Immune-modulating Therapies Research Articles

Toward a Functional Cure for Hepatitis B.

Current treatment of chronic hepatitis B virus (HBV) infection, pegylated interferon-α (pegIFN-α) and nucleos(t)ide analogue (NA), can suppress HBV replication, reverse liver inflammation and fibrosis, and decrease risks of cirrhosis and hepatocellular carcinoma, but hepatitis B surface antigen (HBsAg) loss is rare. Functional HBV cure is defined as undetectable HBsAg and unquantifiable serum HBV DNA for at least 24 weeks after a finite course of therapy. This requires suppression of HBV replication and viral protein production as well as restoration of immune response to HBV. Direct-acting antivirals targeting virus entry, capsid assembly, viral protein production and secretion are in clinical trials. In parallel, immune modulatory therapies to stimulate HBV-specific immune response and to remove immune blockade are being tested. Clinical trials of direct-acting antivirals alone or immune modulatory therapies alone have not been successful in achieving HBV cure. Recent combinations of direct-acting antivirals and immune modulatory therapies have shown promising results particularly with combinations that included pegIFN-α. These results need to be confirmed in larger studies with longer follow-up, and further work is needed to develop simpler regimens with fewer drugs that can be administered orally and safely. While there is a strong desire to develop finite therapies that can achieve HBV cure, safety is paramount and new therapies must provide incremental value compared to standard of care, which is predominantly long-term NA therapy.

Unraveling the immunological landscape in acute pancreatitis progression to sepsis: insights from a Mendelian randomization study on immune cell traits.

Acute pancreatitis (AP) is a severe digestive system disorder with a significant risk of progressing to sepsis, a major cause of mortality. Unraveling the immunological pathways in AP is essential for developing effective treatments, particularly understanding the role of specific immune cell traits in this progression. Employing a bidirectional two-sample Mendelian Randomization (MR) approach, this study first examined the causal relationship between AP and 731 immune cell traits to identify those significantly associated with AP. Subsequently, we explored the causal associations between 731 immune cell traits and sepsis. The analysis utilized extensive genome-wide association studies (GWAS) summary datasets, with a focus on identifying common immune cell traits with statistically significant causal associations between AP and sepsis. Our investigation identified 44 immune cell traits unidirectionally associated with AP and 36 traits unidirectionally associated with sepsis. Among these, CD127 on CD28+ CD45RA- CD8+ T cells emerged as a common mediator, accounting for 5.296% of the increased risk of sepsis in AP patients. This finding highlights the significant role of specific memory CD8+ T cells in the pathophysiology of AP and its progression to sepsis. This study elucidates the critical role of specific immune cell traits, particularly CD127hi memory CD8+ T cells, in the progression of AP to sepsis. Our findings provide a foundation for future research into targeted immune-modulatory therapies, potentially improving patient outcomes in AP-related sepsis and offering new insights into the complex immunological dynamics of this condition.

Abstract 5248: Novel costimulatory domain improves CAR-monocyte activity against tumors

Abstract Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, myeloid cells (particularly M1 macrophages) are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 (GPC3) to treat hepatocellular carcinoma (HCC). The CAR serves a dual purpose as a homing ‘GPS’ signal for trafficking directly to the tumor site, and for directing phagocytosis specifically at targeted tumor cells. We have designed a proprietary CAR molecule (M83 CAR) which contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83 CAR-M cells are not inhibited by the prevalent CD47 checkpoint (‘do not eat me’ signal expressed on tumor cells), which is known to restrict myeloid function in the TME. Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages (M1) enables robust tumor cell phagocytosis, the central mechanism that can result in the following series of events: 1) proinflammatory shift in the TME milieu, 2) recruitment of innate and adaptive immune cells, 3) activation of T-cells against tumor neo-antigens. This is a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Citation Format: Kevin Carbajal, Warren Anderson, Kilsoo Jeon, Grace Reynolds, Prasanna Ekambaram, Beatrice Breckheimer, Anastasya Birger, Jessica Pafford, Matthew Esquivel, Adam Pecoraro, Sumiti Jain. Novel costimulatory domain improves CAR-monocyte activity against tumors [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 5248.

