281 Measuring immune checkpoint inhibitor efficacy using primary patient-derived 3D spheroids

BackgroundCancerous cells can utilize immune checkpoints to escape T-cell-mediated cytotoxicity. Agents that target PD-1, PD-L1 and CTLA4 are collectively deemed immune checkpoint inhibitors (ICIs), and many have been approved for treatment of non-small cell lung cancer (NSCLC) and melanoma. Unfortunately, many patients do not respond to these therapies and often experience disease progression. Immunohistochemistry assays to predict response to ICIs have been inconsistent in their readouts and often patients with low expression levels respond to ICIs. Understanding the determinants of ICI response in individual patients is critical for improving the clinical success of this drug class. Using patient-derived spheroids from NSCLC and melanoma primary tissue, we developed a multi-plexed assay for detecting ICI efficacy.MethodsNine NSCLC and 11 melanoma primary tumor samples were dissociated to single cells, classified for immune checkpoint expression and cell content by flow cytometry, and seeded for spheroid formation. Spheroids were treated with pembrolizumab, nivolumab, atezolizumab, ipilimumab or durvalumab across a range of concentrations and monitored for cytotoxicity at 24-hours and viability at 72-hours by multiplexing CellTox™ Green Cytotoxicity Assay and CellTiter-Glo® 3D Cell Viability Assay. IFNγ and granzyme B secretion was assessed using Luminex technology. ICI response was evaluated by determining the concentration-response relationship for all three read-outs.ResultsIncreased IFNγ and granzyme B were detected for every ICI in one or more patient samples. ICI-induced IFNγ secretion inversely correlated with PD-1+ immune cells. Durvalumab was significantly more cytotoxic for both NSCLC and melanoma spheroids compared to the other ICIs and significantly reduced spheroid viability with mean spheroid survival decreasing to 19.5% for NSCLC and 58.2% for melanoma. We evaluated if there was an association between durvalumab response and cell composition and found that percent spheroid survival significantly correlated with CD8+ T-cells for both NSCLC (r=-0.7920, p=0.0191) and melanoma (r=-0.6918, p=0.0390). Furthermore, CD8+ T-cells correlated with durvalumab-induced granzyme B secretion for NSCLC (r=-0.7645, p=0.0271) and melanoma (r=-0.7419, p=0.0221).ConclusionsIn this study we show ICI-specific increases in immune-related analytes in a concentration-dependent manner for NSCLC and melanoma patient-derived spheroids. We detected spheroid cytotoxicity following short term ICI treatment which closely mirrored decreased spheroid viability at a later timepoint. Finally, we can decipher response mechanisms as exemplified by durvalumab-induced granzyme B secretion correlating with the presence of CD8+ T-cells which results in reduced spheroid viability for both tested cancer indications.

1335. High Serum Levels of Interleukin-10 as a Predictor Factor of In-Hospital Mortality in COVID-19 Patients

Background Since the spread of SARS-CoV-2 worldwide, there has been the need for scores and biomarkers to identify patients at risk of died or requiring admission to the intensive care units (ICU) admission. Interleukin-10 (IL-10) is released as a response to the infection, stimulating inflammatory pathways in the acute phase response. Thus, previous studies have shown that high serum concentrations IL-10 can be identify patients with severe community acquired pneumonia (CAP). Nevertheless, there is a lack of information regarding the capacity of IL-10 to identify severe COVID-19. Thus, the aim of this study was to determine the capacity of IL-10 as a prediction factor for mortality in hospital admitted patients with COVID-19 compared with CAP patients. Methods A prospective observational study was carried out at the Clinica Universidad de La Sabana, Colombia. Patients older than 18 years and old, hospitalized due to COVID 19 or CAP, were included. Patients were stratified into COVID-19 and non-COVID-19 patients. IL-10 levels were quantified in serum samples using the LUMINEX technology. Serum samples were collected within the first 24 hours of hospital admission. Afterward, concentrations of interleukinwere statistically compared among groups. ROC curves were calculated. Results A total of 88 patients with CAP and 152 patients with COVID-19 were enrolled in the study. The median [with IQR] serum concentration of IL-10 were higher in those patients who died (81.1 [30.7-148.9] vs 18.8 [8.3-48.4] p-value < 0.0001). Then, comparing the study group, the median concentration of IL-10 levels among patients deceased by COVID-19 were higher than patients those who survived (85.1 [40-149.8] vs 32.4 [13.9-56.7] p-value < 0.001). In addition, IL-10 levels were higher in patients who survived COVID-19 compared with those who survived CAP (32.4 [13.9-56.7] vs 10.6 [4.9-18] p-value < 0.0001). The area under curve (AUC) ROC of IL-10 to predict mortality risk was 0.754 for all cohort. DeLong′s test comparing ROC curves in COVID-19 and CAP patients had a p= 0.744. Conclusion High serum levels of IL-10 are a good predictor of in-hospital mortality among COVID-19 patients. However, this risk association was not observed in CAP patients. Further studies are needed. Disclosures All Authors: No reported disclosures

