Immune Monitoring Research Articles

Abstract 79: Study of tumor microenvironment of ovarian clear cell carcinoma

Abstract Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material & Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [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 79.

Rubella in Poland in 2020.

Since 2005, rubella has been included in the eradication program coordinated by the World Health Organization (WHO). The elimination of rubella in the country is possible provided that it is achieved by min. 95% immunization status and monitoring of the epidemiological situation, including the recording all suspicions of rubella and conducting laboratory diagnostics in the WHO Reference Laboratory. The aim of the study is to assess the epidemiological situation of rubella in Poland in 2020, taking into account the assessment of rubella vaccination status and the degree of implementation of the WHO rubella elimination program in Poland. Assessment of the epidemiological situation of rubella in Poland based on aggregated reports of suspected rubella cases sent to the National Institute of Public Health NIH - National Research Institute by the Voivodeship Sanitary and Epidemiological Stations, data from the bulletin "Infectious diseases and poisoning in Poland in 2020" and data from the Epibaza system and the bulletin "Preventive vaccination in Poland in 2020". In 2020, 98 cases of rubella were registered, 187 cases less than in 2019 (285 cases). There was also a decrease in the incidence to 0.26 per 100,000, compared to 0.74 per 100,000 in 2019. The highest incidence, iregardless of gender and place of residence, was recorded in the 0-4 age group (2.98 per 100,000). In 2020, no cases of congenital rubella syndrome were reported. In 2020, only one case (1.02%) was classified as a case confirmed in a laboratory test. The remaining 98.98% (97 cases) were diagnosed on the basis of clinical symptoms. In 2020, in Poland, a decrease in the number of rubella was observed. Rubella was registered in 99% on the basis of a general diagnosis, without the required laboratory confirmation, which means that other rash diseases can be registered as rubella.

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio.

Background: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1+ MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation. Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1+ MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1+ MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1+ MDSC tended to be overexpressed by the second week after surgery. Conclusion: The present study showed that NLR, immature neutrophils and LOX-1+ MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients.

Emerging Technologies of Three-Dimensional Printing and Mobile Health in COVID-19 Immunity and Regenerative Dentistry.

The ongoing coronavirus disease 2019 (COVID-19) pandemic highlights the importance of developing point-of-care (POC) antibody tests for monitoring the COVID-19 immune response upon viral infection or following vaccination, which requires three key aspects to achieve optimal monitoring, including three-dimensional (3D)-printed POC devices, mobile health (mHealth), and noninvasive sampling. As a critical tissue engineering concept, additive manufacturing (AM, also known as 3D printing) enables accurate control over the dimensional and architectural features of the devices. mHealth refers to the use of portable digital devices, such as smartphones, tablet computers, and fitness and medical wearables, to support health, which facilitates contact tracing, and telehealth consultations during the pandemic. Compared with invasive biosample (blood), saliva is of great importance in the spread and surveillance of COVID-19 as a noninvasive diagnostic method for virus detection and immune status monitoring. However, investigations into 3D-printed POC antibody test and mHealth using noninvasive saliva are relatively limited. Further exploration of 3D-printed antibody POC tests and mHealth applications to monitor antibody production for either disease onset or immune response following vaccination is warranted. This review briefly describes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and immune response after infection and vaccination, then discusses current widely used binding antibody tests using blood samples and enzyme-linked immunosorbent assays on two-dimensional microplates before focusing upon emerging POC technological platforms, such as field-effect transistor biosensors, lateral flow assay, microfluidics, and AM for fabricating immunoassays, and the possibility of their combination with mHealth. This review proposes that noninvasive biofluid sampling combined with 3D POC antibody tests and mHealth technologies is a promising and novel approach for POC detection and surveillance of SARS-CoV-2 immune response. Furthermore, as key concepts in dentistry, the application of 3D printing and mHealth was also included to facilitate the appreciation of cutting edge techniques in regenerative dentistry. This review highlights the potential of 3D printing and mHealth in both COVID-19 immunity monitoring and regenerative dentistry.