Abstract 5738: Repurposing disulfiram for cancer: A drug delivery and population-based approach

Abstract Purpose: Disulfiram was approved by the FDA for treatment of alcohol abuse in 1951, although for over 40 years there has been evidence supporting disulfiram’s use as an anti-cancer drug. Upon administration, disulfiram is hydrolyzed to form two molecules of DDC, which upon binding Cu forms Cu(DDC)2, the proposed active form against cancer. It was our goal to formulate the water-insoluble Cu(DDC)2 in liposomes to facilitate its repurposing for breast and colon cancers. In addition, we also initiated an ongoing population-based study assessing the protective and treatment effects of disulfiram with cancer. Methods: Cu(DDC)2 liposomes were generated by the thin-film hydration method followed by extrusion, using DSPC and cholesterol (55:45 molar ratio). DDC and CuSO4 were added to the liposomes forming Cu(DDC)2, which was transported inside the liposomes. Anti-tumor activity was evaluated in tumor-bearing mice with or without competent immune systems, and immune system activation was assessed via a cancer vaccination model and the expression of markers of immunogenic cell death (ICD). A study was also initiated for cancer cases and controls in British Columbia (BC Cancer) with at least one prescription of disulfiram from 1996-2022 (PharmaNET), patients treated with immune-modulating therapies (BC Cancer), and outcome of death (Vital Statistics). Results: Liposomes encapsulating Cu(DDC)2 had a size of 100 nm and polydispersity of 0.1, and caused a 40% reduction in tumor burden in a MDA-MB-231 xenograft model. In a syngeneic tumor model of breast cancer the response with immune-competent mice was greater than mice lacking immune function, and a cancer vaccination model and the presence of ATP, HMBG1 and surface-exposed calreticulin indicated the presence of ICD. Conclusions: Cu(DDC)2 was formulated in liposomes facilitating its parenteral administration, and its anti-cancer activity was found to be due in part to activation of the adaptive immune system through ICD. A case-control study is currently being employed to examine the clinical potential of disulfiram, as a protectant and as an adjuvant with immune-activating therapies such as checkpoint inhibitors and anthracyclines to potentially improve efficacy through induction of ICD. Citation Format: Devon Heroux, Ada W. Leung, Roger Gilabert-Oriol, Marcel B. Bally. Repurposing disulfiram for cancer: A drug delivery and population-based approach [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 5738.

Abstract 4256: Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development

Abstract Recent advances in cancer immunotherapy have had a positive impact on the life expectancy of patients for an extensive range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. We developed an innovative alternative, building on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient-derived organoids are generated from normal and malignant tissues and stored as high-quality biobanks that yield highly reproducible results. HUB Organoids® recapitulate complex characteristics of the parental tissue, including molecular heterogeneity, as well as morphological and functional traits. Since cancer progression and response to immunotherapy are governed by immune cell interactions in the tumor microenvironment, we developed an assay in which colorectal (CRC) and non-small cell lung cancer (NSCLC) tumor organoids are co-cultured with paired tumor infiltrating lymphocytes (TIL) that are simultaneously expanded as a source of tumor reactive cells. Paired resources allow for the screening of immune-modulatory therapies under physiologic conditions, not depending on peptide-pulsing and alloreactivity. We quantify tumor organoid killing and TILs reactivity by combining imaging-based analysis of apoptotic cells with flow cytometry to measure cytokine release by cytotoxic TILs. Here, we show that our system is robust and reproducible with an establishment rate of 75%, allowing for expansion of TILs that preserve T cell receptor repertoires. First line expansion of both PDO and TIL components can be used to effectively build coculture models and probe the tumor and immune components, respectively. Our system offers a powerful tool for the development and validation of cancer immunotherapy and can help to stratify cancer patients for susceptibility to various types of immunotherapies. We certify that the research use of each of the HBS marked above was conducted according to the expectations of each institution. Citation Format: Claudia Beaurivage, BanuPriya Sridharan, Sumeyra Mucuk, Kimberly Nadwodny, Derek Poore, Anna Schepers, Farzin Pourfarzad, Sylvia F. Boj. Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development [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 4256.

