK) arrays. In vivo anti-tumor activity of RMC-7977 was determined in two syngeneic mouse melanoma models (SW1 NRASG13D and NRAS#5 NRASQ61R), and tumor immune infiltrate quantified using multiplexed IHC and flow cytometry. Mechanisms of resistance were defined using RNA-seq. Results: RMC-7977 had good activity in vitro against human cancer cell lines and in mouse models of NRAS-mutant melanoma. Time-course studies demonstrated RMC-7977 rapidly inhibited phospho-ERK signaling, which then began to recover after 8 hr of treatment. There was also evidence of adaptive receptor tyrosine kinase signaling by 48 hr. In syngeneic mouse models of NRAS-mutant melanoma, RMC-7977 led to a rapid inhibition of tumor growth, increased expression of tumor MHC class I and PD-L1 and an increase in CD4+ and CD8+ T cell infiltrate into tumors. Therapeutic escape was associated with decreased CD4+ and CD8+ T cell infiltration and increased numbers of immunosuppressive MDSCs. Cells collected from tumors resistant to RMC-7977 in vivo did not show any shift in sensitivity to the inhibitor when re-challenged in vitro, suggesting that the resistance observed in vivo resulted from mechanisms specific to the tumor microenvironment, for example adaptive RTK signaling or immune escape. RNA-seq and IHC analysis suggested that resistance was associated with decreased MHC expression and antigen presentation, as well as reduced co-stimulation of T cells. Combination studies of RMC-7977 with anti-PD-1 are ongoing to identify potential synergistic interactions in these preclinical models. Conclusion: RMC-7977 has anti-tumor activity in syngeneic mouse models of NRAS-mutant melanoma, associated with a robust immune response. Citation Format: Larissa A. Carvalho, Manali S. Phadke, Keiran S. M. Smalley. Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1217.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

MR1 antigen presentation to MAIT cells and other MR1-restricted T cells.

MHC antigen presentation plays a fundamental role in adaptive and semi-invariant T cell immunity. Distinct MHC molecules bind antigens that differ in chemical structure, origin and location and present them to specialized T cells. MHC class I-related protein1 (MR1) presents a range of small molecule antigens to MR1-restricted T (MR1T) lymphocytes. The best studied MR1 ligands are derived from microbial metabolism and are recognized by a major class of MR1T cells known as mucosal-associated invariant T (MAIT) cells. Here, we describe the MR1 antigen presentation pathway: the known types of antigens presented by MR1, the location where MR1-antigen complexes form, the route followed by the complexes to the cell surface, the mechanisms involved in termination of MR1 antigen presentation and the accessory cellular proteins that comprise the MR1 antigen presentation machinery. The current road map of the MR1 antigen presentation pathway reveals potential strategies for therapeutic manipulation of MR1T cell function and provides a foundation for further studies that will lead to a deeper understanding of MR1-mediated immunity.

Interim analysis of a phase II study of nivolumab/ipilimumab plus cabozantinib in patients with unresectable advanced melanoma.