Platelet-derived sCD40L: specific inflammatory marker for early-stage severe acute respiratory syndrome coronavirus 2 infection

Background The SARS-CoV-2 virus is the causing agent of the Coronavirus disease 2019 (COVID-19) characterized by a huge pro-inflammatory response and coagulation disorders that may lead to for its severe forms, in organ failure or even death. As major players of thrombo-inflammation, platelets release large amounts of immunomodulatory molecules and regulate leukocyte and endothelial activity, which are both altered in COVID-19. Altogether, this makes platelets a very likely actor of the thrombo-inflammatory complications of COVID-19. Thus, we propose to identify a platelet inflammatory signature of severe COVID-19 specifically modulated throughout the course of the disease. Methods Luminex technology and enzyme-linked immunosorbent assay were used to assess plasma levels of platelet inflammatory markers in patients with severe acute respiratory syndrome coronavirus 2 infection on admission and for 14 days afterwards. Results In accordance with the observations of other teams, we evidence that the plasma levels of the platelet soluble (s)CD40L is significantly elevated in the early stages of the disease. Interestingly we observe that the plasma level of sCD40L decreases overtime while that of sCD62P increases significantly. Conclusions Our data suggest that there is a platelet signature of inflammatory response to SARS-COv-2 infection which varies overtime and could serve as monitoring biomarkers of patient inflammatory state. Clinical trial registration number: 2020-A01100-39; title: Human Ab Response & immunoMONItoring of COVID-19 Patients, registration date: 05/25/2020; URL of the registry: https://clinicaltrials.gov/ct2/history/NCT04373200?V_5=View.

Identification of Immune Activation Markers in the Early Onset of COVID-19 Infection

This study aimed to determine the specific cytokine profile in peripheral blood during the early onset of COVID-19 infection. This was a cross-sectional exploratory, single center study. A total of 55 plasma samples were studied. Serum samples of adults showing symptoms of COVID-19 infection who were tested positive for SARS-CoV-2 infection (CoV+, n=18) at the COVID-19 outpatient clinic of the Medical University of Vienna were screened for immune activation markers by Luminex technology. Additionally, age and gender-matched serum samples of patients displaying COVID-19 associated symptoms, but tested negative for SARS-CoV-2 (CoV-, n=16) as well as healthy controls (HC, n=21) were analyzed. COVID-19 positive (CoV+) patients showed a specific upregulation of BLC (141; 74-189 pg/mL), SCD30 (273; 207-576 pg/mL), MCP-2 (18; 12-30 pg/mL) and IP-10 (37; 23-96 pg/mL), compared to patients with COVID19-like symptoms but negative PCR test (CoV-), BLC (61; 22-100 pg/mL), sCD30L (161; 120-210 pg/mL), MCP-2 (8; 5-12 pg/mL) and IP-10 (9; 6-12 pg/mL) and healthy controls (HC) (BLC 22; 11-36 pg/mL, sCD30 74; 39-108 pg/mL, MCP-2 6; 3-9. pg/mL, IP-10 = 8; 5-13). The markers APRIL, sIL-2R, IL7, MIF, MIP-1b, SCF, SDF-1a, sTNF-RII were elevated in both CoV+ and CoV- patient groups compared to healthy controls. HGF, MDC and VEGF-A were elevated in CoV- but not CoV+ compared to healthy controls. BLC, sCD30, MCP-2 and IP-10 are specifically induced during early stages of COVID-19 infection and might constitute attractive targets for early diagnosis and treatment of this disease.