Multi-scale geometric network analysis identifies melanoma immunotherapy response gene modules

Melanoma response to immune-modulating therapy remains incompletely characterized at the molecular level. In this study, we assess melanoma immunotherapy response using a multi-scale network approach to identify gene modules with coordinated gene expression in response to treatment. Using gene expression data of melanoma before and after treatment with nivolumab, we modeled gene expression changes in a correlation network and measured a key network geometric property, dynamic Ollivier-Ricci curvature, to distinguish critical edges within the network and reveal multi-scale treatment-response gene communities. Analysis identified six distinct gene modules corresponding to sets of genes interacting in response to immunotherapy. One module alone, overlapping with the nuclear factor kappa-B pathway (NFkB), was associated with improved patient survival and a positive clinical response to immunotherapy. This analysis demonstrates the usefulness of dynamic Ollivier-Ricci curvature as a general method for identifying information-sharing gene modules in cancer.

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

Sepsis in elderly patients: the role of neutrophils in pathophysiology and therapy.

Sepsis is among the most important causes of mortality, particularly within the elderly population. Sepsis prevalence is on the rise due to different factors, including increasing average population age and the concomitant rise in the prevalence of frailty and chronic morbidities. Recent investigations have unveiled a "trimodal" trajectory for sepsis-related mortality, with the ultimate zenith occurring from 60 to 90days until several years after the original insult. This prolonged temporal course ostensibly emanates from the sustained perturbation of immune responses, persevering beyond the phase of clinical convalescence. This phenomenon is particularly associated with the aging immune system, characterized by a broad dysregulation commonly known as "inflammaging." Inflammaging associates with a chronic low-grade activation of the innate immune system preventing an appropriate response to infective agents. Notably, during the initial phases of sepsis, neutrophils-essential in combating pathogens-may exhibit compromised activity. Paradoxically, an overly zealous neutrophilic reaction has been observed to underlie multi-organ dysfunction during the later stages of sepsis. Given this scenario, discovering treatments that can enhance neutrophil activity during the early phases of sepsis while curbing their overactivity in the later phases could prove beneficial in fighting pathogens and reducing the detrimental effects caused by an overactive immune system. This narrative review delves into the potential key role of neutrophils in the pathological process of sepsis, focusing on how the aging process impacts their functions, and highlighting possible targets for developing immune-modulatory therapies. Additionally, the review includes tables that outline the principal potential targets for immunomodulating agents.

P109 Organic Cation Transporter (OCTN)-1 variants shape innate immunity and predict individual response to vedolizumab In ulcerative colitis patients

Abstract Background Limited evidence is available about predictors of response to specific medications for ulcerative colitis (UC). OCTN1, an organic cation transporter, with its variants, could modulate the inflammatory response and potentially modify drug response to immune-modulatory therapy (anti-TNFα and vedolizumab), perhaps predicting individual response. Methods We first confirmed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their clinical features at baseline and clinical response to therapy or disease remission (according to clinical Mayo score) after six weeks (T1) and six months (T2). We finally enrolled a cohort of UC patients in clinical and endoscopic remission (according to full Mayo score) under immune-modulatory therapy (anti-TNFα or vedolizumab) for at least two years. Results OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, 50 starting anti-TNFα and 40 vedolizumab. Patients with 503F genotype showed higher rates of pancolitis as compared to 503L genotype. Among patients with 503L genotype, vedolizumab results highly associated with long-term response (OR 8.92, 95%CI 0.50-11.0; p=0.019), whilst conversely co-presence of 503F genotype and vedolizumab therapy disclosed an opposite role on the long-term response rate (OR 0.02, 95%CI 0.00-0.81; p=0.038).The population of UC patients in clinical and endoscopic remission was composed by 92 patients, 63 under anti-TNFα and 29 under vedolizumab. Of note, similar trends of disease extension were observed across genotypes: higher rates of pancolitis were observed in the 503F population as compared to 503L (62.5% vs 44%). Furthermore, among patients in remission with 503F genotype, the percentage of subjects receiving vedolizumab was sensibly lower than among patients with 503L genotype (87.5% anti-TNFα vs12.5% vedolizumab among 503F genotype; 56% anti-TNFα vs 44% vedolizumab among 503L genotype). Conclusion Mutated OCTN1 genotype (503F) favors an increased IL1β secretion from human stimulated leukocytes, thus suggesting a worse disease course with higher rate of pancolitis and lower response to vedolizumab as compared to 503L.