9563 Background: Nivolumab/ipilimumab (Nivo/Ipi) is a standard therapy in patients with unresectable stage III/IV melanoma, demonstrating an objective response rate (ORR) of 58% and 12-month progression free survival (PFS) rate of 49% [Wolchok J, NEJM 2017]. c-MET/VEGFR2 inhibition with cabozantinib (Cabo) has modest clinical activity in melanoma and can augment antitumor immunity by increasing CD8+ Tcell and macrophage tumor infiltration, decreasing intratumoral regulatory Tcell levels, and increasing tumor cell MHC antigen presentation. Nivo/Ipi plus Cabo showed clinical activity and acceptable toxicity profile in GU malignancies [Apolo AB, JCO 2020]. We performed a study of Nivo/Ipi plus Cabo in advanced melanoma patients aimed at improving efficacy. Methods: We conducted a multicenter phase II study of induction Nivo 3mg/kg IV and Ipi 1mg/kg IV and Cabo 40mg PO daily every 3 weeks for 4 cycles, followed by Nivo 480mg IV plus Cabo 40mg PO daily every 4 weeks for up to 2 years, until disease progression or unacceptable toxicity/patient withdrawal. Patients with unresectable stage III/IV melanoma and no prior anti-PD-1 or CTLA-4 exposure (unless in adjuvant setting > 6 months since last dose) were included. Uveal melanoma excluded. Primary endpoint was 12-month PFS rate. Secondary endpoints included ORR, overall survival (OS), and adverse events (AEs). Interim analysis was preplanned after first 14 subjects were evaluated in a Simon 2 stage design (goal >8 subjects 12-month progression free). Results: As of 1/2023, 14 subjects were enrolled across three cancer centers within the Georgetown-Lombardi Comprehensive Cancer Center Consortium. Median age 66.5 years, all stage IV disease, 10 with cutaneous primary site, 5 with elevated LDH, and 6 with BRAF V600 mutant tumor. Median follow up was 10.6 mos. 12-month PFS rate was 30% (3/10 evaluable subjects); 3 subjects pending PFS evaluation; median PFS 10.3 mos. ORR was 46% (6/13 evaluable subjects) with 2 CR, 4 PR, and 1 SD. Responders had cutaneous (5) or unknown (1) primary sites. 9 patients were alive at last follow up. 12-month OS rate was 67% (6/9 evaluable subjects). Reasons for treatment discontinuation included AEs (4), patient withdrawal (1), and disease progression (6). 3 subjects remain on study drug(s). Treatment related AEs (TRAEs) were observed in 13 subjects; 9 experienced a grade 3-4 TRAE (most frequent were elevated AST/ALT 29% and hypokalemia 14%). Most frequent grade 2 TRAEs were diarrhea (35%) and palmar-planter-erythrodysesthesia (29%). No treatment related deaths occurred. Conclusions: Clinical activity was observed with Nivo/Ipi plus Cabo in advanced melanoma but did not meet the predefined threshold to advance to the second stage of enrollment. TRAE profile was similar to prior reports. Biomarker analyses are planned to identify patient profiles associated with particular benefit from this regimen. Clinical trial information: NCT04091750 .

Abstract 3963: Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinomas are one of the deadliest cancer types with limited response to current therapies. Thus, this study examined changes in the proteome of epithelial cells in pancreatic cancer after neoadjuvant chemotherapy with gemcitabine, to identify new therapeutic targets. Methods: Upon receiving patient consent, fresh pancreatic cancer tissue was received from surgical tumor resections at the University Hospital of Bonn. We mechanically and enzymatically digested and purified the tissue to create single-cell suspensions. After staining the cells against CD45 und CD326, we purified epithelial cells, blood cells, and stroma through FACS. To analyze their proteome, the sorted cells were lysed in TEAB buffer and the protein lysates were measured by the mass spectrometry core facility at the University of Bonn. We then analyzed the data using DAVID and Shiny GO. Results: Gene enrichment analyses showed a significantly reduced expression of calreticulin after neoadjuvant chemotherapy in epithelial cells. A direct comparison between untreated and treated epithelium cancer cells using a heat map showed a decreased expression of actin and enzymes of glycolysis in treated cancer cells. Moreover, untreated epithelial cells showed an increase of proteins related to MHC class Ib protein complex assembly and antigen processing and presentation of endogenous peptide antigen via MHC class. In contrast, treated cells showed a higher presence of proteins for nutrient supply from the blood. Furthermore, untreated stroma cells exhibited an increased number of proteins responsible for mesenchymal migration and for actin filament fragmentation. Conclusion: This study shows that neoadjuvant chemotherapy with gemcitabine leads to distinct changes in the proteome of tumor cells in pancreatic cancers. In particular, a decreased expression of the “eat-me-signal” calreticulin impairs macrophage-mediated cancer cell phagocytosis, thereby indicating a mechanism by which cancer cells protect themselves after chemotherapy. Citation Format: Michael C. Stoffel, Elisabeth BH Dingendorf, Dahlia L. Kittel, Glen Kristiansen, Tristan Lerbs. Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3963.