Assessment of Cardiovascular Risk in Women with Periodontal Diseases According to C-reactive Protein Levels

Cardiovascular diseases (CVD) are highly prevalent non-communicable diseases worldwide. Periodontitis may act as a non-traditional cardiovascular risk (CVR) factor, linked by a low-grade systemic inflammation mediated by C-reactive protein (CRP). Patients with periodontitis reported higher serum CRP levels; however, a CRP systemic and periodontal correlation in gingival crevicular fluid (GCF) and its CVR impact have been barely studied. We aimed to assess the association between periodontal diseases and CVR in a group of adult women, based on serum high-sensitivity CRP (hs-CRP) levels; and secondly, to determine the association between serum and GCF CRP levels. Gingival crevicular fluid and blood samples were obtained from women with periodontitis, gingivitis, and healthy controls. Serum and GCF CRP were determined by turbidimetric method and Luminex technology, respectively. Data were analyzed and adjusted by CVR factors. All women presented moderate CVR, without an evident association between serum hs-CRP levels and periodontal diseases. While serum hs-CRP concentrations did not significantly differ between groups, patients with gingivitis and periodontitis showed higher CRP levels in GCF, which positively correlated to CRP detection in serum.

Comparisons of Vitreal Angiogenic, Inflammatory, Profibrotic Cytokines, and Chemokines Profile between Patients with Epiretinal Membrane and Macular Hole

Objectives. Idiopathic epiretinal membrane (iERM) or idiopathic macular hole (iMH) is frequently used as a “healthy” control in comparison of vitreous cytokines with other vitreoretinal diseases. This study aimed to investigate if there is a difference in vitreal cytokines expression between patients with iERM and iMH. Methods. In this prospective study, all subjects received standard pars plana vitrectomy surgery, and 0.5 ml of native vitreous sample was extracted during the vitrectomy. Luminex technology and enzyme-linked immunosorbent assay were used to profile the concentration of 52 classic angiogenic, inflammatory, and profibrotic cytokines and chemokines. Statistical analyses were performed by the Mann–Whitney U test, followed by multiple comparisons by the Bonferroni correction. Results. Vitreal samples from 13 iERM and 24 iMH were studied. Of the 52 tested cytokines, 41 were similar in expression, and 5 were under the detection limit, while 6 cytokines (MMP-8, Eotaxin, MIP-1a, RANTES, TGF-β2, and IL-4) were differently expressed between two groups ( p < 0.05 ). Nevertheless, these significances disappeared after the adjustment of Bonferroni correction. Conclusion. The tested cytokines showed similar expression between iERM and iMH patients. This indicates that eyes with iERM or iMH can be together served as “healthy” controls.

Predictors of Oral Infection by Mucosal and Cutaneous Human Papillomaviruses in HIV-Infected and Uninfected Men Who Have Sex with Men of the OHMAR Study

Mucosal Human Papillomaviruses (HPVs) play a role in the development of a subset of head and neck cancers. Cutaneous HPVs are abundantly present in the oral cavity. The determinants of these infections have not been extensively investigated. We assessed the correlates of oral infection by alpha and beta and/or gamma HPVs in HIV-infected and uninfected men who have sex with men (MSM). Oral rinse-and-gargles were collected with a mouthwash. Alpha and beta/gamma HPVs were detected using the Linear Array HPV genotyping test and a multiplex PCR combined with Luminex technology, respectively. Multiple logistic regression was performed to identify independent predictors of oral HPV infection. Overall, 193 HIV-uninfected and 117 HIV-infected MSM were enrolled. Among HIV-infected MSM, the only determinant of alpha HPV infection was the number of lifetime oral sex partners (AOR: 8.26, 95% CI: 2.26–30.16). The strongest determinant of beta/gamma HPV infection was represented by practicing condomless receptive oral sex (AOR: 10.76, 95% CI: 1.56–74.17). Age was independently associated with alpha HPV infection in HIV-uninfected MSM. Beta/gamma HPV infection was not associated with sexual behavior in these subjects. In conclusion, predictors of oral infection differ between HIV-infected and uninfected MSM, as well as between alpha and beta/gamma HPVs.