Abstract C013: Analysis of tumor immune microenvironment and clinical outcomes in undifferentiated pancreatic carcinomas with and without osteoclast-like giant cells

Abstract Background: Undifferentiated carcinoma (UC) is a rare (<1%) subtype of pancreatic cancer that was revised in 2010 per WHO classification as UC with or without osteoclast-like giant cells (OGC), affecting interpretation of prior literature comparing UC to pancreatic ductal adenocarcinomas (PDAC). Limited retrospective data suggest UC-OGC occurs at younger age with larger tumor size but the impact on prognosis is unclear, and there no data on the tumor immune microenvironment (TIME). Methods: The aim of the study was to contrast the differences in targeted genomics, TIME and survival between UC and UC-OGC. TIME was assessed via H&E, bulk RNA sequencing (RNA-seq), and multiplex immunofluorescence (mIF). Results: We identified 47 adult consecutive patients with UC (n=32) and UC-OGC (n=15) treated at Michigan Medicine between 2005 and 2021. The demographics, presenting symptoms, tumor size, and CA 19-9 were similar (p>0.05) between the two cohorts. In patients with advanced stage, median overall survival (OS) was 2.1 months (n=19) for UC vs 9.0 months (n=2) for UC-OGC (p=0.42). Thirteen patients from each cohort underwent resection, with median OS 1.84 years vs 11.92 years in UC vs UC-OGC, respectively (p=0.08). Median OS for all patients with UC (n=32) was 0.40 years compared to 10.82 years in UC-OGC (n=15) (p=0.003). The genomic landscape was similar in UC (n=9), UC-OGC (n=5) and PDAC (TCGA; n=159). KRAS and TP53 mutations were the most common alterations identified along with CDKN2A and SMAD4. RNA-seq demonstrated few differentially expressed genes between UC (n=7) and UC-OGC (n=3). Deconvolution with CIBERTSORT-X using the LM22 dataset demonstrated significantly increased antigen-presenting cells (APCs), specifically M2 macrophages, decreased cytotoxic T-cells, and decreased regulatory T cells in UC and UC-OGC tumors compared to PDAC. These findings were confirmed as significant (p<0.5) by multiplex mIF. Though UC (n=13) and UC-OGC (n=5) tumors had many similarities in their TIME, notable differences included decreased regulatory T cells (p=0.36) and a trend toward decreased M2 macrophages in UC-OGC tumors compared to UC (p=0.19). Conclusions: In this institutional series of pancreas UC with and without OGC, median OS in patients with UC-OGC was longer as compared to UC in both advanced and resected patients. Interestingly, there were significant differences in the TIME but not in the genomic profiles. Increased pro-tumorigenic M2 macrophage infiltration and decreased Tregs in the TIME of UC/UC-OGC compared to PDAC suggests a potential role of macrophage inhibitors and other immune-modulatory therapies in combination with chemotherapy for these rare variants of pancreatic cancer. Citation Format: Jamie N. Mills, Valerie Gunchick, Jiaqi Shi, Aaron Udager, Timothy Frankel, Mark M. Zalupski, Vaibhav Sahai. Analysis of tumor immune microenvironment and clinical outcomes in undifferentiated pancreatic carcinomas with and without osteoclast-like giant cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C013.

Pregnancy-related Guillain-Barré syndrome: A Case Series

Introduction: Pregnancy-related Guillain-Barré syndrome (GBS) is a rare autoimmune disorder that affects pregnant women. With an annual incidence ranging from 0.81 to 1.89 cases per 100,000 population, GBS can occur at any trimester of pregnancy, as well as during the postpartum period are susceptible to GBS. The pillars of managing pregnancy-related GBS to improve outcomes include early diagnosis, prompt immune-modulatory therapy, and multidisciplinary input. Case Series: In this study, three case of GBS in pregnancy were reported. The first patient was a 35-year-old woman, G3A1P2 post emergency Transperitoneal Cesarean Section (TPCS), who experienced with lower limb weakness three days before TPCS. After being diagnosed with severe eclampsia and underwent emergency TPCS, her complaint of lower limb weakness worsened. The second patient, a 27-year-old woman, with G2P1A0 experienced weakness in all four limbs. The third patient, a 20-year-old woman with G1P0A0, in the third semester presented with weakness in all four limbs. The electroneurography investigation conducted on these patients supported the diagnosis of GBS, which was subsequently managed with plasma exchange (PE). After the administration of PE, there was observed improvement in the clinical manifestation of GBS. Conclusion: The development of GBS in pregnancy is typically preceded by bacterial or viral infection. Preeclampsia was found to be associated with two folds risk of GBS, which was usually diagnosed based on the neurological examinations with supportive studies, including serological tests, cerebrospinal fluid analysis and electroneurography. The management of pregnancy-related GBS included intravenous immunoglobulin, PE, physiotherapy, and supportive therapy, such as ventilator support.