Abstract WMP112: Type 2 Diabetes Dampens Neuroprotection Against Ischemic Injury By Lipopolysaccharide-Induced Preconditioning

Introduction: Evidence suggests that lipopolysaccharide ( LPS ) preconditioning (PC) provides neuroprotection by inducing tolerance to subsequent lethal ischemia. Ischemic tolerance (IT) declines with age, yet it is unclear whether metabolic disease like type 2 diabetes (T2DM) also interfere with IT. The current study sought to determine whether T2DM reduces neuroprotection induced by LPS PC, and the molecular mechanisms underlying the dampened IT. Methods: Male and female diabetic db/db and control db/+ mice (8-10 months of age) were i.p. injected with Toll-like receptor (TLR)4 ligand LPS at 0.2 mg/kg or saline. Stroke was induced 3 days later by permanent distal occlusion of middle cerebral artery and temporary occlusion of ipsilateral common carotid artery for 60 min. Sensory and motor function was evaluated 1 day after stroke by a combination of tests. Infarct size was determined 3 days after stroke following TTC staining. The effect of LPS PC on the phenotypes of leukocytes in the brain parenchyma after stroke was quantified by Fluorescent activated cell sorter (FACS) 2 days after stroke with a panel of markers for subsets of myeloid cells. Transcriptome was determined by RNAseq of isolated monocytes from peripheral blood mononuclear cells. Differentially expressed genes were examined for distinct biological pathways via enrichment analysis. Results: LPS PC significantly reduced infarct in db/+ mice by one third and improved neurological scores, but it did not protect the db/db mice from stroke. FACS data showed that LPS PC reduced the number of Ly6G+ cells in db/+ but not db/db brains, suggesting that LPS PC curbed stroke-induced infiltration of neutrophils in the brain parenchyma. Pathway analysis suggests that diabetic mice exhibited down-regulated pathways in interferon signaling. The expression of Jak-Stat signaling (such as Stat1), MHC antigen presentation (H2A family), and Toll-like receptor (TLR) signaling (such as Irf7) were also reduced in diabetic mice after stroke. Conclusions: The dampened IT in the diabetic mice induced by LPS-PC is associated with increased neutrophil infiltration after stroke compared to control mice. Ongoing studies will seek toovercome the underlying defective molecular mechanisms in T2DM.

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.

Longitudinal saliva omics responses to immune perturbation: a case study

Saliva omics has immense potential for non-invasive diagnostics, including monitoring very young or elderly populations, or individuals in remote locations. In this study, multiple saliva omics from an individual were monitored over three periods (100 timepoints) involving: (1) hourly sampling over 24 h without intervention, (2) hourly sampling over 24 h including immune system activation using the standard 23-valent pneumococcal polysaccharide vaccine, (3) daily sampling for 33 days profiling the post-vaccination response. At each timepoint total saliva transcriptome and proteome, and small RNA from salivary extracellular vesicles were profiled, including mRNA, miRNA, piRNA and bacterial RNA. The two 24-h periods were used in a paired analysis to remove daily variation and reveal vaccination responses. Over 18,000 omics longitudinal series had statistically significant temporal trends compared to a healthy baseline. Various immune response and regulation pathways were activated following vaccination, including interferon and cytokine signaling, and MHC antigen presentation. Immune response timeframes were concordant with innate and adaptive immunity development, and coincided with vaccination and reported fever. Overall, mRNA results appeared more specific and sensitive (timewise) to vaccination compared to other omics. The results suggest saliva omics can be consistently assessed for non-invasive personalized monitoring and immune response diagnostics.

Hepatic transcriptome study of Taenia asiatica infection in suckling pigs

Taenia asiatica is a crucial Taenia that is prevalent in East and Southeast Asia. Domestic pigs and wild boars are essential intermediate hosts for Taenia. Cysticercus larvae are mainly parasitic in the liver of domestic pigs. The Taenia asiatica was collected from Liangmu Township, Duyun City, Guizhou Province. Twelve Yorkshire Suckling pigs of 20 days of age were randomly divided into an experimental and control group of 6 pigs each. RNA sequencing (RNA-seq) technology was used to detect the expression differences of the mRNA transcriptomes in the liver of the experimental and control group at different infection times. Differential genes were analyzed by bioinformatics and verified by Real Time-PCR(RT-PCR). On the 15th and 75th days after infection, 152 and 558 differentially expressed genes were detected in the liver of the experimental group, respectively, accounting for 0.85% and 3.12% of all identified transcribed RNA genes, respectively. Through GO and KEGG related bioinformatics analysis, it was found that these differentially expressed genes are involved in the immune response, material metabolism, fibrosis, and tissue proliferation and repair of suckling pig liver, and related to MHC antigen processing and presentation, cytochrome P450, transforming growth factor-beta (TGF-β) signaling pathway and so on. Cysticercus asiatica parasites cause significant differential gene expression in the liver of suckling pigs. Specific differentially expressed genes are involved in biological processes such as liver metabolism, immune response, and tissue repair or regeneration in suckling pigs. The immune evasion is related to the immuno-suppressive response of the intermediate host.

