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BackgroundThe NLRP3 inflammasome is a critical multi-molecular platform involved in mediating innate immune responses, and could play a role in Adult onset Still’s disease (AOSD). Upon NLRP3 inflammasome activation, a large protein complex assembles, resulting in the re-localisation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), secretion of IL-1β and IL-18, and ASC aggregation into a single inflammasome speck. ASC specks are considered as potential biomarkers for inflammatory disorders, with subsequent release into the bloodstream during pyroptosis. Somatic variants in NLRP3 have also been observed in phenocopies of autoinflammatory disorders and their role in AOSD has been postulated.ObjectivesTo investigate the role of NLRP3-derived ASC protein specks as a potential disease activity biomarker andNLRP3genetic variants in the pathogenesis of AOSD.MethodsSera from 103 AOSD patients [treatment naïve, on treatment, and patients who previously failed several bDMARDs and were involved in CONSIDER clinical trial (n=30)] were analysed. These were compared to Systemic Juvenile Idiopathic Arthritis (sJIA, n=14), Cryopyrin Associated Periodic Syndrome (CAPS, n=9), Schnitzler’s syndrome (n=10) and Familial Mediterranean Fever (FMF, n=27) patients. Extracellular NLRP3-derived ASC specks were identified using flow cytometry (double positivity for ASC-PE and NLRP3-APC), gating for events around 1μm in size, in sera. To determine if ASC/NLRP3 speck levels provide additional biological information, the correlation between the specks and known biomarkers (such as IL-18 and C-reactive protein (CRP)) were analysed in sera. This involved cytokine profiling, using 13-plex Inflammatory LEGENDplex assay and high-sensitivity CRP ELISAs. Deep whole exome sequencing (WES x100) (analysis restricted to autoinflammatory panel) was carried out on DNA from the 30 CONSIDER trial patients.ResultsIn serological analyses, extracellular ASC/NLRP3 speck levels were increased in AOSD patients compared to 32 healthy control (HC) sera (p<0.01, Figure 1A). ASC/NLRP3 levels defined three subgroups of AOSD patients (low, moderate and high). High ASC/NLRP3 levels were present in all pre-treatment sera from CONSIDER trial patients (p< 0.001), compared to HC, suggesting their role may be dependent on the stage of the disease process. Interestingly, these patients were still responsive to canakinumab, showing significant reduction in extracellular ASC/NLRP3 levels in CONSIDER clinical trial cohort (baseline to week 12), compared to placebo (p<0.01) (Figure 1B) [1]. There was no correlation between ASC/NLRP3 specks and CRP levels (Figure 1C) or ASC/NLRP3 and total IL-18. No germline or somatic variants inNLRP3were identified from patients in the CONSIDER cohort despite very high levels of ASC/NLRP3 specks being detected in their serum.ConclusionThis study involved development of an assay that quantifies extracellular ASC specks as a biomarker of NLRP3 activation, to improve diagnoses and classification of SAIDs, particularly those with sporadic or unknown causes, such as AOSD. Increased levels of extracellular NLRP3-derived ASC specks were found in sera of AOSD compared to HC and autoinflammatory disease controls. Our findings also demonstrate heterogeneity within AOSD cohorts, with high-ASC speck levels in therapy-resistant CONSIDER trial patients suggesting the role of ASC specks may be dependent on the particular stage of the disease. Further analysis of rare somatic variants in genes associated with myelodysplastic syndrome, another condition associated with elevated ASC/NLRP3 specks and systemic inflammation, is currently underway, since this may provide an alternative explanation for our findings.Reference[1]Kedor C, Listing J, Zernicke J, ….. Feist E. Canakinumab for Treatment of Adult-Onset Still’s Disease to Achieve Reduction of Arthritic Manifestation (CONSIDER): phase II, randomised, double-blind, placebo-controlled, multicentre, investigator-initiated trial. Ann Rheum Dis. 2020;79(8):1090-1097.AcknowledgementsImmunAID, University of Leeds (LIRMM), CONSIDER clinical trial team and SOBI.Disclosure of InterestsJoanne Topping: None declared, James Poulter: None declared, Fatima Nadat: None declared, Clive Carter: None declared, Jan Zernicke Grant/research support from: GALAPAGOS Biopharma Germany GmbH. The CONSIDER clinical trial study was sponsored by Novartis., ImmunAID consortium Speakers bureau: ImmunAID is a European consortium of researchers whose members have been paid speakers for (but not limited to); AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris, Takeda, BioCryst, CSL