Adenoviral Penton and Hexon Proteins Are Equivalent Immunogenic Targets of Virus-Specific T Cells after Hematopoietic Stem Cell Transplantation in Children

Human adenovirus (HAdV) infection is a serious complication that can lead to significant morbidity and mortality, especially in immunocompromised pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Control and elimination of HAdV requires the presence of the respective antiviral T cells, and adoptive transfer of virus-specific T cells has become an important new treatment option for patients refractory to antiviral treatment. Although the adenoviral capsid protein hexon is known to be a major immunodominant T cell target across HAdV species, up to 30% of HAdV-seropositive donors show no T cell responses to the overlapping peptide pool spanning the entire protein. Our group recently verified the capsid protein penton as a second immunodominant target in HAdV infection. Here we aimed to investigate the prevalence of both penton-specific and hexon-specific HAdV T cells and their impact in virus control after HSCT. We analyzed the prevalence and characteristics of HAdV-specific T cells in 33 consecutive pediatric patients with HAdV reactivation following allogeneic HSCT and correlated them with viral load analysis. Our study demonstrates that penton is an important immunodominant target antigen of HAdV reactivation/ infection after HSCT in most patients. We demonstrate that in the majority of patients, both penton- and hexon-specific T cells appear at similar time intervals after transplantation. Despite the prevalence for either hexon-specific or penton-specific T cells in individual patients, we were unable to attribute the predominance to specific HLA types or HAdV serotypes. The occurrence of HAdV-specific T cells was closely linked to viral control, arguing for immune monitoring strategies to tailor antiviral treatment and adoptive T cell therapy.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Epigenetic immune monitoring for COVID-19 disease course prognosis.

The course of COVID-19 is associated with severe dysbalance of the immune system, causing both leukocytosis and lymphopenia. Immune cell monitoring may be a powerful tool to prognosticate disease outcome. However, SARS-CoV-2 positive subjects are isolated upon initial diagnosis, thus barring standard immune monitoring using fresh blood. This dilemma may be solved by epigenetic immune cell counting. In this study, we used epigenetic immune cell counting by qPCR as an alternative way of quantitative immune monitoring for venous blood, capillary blood dried on filter paper (dried blood spots, DBS) and nasopharyngeal swabs, potentially allowing a home-based monitoring approach. Epigenetic immune cell counting in venous blood showed equivalence with dried blood spots and with flow cytometrically determined cell counts of venous blood in healthy subjects. In venous blood, we detected relative lymphopenia, neutrophilia, and a decreased lymphocyte-to-neutrophil ratio for COVID-19 patients (n =103) when compared with healthy donors (n = 113). Along with reported sex-related differences in survival we observed dramatically lower regulatory T cell counts in male patients. In nasopharyngeal swabs, T and B cell counts were significantly lower in patients compared to healthy subjects, mirroring the lymphopenia in blood. Naïve B cell frequency was lower in severely ill patients than in patients with milder stages. Overall, the analysis of immune cell counts is a strong predictor of clinical disease course and the use of epigenetic immune cell counting by qPCR may provide a tool that can be used even for home-isolated patients.

Routine EWS Fusion Analysis in the Oncology Clinic to Identify Cancer-Specific Peptide Sequence Patterns That Span Breakpoints in Ewing Sarcoma and DSRCT.