Abstract 4566: Performance of neoantigen prediction for the design of TG4050, a patient specific neoantigen cancer vaccine

Abstract The development of therapeutic cancer vaccines to immunize against tumor antigens constitutes a promising modality. Mutation associated antigens are considered major targets given their specificity to tumor cells. These mutations are specific to the patients and require a tailor-made vaccine targeting mutations identified in each tumor. Many mutations are identified in the tumoral genome in most patients, but only a small fraction (around 1%) is suitable as vaccine target. Herein, we report data documenting the prediction performance of the algorithm used for the design of TG4050, a clinical stage patient specific viral-based neoantigen vaccine. We have trained a set of independent machine learning algorithms to score each candidate neoantigen for several steps of the MHC antigen presentation pathway, including MHC binding, intracellular processing, similarity to self, and likelihood to elicit a T-cell response in peptide stimulated ELISPOT. Further, we have developed a novel graph neural network to combine all these scores to predict the likelihood that a neoantigen will elicit a T-cell response while also incorporating patient-specific factors, such as expression level and conservation of the mutation across different clones. To validate the system, we collected samples from 6 patients diagnosed with NSCLC, sequenced healthy and tumor tissue, identified mutations and ranked them using our algorithm; then, to evaluate immunogenicity, we focused our analysis on CD8+ T cell and measured the frequency of IFN γ+ cells against predicted peptides in autologous PBMC. Immunogenicity of peptides was assayed in 5 pools then deconvoluted against individual peptides. From 3339 to 4782 somatic variants were detected in tumor tissue samples. After applying technical filtering, removing synonymous mutations, and filtering on transcript expression we detected a median of 281 (192-471) expressed tumor mutations resulting in a median of 2767 candidate class I epitopes (1769 - 4573). The model resulted in high accuracy allowing us to identify peptides with pre-existing ex vivo immunogenic responses in 5 out of 6 patients. Immunogenicity of peptide pools was correlated with ranking by the algorithm. Immunogenicity of the 6 top ranking individual epitopes in each patient showed a median of 5 (2-6) immunogenic peptides resulting in a 77% of true positive rate (TP). It should be noted that when no response was detected, it cannot be excluded that a response could be primed by a vaccine. In a similar setting, the netMHC 4.0 algorithm yielded a TP of 30% and only identified 39% of positive calls of our algorithm. We demonstrate that the prediction algorithm is accurate in identifying immunogenic cancer mutations even among a large set of candidates. Ongoing TG4050 clinical studies (NCT03839524 and NCT04183166) will allow further validation of the antitumor activity of the elicited immune response. Citation Format: Brandon Malone, Caroline Tosch, Benoit Grellier, Kousuke Onoue, Timo Sztyler, Karola Rittner, Yoshiko Yamashita, Eric Quemeneur, Kaidre Bendjama. Performance of neoantigen prediction for the design of TG4050, a patient specific neoantigen cancer vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4566.

A proposed treatment for pathogenic enveloped viruses having high rates of mutation or replication.