Plasmodium falciparum and helminth coinfections increase IgE and parasite-specific IgG responses

Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites since they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (p≤ 0.05). There was a positive association between exposure/infection to P. falciparum and exposure/infection to helminths or the number of helminth species, and vice versa (p≤ 0.001). In addition, children coexposed/coinfected tended (p= 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host.

POS0176 COMBINING CXCL5 WITH CONVENTIONAL THERAPY PROVIDES DURABLE IMMUNOSUPPRESSION IN MURINE LUPUS NEPHRITIS

Background:Lupus nephritis (LN) is a condition arising from abnormal immune responses to internal organs. Patients with LN suffer from severe morbidity and mortality1, 2. Despite the aggressive regimen, 20-40% of patients do not respond to current conventional therapy. CXCL5, as a potent chemoattractant and activator of neutrophils3, demonstrates strong immunosuppression in the pre-clinical mouse model of graft versus host disease (GvHD)4 and lupus nephritis (LN) by intravenous administration.Objectives:In this study, we aim to evaluate whether the therapeutic effect of conventional therapy could be further improved by combination therapy with CXCL5.Methods:Ten doses of exogenous CXCL5 (3ug/kg, biweekly) together with conventional therapy (methylprednisolone (MP, intravenous (IV) injection with 8.3mg/kg/day at day-1, day-2 and day-3) + cyclophosphamide (CP, IV injection with 0.5g/BSA at day-4, monthly for 5 doses)) were administered to 8-week-old Faslpr mice by IV injection. Mice were monitored for 64 weeks. Splenic immune profile at 3 weeks post treatment (PT) was measured by flow cytometry. Circulating cytokine profile were detected by Luminex technology. Renal function was evaluated by urinary spot albumin creatinine ratio. In situ renal immune cell infiltration and complement 3 deposition were detected by Haematoxylin and Eosin (H&E) and immunohistochemistry staining.Results:Comparing to control mice (dPBS: 0% at 28 weeks PT), mice survival was improved to 100% at 40 weeks PT and 55.6% at 64 weeks PT by combination therapy of CXCL5 and conventional therapy (MP + CP) (p<0.0001). The accumulation of autoantibody (anti-dsDNA) and proteinuria were reduced 61.2-fold at 32 weeks PT (p=0.004) and 83.5-fold at 28 weeks PT (p=0.03) respectively. Both autoantibody and proteinuria were maintained at low level for 64 weeks. The classification of LN was significantly reduced at 10 weeks PT and equivalent to the classes we observed in pre-onset mice (p=0.004). Although combination therapy was not able to promote Tregs, it reduced both innate (neutrophils and macrophages) and adaptive (TH1, TH2 and TH17 cells and B cells) immunities significantly. Concomitantly, the serum level of endogenous CXCL5 was boosted up by exogenous administration from 74.2 +/- 53.9 pg/ml to 254.1 +/- 147.1 pg/ml at 8 weeks PT (p=0.05) and this relative high concentration was maintained for 48 weeks.Conclusion:Combining CXCL5 with conventional therapy provides effective and durable immunosuppression in murine LN and it may provide a new option for LN therapy.References:[1]Almaani S, Meara A, Rovin BH. Update on Lupus Nephritis. Clin J Am Soc Nephrol. May 8 2017;12(5):825-835. doi:10.2215/cjn.05780616.[2]Touma Z, Gladman DD. Current and future therapies for SLE: obstacles and recommendations for the development of novel treatments. Lupus Sci Med. 2017;4(1):e000239. doi:10.1136/lupus-2017-000239.[3]Koltsova EK, Ley K. The mysterious ways of the chemokine CXCL5. Immunity. Jul 23 2010;33(1):7-9. doi:10.1016/j.immuni.2010.07.012.[4]Fan X, Guo D, Cheung AMS, et al. Mesenchymal Stromal Cell (MSC)-Derived Combination of CXCL5 and Anti-CCL24 Is Synergistic and Superior to MSC and Cyclosporine for the Treatment of Graft-versus-Host Disease. Biol Blood Marrow Transplant. Jun 5 2018;doi:10.1016/j.bbmt.2018.05.029.Disclosure of Interests:None declared