Chimeric Antigen Receptor (CAR)-Based Cell Therapy for Type 1 Diabetes Mellitus (T1DM); Current Progress and Future Approaches.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that destroys insulin-producing pancreatic β-cells. Insulin replacement therapy is currently the mainstay of treatment for T1DM; however, treatment with insulin does not ameliorate disease progression, as dysregulated immune response and inflammation continue to cause further pancreatic β-cell degradation. Therefore, shifting therapeutic strategies toward immunomodulating approaches could be effective to prevent and reverse disease progression. Different immune-modulatory therapies could be used, e.g., monoclonal-based immunotherapy, mesenchymal stem cell, and immune cell therapy. Since immune-modulatory approaches could have a systemic effect on the immune system and cause toxicity, more specific treatment options should target the immune response against pancreatic β-cells. In this regard, chimeric antigen receptor (CAR)-based immunotherapy could be a promising candidate for modulation of dysregulated immune function in T1DM. CAR-based therapy has previously been approved for a number of hematologic malignancies. Nevertheless, there is renewed interest in CAR T cells' " off-the-shelf " treatment for T1DM. Several pre-clinical studies demonstrated that redirecting antigen-specific CAR T cells, especially regulatory CAR T cells (CAR Tregs), toward the pancreatic β-cells, could prevent diabetes onset and progression in diabetic mice models. Here, we aim to review the current progress of CAR-based immune-cell therapy for T1DM and the corresponding challenges, with a special focus on designing CAR-based immunomodulatory strategies to improve its efficacy in the treatment of T1DM.

Polygenic HLA-Class I and HLA-Class II Models Define Various Types of Lymphoma

Introduction: Human leukocyte antigen (HLA) plays a major role in the interaction between the immune system and oncogenic process in various types of tumors including lymphoma. Mono-allelic and bi-allelic loss of the HLA-I has been reported in diffuse large B-cell lymphoma (DLBCL) and other types of lymphoma. Poor outcome was also reported with the loss of the HLA-I system in lymphoma. Since HLA-I and HLA-II are both critical for healthy immune response and both can be expressed in the lymphoma cells as well as in the lymphoma responding microenvironment, analyzing both systems is crucial for deciphering the role of the HLA system in lymphoma. Toward this goal, we evaluated the expression level of various HLA-I and HLA-II genes and compared these levels between subtypes of lymphoma. We quantified RNA expression levels of 15 HLA-I and HLA-II genes using next generation sequencing (NGS) and used machine learning algorithm (random forest) to evaluate the depth of the significance in variation of the expression profiles between various types of lymphoma. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue from 99 diffuse large B-cell lymphoma (DLBCL), 79 T-cell lymphoma, 40 follicular lymphoma, 28 mantle cell lymphoma, and 14 Hodgkin lymphoma samples. RNA sequencing was performed using a targeted hybrid capture panel. The sequenced HLA-I genes were HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-H. The sequenced HLA-II genes were HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB5, and HLA-DRB6. Salmon v1.4.0 software was used for expression quantification (TPM). Random forest machine learning system was used for predicting subtypes. Two thirds of samples were used for training the random forest algorithm and one third was used for testing. Results: RNA levels of individual HLA-I and HLA-II genes varied mildly between various B-cell lymphoma subtypes without specific pattern, especially after adjusting for multiple testing. However, T-cell lymphoma showed overall significantly (P< 0.0001) higher HLA-I (B, C, and F) and lower HLA-II (DRB1 and DRB5) expression levels as compared with B-cell lymphoma. In contrast, using machine learning algorithm to evaluate combination of HLA-I and HLA-II expression profiles showed discrete and significant differences between various types of lymphoma. The levels of expression of HLA-I and HLA-II genes were adequate to distinguish between DLBCL and follicular lymphoma with AUC of 0.778 (95% CI: 0.46-1.00), and between DLBCL and T-cell lymphoma with AUC of 0.833 (95% CI: 0.624-1.00). The number of Hodgkin lymphoma and mantle cell cases was small but the random forest distinguished between DLBCL and Hodgkin lymphoma with AUC of 0.833 (95% CI: 0.294-1.00), and between DLBCL and mantle cell lymphoma with AUC of 0.778 (95% CI: 0.416-1.00). Conclusions: This data confirms the polygenic effects of the HLA-I and HLA-II on various types of lymphoma. The HLA-I and HLA-II combined expression profiles are significantly different between various types of lymphoma. More importantly, this data suggests that immune modulating therapy should consider the polygenic effects of both the HLA-I and HLA-II systems.