(1) Background: EWS fusion genes are associated with Ewing sarcoma and other Ewing family tumors including desmoplastic small round tumor, DSRCT. We utilize a clinical genomics workflow to reveal real-world frequencies of EWS fusion events, cataloging events that are similar, or divergent at the EWS breakpoint. (2) Methods: EWS fusion events from our next-generation sequencing panel (NGS) samples were first sorted by breakpoint or fusion junctions to map out the frequency of breakpoints. Fusion results were illustrated as in-frame fusion peptides involving EWS and a partner gene. (3) Results: From 2471 patient pool samples for fusion analysis at the Cleveland Clinic Molecular Pathology Laboratory, we identified 182 fusion samples evolved with the EWS gene. They are clustered in several breakpoints: chr22:29683123 (65.9%), and chr22:29688595 (2.7%). About 3/4 of Ewing sarcoma and DSRCT tumors have an identical EWS breakpoint motif at Exon 7 (SQQSSSYGQQ-) fused to a specific part of FLI1 (NPSYDSVRRG or-SSLLAYNTSS), ERG (NLPYEPPRRS), FEV (NPVGDGLFKD) or WT1 (SEKPYQCDFK). Our method also worked with Caris transcriptome data, too. Our primary clinical utility is to use this information to identify neoantigens for therapeutic purposes. (4) Conclusions and future perspectives: our method allows interpretation of what peptides result from the in-frame translation of EWS fusion junctions. These sequences, coupled with HLA-peptide binding data, are used to identify potential sequences of cancer-specific immunogenic peptides for Ewing sarcoma or DSRCT patients. This information may also be useful for immune monitoring (e.g., circulating T-cells with fusion-peptide specificity) to detect vaccine candidates, responses, or residual disease.

A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8+, CD4+), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells.Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.

Monitoring of SARS-CoV-2 Infection in Ragusa Area: Next Generation Sequencing and Serological Analysis

The coronavirus disease 19 (COVID-19) post pandemic evolution is correlated to the development of new variants. Viral genomic and immune response monitoring are fundamental to the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since 1 January to 31 July 2022, we monitored the SARS-CoV-2 variants trend in Ragusa area sequencing n.600 samples by next generation sequencing (NGS) technology: n.300 were healthcare workers (HCWs) of ASP Ragusa. The evaluation of anti-Nucleocapside (N), receptor-binding domain (RBD), the two subunit of S protein (S1 and S2) IgG levels in 300 exposed vs. 300 unexposed HCWs to SARS-CoV-2 was performed. Differences in immune response and clinical symptoms related to the different variants were investigated. The SARS-CoV-2 variants trend in Ragusa area and in Sicily region were comparable. BA.1 and BA.2 were the most representative variants, whereas the diffusion of BA.3 and BA.4 affected some places of the region. Although no correlation was found between variants and clinical manifestations, anti-N and anti-S2 levels were positively correlated with an increase in the symptoms number. SARS-CoV-2 infection induced a statistically significant enhancement in antibody titers compared to that produced by SARS-CoV-2 vaccine administration. In post-pandemic period, the evaluation of anti-N IgG could be used as an early marker to identify asymptomatic subjects.

Systemic Lupus Erythematosus Pathogenesis: Interferon and Beyond.

Autoreactive B cells and interferons are central players in systemic lupus erythematosus (SLE) pathogenesis. The partial success of drugs targeting these pathways, however, supports heterogeneity in upstream mechanisms contributing to disease pathogenesis. In this review, we focus on recent insights from genetic and immune monitoring studies of patients that are refining our understanding of these basic mechanisms. Among them, novel mutations in genes affecting intrinsic B cell activation or clearance of interferogenic nucleic acids have been described. Mitochondria have emerged as relevant inducers and/or amplifiers of SLE pathogenesis through a variety of mechanisms that include disruption of organelle integrity or compartmentalization, defective metabolism, and failure of quality control measures. These result in extra- or intracellular release of interferogenic nucleic acids as well as in innate and/or adaptive immune cell activation. A variety of classic and novel SLE autoantibody specificities have been found to recapitulate genetic alterations associated with monogenic lupus or to trigger interferogenic amplification loops. Finally, atypical B cells and novel extrafollicular T helper cell subsets have been proposed to contribute to the generation of SLE autoantibodies. Overall, these novel insights provide opportunities to deepen the immunophenotypic surveillance of patients and open the door to patient stratification and personalized, rational approaches to therapy. Expected final online publication date for the Annual Review of Immunology, Volume 41 is April 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Identification of proteinase 3 autoreactive CD4+T cells and their T-cell receptor repertoires in antineutrophil cytoplasmic antibody–associated vasculitis