Several enveloped viruses, particularly some RNA viruses, have high rates of mutation or replication, which can make them virulent pathogens in humans and other mammals. A proposed treatment could use synthesized proteins to mask pathogenic viral surface proteins to quickly induce an immune attack on specific enveloped viruses by using existing immune cells. One treatment could inject dual‐protein ligand masks into patients' bloodstreams to mask pathogenic surface proteins used to infect mammalian cells. The mammalian immune system already uses an analogous, more complex structure called a pentraxin to neutralize some pathogens by connecting their surface proteins to immune cells. And several types of antiviral peptides have already experimentally demonstrated effectiveness in blocking various viral pathogen infections. These treatments offer advantages, especially for currently untreatable viral pathogens. Furthermore, using dual‐protein ligands and the antigenic memory of some sub‐populations of NK cells would also allow the creation of defacto vaccines based on a host's NK cells, instead of vaccines utilizing CD4 and CD8 α:β T cells, which are limited by the requirement of MHC presentation of the target antigens to α:β T cells. Targeted NK cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having pathogen downregulated MHC antigen presentation. Eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen vs host dominance are also listed, which should be applicable to viral and non‐viral pathogens.

PD-L1/L2 protein levels rapidly increase on monocytes via trogocytosis from tumor cells in classical Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL)-characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells-tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called "trogocytosis" from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.

Abstract MS1-1: Immunotherapy in breast cancer: enhancing response to checkpoint blockade

Abstract Historically, breast tumors have been categorized as non-immunogenic in part due to a paucity of tumor infiltrating lymphocytes (TIL) when compared to other histologies. Multiple studies have now demonstrated an endogenous immune response against breast cancer with tumors having TIL present in the tumor and surrounding stroma. The extent of TIL infiltrate is dictated by the subtype of disease being highest in triple negative breast cancer (TNBC) and lowest in hormone receptor positive, HER2 negative (HR+/HER2-) disease. Recent studies have shown that immunotherapy, in the form of immune checkpoint blockade, can be effective in treating breast cancer patients. Specifically, the IMpassion 130 trial, a phase III study that enrolled over 900 patients with metastatic TNBC and randomized them to the immune checkpoint blockade agent atezolizumab plus nab-paclitaxel versus placebo plus nab-paclitaxel, demonstrated benefit to the addition of immunotherapy with respect to progression free survival as well as overall survival; a difference that was greater in patients with PD-L1 positive disease. Based on these data, the combination of atezolizumab plus nab-paclitaxel was approved for use in patients with PD-L1+ metastatic TNBC. More recently, data from a large phase III trial evaluating the use of the immune checkpoint blockade agent pembrolizumab in the neoadjuvant setting for TNBC patients showed a significant improvement in the rates of pathologic complete response. Based in part on these successes, there is significant enthusiasm for using immunotherapy to treat breast cancer patients. It must be recognized however, that a minority of patients respond. Additional work must therefore be done to expand the number of patients with TNBC who respond as well as to identify strategies for successfully employing immunotherapy for HER2+ and HR+ positive breast cancer. Development of these strategies requires understanding of the cancer immunity cycle and how it can be altered to enhance endogenous immune responses to breast cancer as well as improved understanding of the immune aspects of the tumor microenvironment and how standard of care therapies alter that microenvironment. As an example, there is emerging data showing that PARP inhibition leads to cytoplasmic DNA which activates the STING pathway leading to increased expression of release of type 1 interferons, increased MHC expression and antigen presentation and increased infiltration of effector T cells. Ongoing trials are therefore evaluating those agents in combination with immunotherapy. Our lab has shown that PARP inhibition further modifies the tumor microenvironment suggesting additional therapeutic targets. In addition, there is mounting evidence regarding potential mechanisms behind why some subtypes of breast cancer mount a robust immune response while others remain immunologically “cold”. Differences in antigen processing and presentation likely contribute and understanding how HR+ tumors “hide” from immune recognition could contribute to developing strategies to make these tumors susceptible to immunotherapy. Finally, our group has recently published data demonstrating a difference in susceptibility to immune checkpoint blockade based on age suggesting that a patient’s systemic immune status could predict therapeutic response and potentially be manipulated to enhance that response. Citation Format: E Mittendorf. Immunotherapy in breast cancer: enhancing response to checkpoint blockade [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr MS1-1.