POS0417 EXOGENOUS CXCL5 RESTORES ENDOGENOUS BLOOD-TISSUE CHEMOKINE GRADIENT TO IMPROVE SURVIVAL IN MURINE LUPUS

Background:Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease that is potentially fatal. There is an unmet need to improve current therapies. In patients with SLE, we observed that serum CXCL5 levels were significantly lower than healthy control subjects and negatively correlated with disease activity(1-9).Objectives:The aim of this study is to elucidate the effect of supplemental serum CXCL5 in abrogating the pathological processes of SLE.Methods:Ten doses of exogenous CXCL5 (3µg/kg) was administered to 16-week-old Faslpr mice weekly by intravenous injection. Mice were monitored for 10 weeks. Splenic immune profile was measured by flow cytometry. Circulating cytokine and immunoglobulin profile were detected by Luminex technology. Renal function was evaluated by urinary spot albumin creatinine ratio. In situ renal immune cell infiltration and complement 3 deposition were detected by Haematoxylin and Eosin (H&E) and immunohistochemistry staining. The molecular pathways involved were examined by RNA sequencing.Results:In Faslpr mice, intravenous administration of exogenous CXCL5 significantly improved mouse survival with concomitant reduction of autoantibody secretion, proteinuria, complement 3 deposition, neutrophil infiltration and lupus nephritis classes. Through evaluating the changes of immune profile, cytokine profile and molecular pathways, we found that intravenous CXCL5 reduced inflammation via an orchestral effect of regulating neutrophil trafficking and modulating helper T cell-mediated immune response. Pharmacokinetic and real-time Polymerase Chain Reaction studies further demonstrated that this orchestration was triggered by a cascade reaction - restoring vascular under-expressed CXCL5 by an exogenous stimulation, re-establishing the normal serum levels of endogenous CXCL5 and reverting the CXCL5 chemokine gradient between inflamed tissues and blood circulation.Conclusion:Managing the dysregulation of CXCL5 by exogenous supplement may provide a new option for SLE therapy.References:[1]Dufies M, Grytsai O, Ronco C, et al. New CXCR1/CXCR2 inhibitors represent an effective treatment for kidney or head and neck cancers sensitive or refractory to reference treatments. Theranostics. 2019;9(18):5332-5346. doi:10.7150/thno.34681[2]Yildirim K, Colak E, Aktimur R, et al. Clinical Value of CXCL5 for Determining of Colorectal Cancer. Asian Pac J Cancer Prev. Sep 26 2018;19(9):2481-2484. doi:10.22034/apjcp.2018.19.9.2481[3]Wu K, Yu S, Liu Q, Bai X, Zheng X. The clinical significance of CXCL5 in non-small cell lung cancer. Onco Targets Ther. 2017;10:5561-5573. doi:10.2147/ott.s148772[4]Zhao J, Ou B, Han D, et al. Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3beta/beta-catenin pathways. Mol Cancer. Mar 29 2017;16(1):70. doi:10.1186/s12943-017-0629-4[5]Han KQ, Han H, He XQ, et al. Chemokine CXCL1 may serve as a potential molecular target for hepatocellular carcinoma. Cancer Med. Oct 2016;5(10):2861-2871. doi:10.1002/cam4.843[6]Pappa CA, Tsirakis G, Kanellou P, et al. Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma. Cytokine. Dec 2011;56(3):616-20. doi:10.1016/j.cyto.2011.08.034[7]Zhang L, Li H, Ge C, et al. CXCL3 contributes to CD133(+) CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation. Sci Rep. Jun 3 2016;6:27426. doi:10.1038/srep27426[8]Matsubara J, Honda K, Ono M, et al. Reduced plasma level of CXC chemokine ligand 7 in patients with pancreatic cancer. Cancer Epidemiol Biomarkers Prev. Jan 2011;20(1):160-71. doi:10.1158/1055- 9965.epi-10-0397[9]Ma Y, Ren Y, Dai ZJ, Wu CJ, Ji YH, Xu J. IL-6, IL-8 and TNF-alpha levels correlate with disease stage in breast cancer patients. Adv Clin Exp Med. May-Jun 2017;26(3):421-426. doi:10.17219/acem/62120Disclosure of Interests:None declared