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease involving autoreactivity to proteinase 3 (PR3) as demonstrated by presence of ANCAs. While autoantibodies are screened for diagnosis, autoreactive T cells and their features are less well-studied. Here, we investigated PR3-specific CD4+T cell responses and features of autoreactive T cells in patients with PR3-AAV, using a cohort of 72 patients with either active or inactive disease. Autoreactive PR3-specific CD4+T cells producing interferon γ in response to protein stimulation were found to express the G-protein coupled receptor 56 (GPR56), a cell surface marker that distinguishes T cells with cytotoxic capacity. GPR56+CD4+T cells were significantly more prominent in the blood of patients with inactive as compared to active disease, suggesting that these cells were affected by immunosuppression and/or that they migrated from the circulation to sites of organ involvement. Indeed, GPR56+CD4+T cells were identified in T-cell infiltrates of affected kidneys and an association with immunosuppressive therapy was found. Moreover, distinct TCR gene segment usage and shared (public) T cell clones were found for the PR3-reactive TCRs. Shared T cell clones were found in different patients with AAV carrying the disease-associated HLA-DP allele, demonstrating convergence of the autoreactive T cell repertoire. Thus, we identified a CD4+T cell signature in blood and in affected kidneys that display PR3 autoreactivity and associates with T cell cytotoxicity. Our data provide a basis for novel rationales for both immune monitoring and future therapeutic intervention in PR3-AAV.

Clinical application of immune repertoire sequencing in solid organ transplant.

Measurement of T cell receptor (TCR) or B cell receptor (BCR) gene utilization may be valuable in monitoring the dynamic changes in donor-reactive clonal populations following transplantation and enabling adjustment in therapy to avoid the consequences of excess immune suppression or to prevent rejection with contingent graft damage and to indicate the development of tolerance. We performed a review of current literature to examine research in immune repertoire sequencing in organ transplantation and to assess the feasibility of this technology for clinical application in immune monitoring. We searched MEDLINE and PubMed Central for English-language studies published between 2010 and 2021 that examined T cell/B cell repertoire dynamics upon immune activation. Manual filtering of the search results was performed based on relevancy and predefined inclusion criteria. Data were extracted based on study and methodology characteristics. Our initial search yielded 1933 articles of which 37 met the inclusion criteria; 16 of these were kidney transplant studies (43%) and 21 were other or general transplantation studies (57%). The predominant method for repertoire characterization was sequencing the CDR3 region of the TCR β chain. Repertoires of transplant recipients were found to have decreased diversity in both rejectors and non-rejectors when compared to healthy controls. Rejectors and those with opportunistic infections were more likely to have clonal expansion in T or B cell populations. Mixed lymphocyte culture followed by TCR sequencing was used in 6 studies to define an alloreactive repertoire and in specialized transplant settings to track tolerance. Methodological approaches to immune repertoire sequencing are becoming established and offer considerable potential as a novel clinical tool for pre- and post-transplant immune monitoring.

A novel immune monitoring platform for patients with gastrointestinal cancer.