NNAlign_MA; MHC Peptidome Deconvolution for Accurate MHC Binding Motif Characterization and Improved T-cell Epitope Predictions

The set of peptides presented on a cell's surface by MHC molecules is known as the immunopeptidome. Current mass spectrometry technologies allow for identification of large peptidomes, and studies have proven these data to be a rich source of information for learning the rules of MHC-mediated antigen presentation. Immunopeptidomes are usually poly-specific, containing multiple sequence motifs matching the MHC molecules expressed in the system under investigation. Motif deconvolution -the process of associating each ligand to its presenting MHC molecule(s)- is therefore a critical and challenging step in the analysis of MS-eluted MHC ligand data. Here, we describe NNAlign_MA, a computational method designed to address this challenge and fully benefit from large, poly-specific data sets of MS-eluted ligands. NNAlign_MA simultaneously performs the tasks of (1) clustering peptides into individual specificities; (2) automatic annotation of each cluster to an MHC molecule; and (3) training of a prediction model covering all MHCs present in the training set. NNAlign_MA was benchmarked on large and diverse data sets, covering class I and class II data. In all cases, the method was demonstrated to outperform state-of-the-art methods, effectively expanding the coverage of alleles for which accurate predictions can be made, resulting in improved identification of both eluted ligands and T-cell epitopes. Given its high flexibility and ease of use, we expect NNAlign_MA to serve as an effective tool to increase our understanding of the rules of MHC antigen presentation and guide the development of novel T-cell-based therapeutics.

MON-218 Inflammatory Role of Sex Steroids in Hidradenitis Suppurativa: An Androgenic Phenotype

Hidradenitis Suppurativa (HS) is an autoinflammatory skin disease more common in women and associated with hyper-androgenic disorders such as polycystic ovary syndrome. HS is characterized by chronic recurrent deep-seated painful nodules and draining tunnels in skin areas that follow the distribution of pubertal terminal hair growth. In women, HS disease activity varies with hormonal fluctuation across the menstrual cycle and pregnancy. Anti-androgens have shown promise in treatment of HS, but the pathophysiologic link between sex hormones and HS is unclear. We sought to further elucidate the role of sex hormones in the pathogenesis of HS. Objective: To determine if there is differential estrogen and androgen regulated gene expression in lesional compared to non-lesional skin transcriptome in HS patients. Methods: (a) We analyzed mRNA microarray data from lesional and non-lesional HS skin (GEO, GSE72702) (Blok JL, Br J Dermatol. 2016;174(6):1392). Inflammatory lesional skin from affected axilla or groin and non-lesional skin from the upper arm or leg was obtained from 17 patients with moderate to severe HS. We then examined differential expression of hormone regulated genes identified in existing androgen and estrogen gene expression signatures from prostate cancer cells [(VCaP treated with dihydrotestosterone (DHT)] and breast cancer cells [MCF7 treated with 17 beta-estradiol, (E2)] in the lesional and non-lesional skin transcriptomes of the HS patients. We identified overlap between the sets of genes that were either both upregulated or both repressed in HS lesions and the hormone-responsive prostate and breast cancer cell line models. Significance of differential gene expression was defined as FDR-adjusted p-value for two-tailed Student t-test less than 0.05. (b) After overlapping genes were identified, we used GeneGo Metacore pathway analysis software to explore the functional significance of the identified genes. Results: (a) Of the 3379 E2-upregulated probe sets in MCF7 breast cancer cells, 647 were significantly upregulated and 584 of 2871 E2-downregulated probe sets were significantly downregulated in HS lesions. Of the 1328 DHT-upregulated probe sets in prostate cancer cells, 341 were significantly upregulated and 196 of the 1251 DHT-downregulated probe sets were significantly downregulated in HS lesions. (b) Initial pathway analysis indicates that DHT upregulated genes in HS lesions are strongly enriched in innate immunity pathways, specifically, type 1 interferon signaling and class I MHC antigen presentation. Conclusion: There is substantial overlap between the transcriptional programme of sex steroids and transcriptional changes observed in HS lesional skin. In particular, androgen target genes are significantly enriched in innate immunity pathways, suggesting a functional link between the androgen signaling and the inflammatory process in HS lesions.