Tumor immune evasion: insights from CRISPR screens and future directions.

Despite the clinical success of cancer immunotherapies including immune checkpoint blockade and adoptive cellular therapies across a variety of cancer types, many patients do not respond or ultimately relapse; however, the molecular underpinnings of this are not fully understood. Thus, a system-level understating of the routes to tumor immune evasion is required to inform the design of the next generation of immunotherapy approaches. CRISPR screening approaches have proved extremely powerful in identifying genes that promote tumor immune evasion or sensitize tumor cells to destruction by the immune system. These large-scale efforts have brought to light decades worth of fundamental immunology and have uncovered the key immune-evasion pathways subverted in cancers in an acquired manner in patients receiving immune-modulatory therapies. The comprehensive discovery of the main pathways involved in immune evasion has spurred the development and application of novel immune therapies to target this process. Although successful, conventional CRISPR screening approaches are hampered by a number of limitations, which obfuscate a complete understanding of the precise molecular regulation of immune evasion in cancer. Here, we provide a perspective on screening approaches to interrogate tumor-lymphocyte interactions and their limitations, and discuss further development of technologies to improve such approaches and discovery capability.

The Impact of COVID-19 on Health Anxiety and Perceived Stress Among Persons with IBD: A Population-representative Study.

The Corona Virus Immune Disease-2019 (COVID-19) pandemic has broadly impacted the mental health of individuals worldwide, especially with restrictions including social distancing and quarantining. Persons with IBD are at increased risk of mental health disorders. The aim was to understand how the COVID-19 pandemic affected adults with a chronic health issue, inflammatory bowel disease (IBD), using a population-based sample. A survey study in Manitoba was conducted during the second COVID-19 wave in autumn 2020. We assessed proportions of health anxiety and perceived stress using validated measures, as well as stressors during the COVID-19 pandemic. We used univariable and multivariable logistic regression analysis to assess predictors of perceived stress and health anxiety. A total of 1,384 (47.1%) persons responded, with a mean age of 58.0, and 46.9% had Crohn's disease. Almost three quarters (73.7%) had increased stress, with 37.7% having increased stress about their IBD, and 33.6% worried about their IBD worsening. 46.0% felt increased stress about accessing their doctor or nurse, and 56.5% felt they had good access to their gastroenterologist. Elevated stress and health anxiety were seen in 63.5% and 17.2% of respondents, respectively. Younger age and being on immune-modifying therapy (IMT) was predictive of increased health anxiety and stress. Longer IBD duration was associated with decreased stress, including those on IMT. Persons with IBD reported high rates of stress and health anxiety early in the COVID-19 pandemic, especially among those on IMT. It will be important to determine how this evolved over time and to what extent this impacted on disease course.