424 Background: Understanding the impact of anticancer therapies on immune system is critical to successful design and incorporation of immunotherapies. We developed a novel immune monitoring platform that measures the relative amount of circulating effector T cells and antitumor cytotoxicity of circulating immune cells. We previously showed that this assay can differentiate patients’ clinical response to anti-PD-1 therapy. Here, we applied this assay to patients with gastric or gastroesophageal junction adenocarcinoma (GGEA) who received chemoradiation or chemotherapy-based therapies. Methods: Patients underwent peripheral blood collection at baseline and during follow-up. Levels of circulating effector T cells (CX3CR1+NKG7+/CD3+CD8+) were quantified by flow cytometry using peripheral blood mononuclear cells (PBMCs). Antitumor cytotoxicity of circulating immune cells was evaluated by co-culturing CD3/CD28 activated PBMCs with calcein-AM labeled GGEA tumor cells (FLO-1). Antitumor cytotoxicity of individual patients’ PBMCs was calculated based on the amount of calcein released by dead tumor cells. Results: In a pilot study of 8 patients with mismatch repair-proficient (pMMR) GGEA, the changes in the frequency of effector T cells appeared to be associated with radiographic response to treatment based on RECIST (3 partial response [PR], 3 stable disease [SD], and 2 progressive disease [PD]). The median interval between sequential blood collections was 64 days and an increase in effector T cells was observed in patients with PD. Antitumor cytotoxicity was evaluated in 4 patients who did not receive targeted therapies or immune checkpoint inhibitors. Two of these patients (a, b) had blood collected at the time of diagnosis (treatment naïve) and after neoadjuvant therapy. One developed pulmonary metastases after chemoradiation while the other had significant tumor shrinkage after chemotherapy. The decrease in antitumor cytotoxicity of PBMCs was less in the patient with PD (-19.4%) than the one with PR (-48.5%). Two patients (c, d) had blood collected twice while receiving the same chemotherapy for metastatic disease (not treatment naïve). The decrease in antitumor cytotoxicity of PBMCs was less in the patient with SD (-0.3%) than the one with PR (-14.5%). Conclusions: We demonstrated the feasibility of using PBMCs to monitor immune response to anticancer therapies. Our data raise the possibility that the amount of circulating effector T cells and antitumor cytotoxicity of PBMCs may be associated with tumor burden in patients with pMMR solid tumors. [Table: see text]

P215 Deep immune phenotyping of unconventional T cells reveals a depletion in Mucosa Associated T Cells (MAIT) in IBD patients with extraintestinal manifestations

Abstract Background Extraintestinal manifestations (EIM) are common in IBD and they affect importantly the quality of life of patients. Although the pathogenesis of EIM is still poorly understood, an altered immune response involving T cells has been proposed. In addition, the presence of EIM during disease course will impact the therapeutic choices with new biologics. In the present work we aim to characterize T cell populations -with focus on Uncoventional T cells- in IBD patients presenting EIM using advance flow cytometry and proteomics. Methods A total of 184 individuals (106 F/78 M) and were prospectively included: 149 IBD patients (84 UC/65 CD) and 25 healthy matched controls. Biologic samples including Peripheral Blood Mononuclear Cells (PBMC) and plasma were consecutively stored and classified in an established cross sectional IBD biobank at the Norwegian University of Science and Technology. Patient characteristics including age, sex, smoking status, current medication, Montreal Classification phenotype and inflammation biomarkers (CRP) and fecal Calprotectin were recorded. PBMC were stained with antibodies and acquired using BD FACS Symphony flow cytometer using automatic compensations and Multiplex ELISA for 65 proteins was performed using Immune Monitoring 65-Plex Human ProcartaPlex™ Panel at Singapore Immunology Network (SIgN), as previously described. Statistical analysis was performed using GraphPad Prism (San Diego, CA). p≤ 0.05 was considered statistically significant. Results Of the 184 IBD patients included, the presence or not of EIM was registered in 149 (mean age 37 +/-7.2; 86 female) of which 84 had UC and 65 CD. Of them, 21 (14.1%) presented EIM at inclusion. We analyzed the differences in all subsets of unconventional T cells: gammadelta T cells (VD1, VD2, VD3), MAIT cells, innate NKT cells and CD4/CD8 positive T cells between IBD patients with or without EIM (Fig1A). MAIT cells were selectively depleted in patients with EIM (p<0.01), while there was no statistically significant change in the ratios of other T cell subpopulations. We then studied the concentrations of plasma proteins between the two groups. We found a significant increase in IL-2, GM-CSF, IL-6, Eotaxin, Leukemia Inhibitory Factor Protein (LIF), TNF-RII and Monocyte Chemoattractant Protein-1 (MCP), all p<0.05 in patients with EIM (Fig1B). In order to address the potential use of MAIT as marker of EIM we constructed a ROC curve showing an AUC 0.752 (95% CI 0.639-0.865, p= 0.0002) Fig. 1C Conclusion MAIT cell depletion associated strongly with the presence of EIM and could be used as a clinical marker for EIM in IBD. A deeper understanding of its role in the pathogenesis of EIM, as well as an external validation as a marker, deserves further investigation.