Modulating T Cell Responses via Autophagy: The Intrinsic Influence Controlling the Function of Both Antigen-Presenting Cells and T Cells

Autophagy is a homeostatic and inducible process affecting multiple aspects of the immune system. This intrinsic cellular process is involved in MHC-antigen (Ag) presentation, inflammatory signaling, cytokine regulation, and cellular metabolism. In the context of T cell responses, autophagy has an influential hand in dictating responses to self and non-self by controlling extrinsic factors (e.g., MHC-Ag, cytokine production) in antigen-presenting cells (APC) and intrinsic factors (e.g., cell signaling, survival, cytokine production, and metabolism) in T cells. These attributes make autophagy an attractive therapeutic target to modulate T cell responses. In this review, we examine the impact autophagy has on T cell responses by modulating multiple aspects of APC function; the importance of autophagy in the activation, differentiation and homeostasis of T cells; and discuss how the modulation of autophagy could influence T cell responses.

RIP2 Is a Critical Regulator for NLRs Signaling and MHC Antigen Presentation but Not for MAPK and PI3K/Akt Pathways.

RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as “Antigen processing and presentation” and “NOD-like receptor signaling pathway” and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.

Transcriptomic responses of water buffalo liver to infection with the digenetic fluke Fasciola gigantica

BackgroundFasciola gigantica, the tropical liver fluke, infects buffaloes in Asian and African countries and causes significant economic losses and poses public health threat in these countries. However, little is known of the transcriptional response of buffaloes to infection with F. gigantica. The objective of the present study was to perform the first transcriptomic analysis of buffalo liver infected by F. gigantica. Understanding the mechanisms that underpin F. gigantica infection in buffaloes will contribute to our ability to control this parasite.MethodsWe challenged buffaloes with 500 viable F. gigantica metacercariae and collected liver samples through a time course at 3, 42 and 70 days post-infection (dpi). Then, we performed gene expression analysis on liver samples using RNA sequencing (RNA-Seq) Illumina technology and confirmed the RNA-Seq data by quantitative RT-PCR analysis.ResultsTotals of 496, 880 and 441 differentially expressed transcripts were identified in the infected livers at 3, 42 and 70 dpi, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that transcriptional changes in the liver of infected buffaloes evolve over the course of infection. The predominant response of buffaloes to infection was mediated by certain pathways, such as MHC antigen processing and presentation, Toll-like receptor 4 (TLR4), transforming growth factor beta (TGF-β), and the cytochrome P450. Hepatic drug metabolizing enzymes and bile secretion were also affected.ConclusionsFasciola gigantica can induce statistically significant and biologically plausible differences in the hepatic gene expression of infected buffaloes. These findings provide new insights into the response of buffaloes to F. gigantica over the course of infection, which may be useful in determining pathways that can modulate host-parasite interaction and thus potentially important for clearance of the parasite.

Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli.

Bovine mastitis is a widespread disease in dairy cows, and is often caused by bacterial mammary gland infection. Mastitis causes reduced milk production and leads to excessive use of antibiotics. We present meta-analysis of transcriptional profiles of bovine mastitis from 10 studies and 307 microarrays, allowing identification of much larger sets of affected genes than any individual study. Combining multiple studies provides insight into the molecular effects of Escherichia coli infection in vivo and uncovers differences between the consequences of E. coli vs. Staphylococcus aureus infection of primary mammary epithelial cells (PMECs). In udders, live E. coli elicits inflammatory and immune defenses through numerous cytokines and chemokines. Importantly, E. coli infection causes downregulation of genes encoding lipid biosynthesis enzymes that are involved in milk production. Additionally, host metabolism is generally suppressed. Finally, defensins and bacteria-recognition genes are upregulated, while the expression of the extracellular matrix protein transcripts is silenced. In PMECs, heat-inactivated E. coli elicits expression of ribosomal, cytoskeletal and angiogenic signaling genes, and causes suppression of the cell cycle and energy production genes. We hypothesize that heat-inactivated E. coli may have prophylactic effects against mastitis. Heat-inactivated S. aureus promotes stronger inflammatory and immune defenses than E. coli. Lipopolysaccharide by itself induces MHC antigen presentation components, an effect not seen in response to E. coli bacteria. These results provide the basis for strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics.

Identification of potential immunotherapeutic targets in antigen presentation and costimulation networks

Meeting abstracts Anti-tumor immune responses are often hampered by an excess of coinhibition of T cells, but also by a lack of MHC antigen presentation and costimulation. The latter are not yet successfully exploited as targets for immunotherapy, while augmenting MHC and costimulatory molecules