TMIC-84. A DEEP LEARNING-BASED METHOD FOR RAPID, PATIENT-SPECIFIC ASSAY OF MACROPHAGE INFILTRATION IN HIGH-GRADE GLIOMA USING LABEL-FREE STIMULATED RAMAN HISTOLOGY

Abstract BACKGROUND 5-Aminolevulinic acid (5-ALA), the most widely used fluorophore in image-guided glioma surgery, identifies tumor-associated macrophages (TAM) in high-grade glioma (HGG) tissue. Infiltrating macrophages, the predominant immune cells in glioma, are highly implicated in tumor progression, recurrence, and treatment response – particularly in immunotherapy. Using a unique, paired dataset of label-free stimulated Raman histology (SRH) and two-photon microscopy (TPEF) images sharing one-to-one spatial resolution, we developed a deep-learning approach to automatically quantify TAM infiltration from intraoperatively acquired SRH images. METHODS We compiled a dataset of 906 paired whole-slide SRH/TPEF images from 79 patients with HGG. We first trained a pix2pix generative adversarial network to convert raw, label-free SRH into synthetic TPEF with identifiable macrophages. Next, we trained a MaskR-CNN model to locate and segment TAMs. The pix2pix network was trained using 5,531 hand-picked 300-by-300-pixel fields-of-view (FOV) best exemplifying TAMs. We used a human-in-the-loop approach to train the segmentation network on 1,000 hand-labeled FOVs. RESULTS Macrophage segmentation from purely histologic data was near-identical to the fluorescence-based ground truth, with a mean dice score of 90.3%. Predicted TAM density was highly correlated with r=0.735. Subgroup analyses of TAM density in 346,836 non-overlapping FOVs revealed significantly lower TAM density in IDH-mutant (p< 0.01) and MGMT-hypermethylated (p=0.03) tumors. Our end-to-end algorithm uses fresh, unlabeled tissue specimens in the operating room and takes just three minutes to analyze whole-slide SRH images. CONCLUSION We demonstrate how deep neural networks can be used to rapidly and quantitatively evaluate macrophage infiltration in HGG in the operating room. Our software enables analysis of glioma patients’ tumor immune environment without immunohistochemistry or fluorescent labels, and may be leveraged to study the effects of immune-modulating therapies in the tumor microenvironment. Future work will focus on evaluating macrophage density and distribution as biomarkers for response to immunotherapies showing efficacy in glioma patients.

Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm.

Over the past years, inflammatory bowel disease (IBD) treatment has become more targeted, anticipating the use of immune-modifying therapies at an earlier stage. During the treatment process prevention and management of viral infections hold significant importance. The protective role of favipiravir on enterocytes which are affected by inflammation is still unknown. We aim to analyze the effects of favipiravir on enterocytes after an inflammatory condition. We conducted a 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to assess the cytotoxicity of favipiravir on intestinal epithelioid cells (IEC-6). To mimic the inflammation model in cell culture conditions, we exposed IEC-6 cells to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). The cells were categorized into four groups: control, inflammation model, application of favipiravir before inflammation (prophylactic), and application of favipiravir after inflammation (treatment). We assessed the presence and distribution of caspase 1, caspase 3, interleukin6 (IL6), interleukin 8 (IL8), mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase 1 (RIPK1), and TNF-α using indirect immunoperoxidase staining. TNF-α and IL8 levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in a culture medium. Caspase 1 was observed to be strong (+++) in the treatment group and weak (+) in the prophylactic group compared to the inflammation group. Caspase 3 was weak (+) in the inflammation group, and it was strong (+++) in the prophylactic and treatment group, the increase in the treatment group was significant. Therefore administering favipiravir before inducing inflammation appears to control the inflammatory caspase pathway in intestinal enterocytes, protecting them from inflammatory responses, while the caspase 3-dependent apoptotic pathway may not be active in enterocytes during inflammation. IL6 and IL8 were negative (-) in control, IL6 was weak (+) in inflammation and favipiravir treated groups; IL8 increased significantly in favipiravir groups compared to control and inflammation groups. Consequently, favipiravir may trigger IL6 release, initiating the inflammatory pathway and potentially enhancing IL8 interactions with other cytokines. TNF-α immunoreactivity was strong (+++) in the inflammation group, while it was moderate (++) in favipiravir-administered groups. MLKL immunoreactivity was strong (+++) in all groups, RIPK1 was weak (+) in control, strong (+++) in the inflammation and treatment group, moderate (++) in the prophylactic group, and the increase in inflammation and treatment group was significant compared to control. Our findings suggest that in the treatment group, necroptosis was triggered by increased MLKL and RIPK1, key players in inflammation and cell death. After immunocytochemical evaluation, our findings suggest that, after the onset of inflammation, favipiravir may play a role in cell death by increasing necroptosis rather than apoptosis.