P575 Effect of etrasimod on immune cell subsets in colonic tissue of patients with ulcerative colitis: immunophenotyping analysis of colon biopsy samples from the phase 3 ELEVATE UC 52 and ELEVATE UC 12 trials

Abstract Background Etrasimod is an investigational, once-daily, oral, selective sphingosine 1-phosphate receptor 1,4,5 (S1P1,4,5) modulator in development for the treatment of moderately to severely active ulcerative colitis (UC). Etrasimod reversibly sequesters specific lymphocyte subsets in lymph nodes, reducing circulating lymphocytes and resulting in fewer immune cells available to traffic to the gastrointestinal tract. To better understand how etrasimod reduces inflammation in UC, the phase 3 ELEVATE UC 52 and ELEVATE UC 12 trials included a per protocol exploratory immunophenotyping biomarker analysis in fixed-formalin paraffin-embedded (FFPE) colon biopsies. Methods In ELEVATE UC 52 (NCT03945188) and ELEVATE UC 12 (NCT03996369), patients (16-80 years) with moderately to severely active UC were randomised 2:1 to once-daily etrasimod 2 mg or placebo (PBO). Characterization of immune cell types in colon biopsies were assessed at baseline, week 12, and week 52 (ELEVATE UC 52 only) by Epiontis ID, an epigenetic immune monitoring method. Biomarker levels for each lymphocyte subset were evaluated between responders vs nonresponders, defined by achievement of clinical efficacy criteria within each treatment group, using t-tests and nominal P values. Results Colon biopsies were collected and analysed from 276 subjects in ELEVATE UC 52 (179 etrasimod, 97 PBO) and 150 subjects in ELEVATE UC 12 (97 etrasimod, 53 PBO). Etrasimod induced significant changes from baseline (nominal P value <0.05) in colonic T- and B-cell subsets at week 12 in ELEVATE UC 52 and ELEVATE UC 12 (Figure 1), which were maintained through week 52 in ELEVATE UC 52. At week 12, etrasimod induced significant reductions from baseline (nominal P value <0.05) in colonic T- and B-cell subsets expressing IL17A, FOXP3, CCR7, CXCR3, and LRP5A in responders vs nonresponders (Table 1). At week 12, etrasimod, but not PBO, induced significant reductions from baseline (nominal P value <0.05) in colonic CD8B-expressing T cells in responders vs nonresponders in ELEVATE UC 52; these trends were maintained through week 52. Significant effects on CXCR3, FOXP3, and LRP5A were observed in responders vs nonresponders in ELEVATE UC 12. Conclusion In this per protocol exploratory analysis of two independent phase 3 trials, etrasimod induced changes from baseline in colonic tissue T- and B-cell subsets in subjects with UC, with nominally significant reductions observed in those who met response criteria. The reduction in activated immune cells in colonic tissue in responders suggests etrasimod induces a reduction in local inflammation that could contribute to remission. These findings are the first to report changes to immune cell subsets in colonic tissue in response to a S1P receptor modulator.

Pathogen-agnostic immune biomarkers that predict infection after solid organ transplantation.

Solid organ transplant recipients (SOTRs) remain at high risk for infection throughout their post-transplant course. Dosing of immunosuppressive medications, strategies that prevent infection, and choice of empiric antimicrobial treatment could be optimized by a better understanding of an individual patient's risk for infectious complications. Diagnostic tests that qualitatively or quantitatively measure the function of the immune system and/or its response to infection may be useful for individualized management decisions. Numerous studies have identified an association between infectious outcomes after solid organ transplantation (SOT) and the results of a variety of non-pathogen-specific or "pathogen-agnostic" immune monitoring tests. These biomarkers include humoral immune markers, functional or quantitative assessments of cellular immunity, transcriptomic-based diagnostics, and replication of viruses within the human virome, which have been used to predict or diagnose a variety of different infectious diseases complicating SOT. In this narrative review, we discuss several host-derived immune biomarkers that show promise for either predicting or diagnosing infection among SOTRs. However, additional studies are needed to determine the optimal use of immune response testing. Whether immune biomarkers contribute added benefits to current standard clinical care has not yet been determined. Testing must be validated across a range of clinical scenarios, including surveillance to predict infection risk and diagnosis of active infection at various time points post transplant.

Radiation-induced circulating myeloid-derived suppressor cells induce systemic lymphopenia after chemoradiotherapy in patients with glioblastoma.

Severe and prolonged lymphopenia frequently occurs in patients with glioblastoma after standard chemoradiotherapy and has been associated with worse survival, but its underlying biological mechanism is not well understood. To address this, we performed a correlative study in which we collected and analyzed peripheral blood of patients with glioblastoma (n=20) receiving chemoradiotherapy using genomic and immune monitoring technologies. RNA sequencing analysis of the peripheral blood mononuclear cells (PBMC) showed an elevated concentration of myeloid-derived suppressor cell (MDSC) regulatory genes in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Additional analysis including flow cytometry and single-cell RNA sequencing further confirmed increased numbers of circulating MDSC in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Preclinical murine models were also established and demonstrated a causal relationship between radiation-induced MDSC and systemic lymphopenia using transfusion and depletion experiments. Pharmacological inhibition of MDSC using an arginase-1 inhibitor (CB1158) or phosphodiesterase-5 inhibitor (tadalafil) during radiation therapy (RT) successfully abrogated radiation-induced lymphopenia and improved survival in the preclinical models. CB1158 and tadalafil are promising drugs in reducing radiation-induced lymphopenia in patients with glioblastoma. These results demonstrate the promise of using these classes of drugs to reduce treatment-related lymphopenia and immunosuppression.

Expression and significance of IL-6 and IL-8 in canine mammary gland tumors

Mammary gland tumors are the most common malignant diseases which seriously threaten the health of women and female dogs. There is a lack of an effective tumor marker which can effectively assist in the early diagnosis, and prognosis of mammary gland tumors in veterinary clinical medicine. IL-6, and IL-8 as immunosuppressive factors may stimulate tumor cells growth, contribute to loco-regional relapse and metastasis that might be utilized as a marker for immunity status and monitoring of the course of tumor. The present study aimed to investigate the expression of serum/tissue IL-6, IL-8 and IL-10 in canine mammary gland tumors using Enzyme linked immunosorbent assay(ELISA), Western blot and Immunohistochemistry assay(IHC) to determine whether it is associated with tumor progression. The results showed that levels of IL-6, IL-8 and IL-10 in serum were higher in malignant tumor group than that in benign tumor and control group; the expression levels of IL-6 and IL-8 were significantly elevated in grade III than in grade I and II and was related to metastasis. Likewise, IL-6 and IL-8 were also highly expressed in malignant tumor tissues. Elevated expression of IL-6 was associated with histopathological grade and metastases in malignant tumors. Moreover, high expression of IL-6 occurred in the Basal-like subtypes whereas high expression of IL-8 occurred in the Luminal B subtypes. The results of this study indicated that changes of IL-6 and IL-8 in the tumor microenvironments were closely related to the diseases status and may be used as a potential diagnostic or biomarker in canine mammary gland tumors.

The need and benefit of immune monitoring to define patient and disease heterogeneity, mechanisms of therapeutic action and efficacy of intervention therapy for precision medicine in type 1 diabetes.

The current standard of care for type 1 diabetes patients is limited to treatment of the symptoms of the disease, insulin insufficiency and its complications, not its cause. Given the autoimmune nature of type 1 diabetes, immunology is critical to understand the mechanism of disease progression, patient and disease heterogeneity and therapeutic action. Immune monitoring offers the key to all this essential knowledge and is therefore indispensable, despite the challenges and costs associated. In this perspective, I attempt to make this case by providing evidence from the past to create a perspective for future trials and patient selection.