COVID-19 encephalopathy with severe neurological symptoms: a clinical case presentation with literature review

COVID-19 infection is affecting more and more people around the world, and as the number of recovered patients increases, so does the knowledge on the potential clinical signs of the disease. Although SARS-CoV-2 virus is commonly associated with damage to the respiratory system, it has been observed that about half of patients with COVID-19 infection may also develop various neurological symptoms such as anosmia, dysgeusia, headache, myalgia, or dizziness. Encephalopathy is singled out as one of the most severe complications of the central nervous system caused by SARS-CoV-2 virus and associated with longer duration of the disease, increased disability, and mortality. Acute encephalopathy is a disorder of the brain that clinically occurs with a sudden change in the level of consciousness in otherwise healthy patients before the onset of symptoms. Risk factors include older age, male gender, quicker hospitalization after the onset of symptoms, and chronic illnesses. In exceptional cases, encephalopathy may be an early or even a major symptom of COVID-19 in young patients. The pathogenesis of COVID-19 encephalopathy is not fully understood. However, the most likely etiology of encephalopathy is multifactorial: systemic disease response, inflammation, coagulopathy, direct viral neuroinvasion, endoartheritis, and possibly post-infectious autoimmune mechanisms. For patients with suspected changes in the level of consciousness due to coronavirus infection, it is recommended to perform a thorough examination of the cerebrospinal fluid (CSF), head imaging with a preference for magnetic resonance imaging (MRI), and electroencephalography (EEG) It is worth noting that blood or imaging tests often do not show specific changes in patients with encephalopathy. As revealed by some studies of CSF examinations, cytosis is usually absent or very low while the protein concentration remains normal. It is important to note that SARS-CoV-2 is detected in the cerebral fluid only in isolated cases. Although the EEG of patients with COVID-19 are often normal, they sometimes show specific encephalopathic changes including excessive generalized frontal delta waves, triphasic waves and lower amplitude alpha and beta waves. The MRI describes a spectrum of neurovisual abnormalities, the most common of which are foci of leukoencephalopathy, changes in diffusion restriction imaging in the white, rarely in the gray matter, signs of microhaemorrhage and leptomeningitis. Treatment for COVID-19 encephalopathy includes supportive care and symptomatic treatment. Some studies have shown that immune modulation therapy, including high-dose corticosteroids and intravenous immunoglobulins, is effective in some severely ill patients.

Rheumatological manifestations in patients with malignancies: Relation to immune modulation therapy

Rheumatological manifestations in patients with malignancies: Relation to immune modulation therapy

COVID-19 illness: Different comorbidities may require different immunological therapeutic targets.

The SARS-CoV-2 pandemic has led to more than 6,870.000 deaths worldwide. Despite recent therapeutic advances, deaths in Intensive Care Units still range between 34 and 72%, comprising substantial unmet need as we move to an endemic phase. The general agreement is that in the first few days of infection, antiviral drugs and neutralizing monoclonal antibodies should be adopted. When the patient is hospitalized and develops severe pneumonia, progressing to a systemic disease, immune modifying therapy with corticosteroids is indicated. Such interventions, however, are less effective in the context of comorbidities (e.g., diabetes, hypertension, heart failure, atrial fibrillation, obesity and central nervous system-CNS diseases) which are by themselves associated with poor outcomes. Such comorbidities comprise common and some distinct underlying inflammatory pathobiology regulated by differential cytokine taxonomy. Searching in the PubMed database, literature pertaining to the biology underlying the different comorbidities, and the data from the studies related to various immunological treatments for the Covid-19 disease were carefully analyzed. Several experimental and clinical data have demonstrated that hypertension and atrial fibrillation present an IL-6 dependent signature, whereas diabetes, obesity, heart failure and CNS diseases may exhibit an IL-1a/b predominant signature. Distinct selective cytokine targeting may offer advantage in treating severe COVID-19 illness based on single or multiple associated comorbidities. When the patient does not immediately respond, a broader target range through JAKs pathway inhibitors may be indicated. Herein, we discuss the biological background associated with distinct comorbidities which might impact the SARS-CoV-2 infection course and how these should to be addressed to improve the current therapeutic outcome.