Behring., Consultant of: ImmunAID is a European consortium of researchers whose members have been paid consultants for (but not limited to); AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris, Takeda, CSL Behring, BioCryst, KalVista., Grant/research support from: ImmunAID is a European consortium of researchers whose members have received financial grants from (but not limited to); AbbVie, CSL Behring, Lilly, MSD, Novartis and Pfizer., Jürgen Rech Speakers bureau: Sobi and Novartis., Paid instructor for: Sobi and Novartis., Consultant of: Sobi and Novartis., Grant/research support from: Sobi and Novartis., Bruno Fautrel Speakers bureau: AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris., Consultant of: AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris., Grant/research support from: AbbVie, Lilly, MSD and Pfizer., Eugen Feist Speakers bureau: Abbvie, Galapagos, Lilly, Novartis, Pfizer, Sanofi, Sobi., Consultant of: Consultant for Abbvie, Galapagos, Lilly, Novartis, Sanofi., Grant/research support from: Galapagos, Lilly, Novartis, Pfizer. The CONSIDER clinical trial study was sponsored by Novartis., Michael McDermott: None declared, Sinisa Savic Speakers bureau: Novartis, Sobi, Takeda, BioCryst, CSL Behring., Consultant of: Novartis, SOBI, Takeda, CSL Behring, BioCryst, KalVista., Grant/research support from: Novartis, CSL Behring.

Immune-mediated rejection following renal transplantation is essentially centred around two primary mechanisms. The presence of preformed antibodies against donor human leucocyte antigen (HLA) in sensitised recipients results in hyperacute rejection. Non-self HLA can also trigger a cell-mediated immune response in which a key event is the direct activation of CD4 + T-cells by non-self HLA class II molecules expressed on antigen-presenting cells present in the donor organ. Furthermore, recent reports have highlighted an association between increased MHC class I-related chain A/B (MICA/B) expression in graft tissue and immunological tissue damage and rejection following renal transplantation (1) . Protocols that decrease the expression of MICA/B or class II HLA proteins either pre-or post transplantation would be a potentially useful approach in a live donor setting. Whilst considerable efforts are made to control the recipient's immune system post transplant little attention is paid to this aspect in the donor pre-transplant. There are, however, a number of reports suggesting that pharmacological agents (e.g. statins) (2) as well as dietary products (most notably fish oils) (3) reduce the expression of HLA class II expression. Whilst administering statins to healthy donors before the transplant may be difficult to justify, the use of dietary products such as fish oils may be both beneficial and appropriate. The aim of the present study was to develop an in vitro assay in which the effects of potential immunomodulators on MICA/B and HLA-DR expression were measured. To this end, human T lymphocytes were stimulated with phytohaemagglutinin (PHA) and HLA-DR and MICA/B expression were measured by antibody staining and two-colour flow cytometry. Incubation of peripheral blood mononuclear cells with PHA for 3-5 d led to consistently increased HLA-DR expression. This was found to be the case both in the frequency (% positive) of CD3 + T-cells expressing HLA-DR and in the mean channel fluorescence (MCF) that estimates antigen density (Table). The expression of MICA/B following PHA stimulation was found to be considerably lower than HLA-DR and was considered to be too inconsistent for further consideration in this assay (Table). The kinetics of expression and the required concentration of PHA to give optimal expression as well as alternative stimulators (anti-CD3 stimulation) were also investigated. This in vitro system is now being used to assess the ability of dietary products to down regulate the expression of class II HLA-DR expression on T-cells following stimulation.

IntroductionWe have previously demonstrated an association between post-operative wound complications and systemic breast cancer recurrence (p&lt;0.0001), Murthy et al (2007) Br J Cancer 97, 1211-7. The aim of this study was to examine the potential role of the immune system in establishing this association, and therefore whether immune factors might be used to predict either wound complications or cancer recurrences.MethodsPatients with primary operable breast cancer were prospectively recruited to the study. Serial blood investigations were performed pre-operatively, peri-operatively and post-operatively. Absolute numbers of various lymphocyte cell populations were measured using multi-colour flow-cytometry including CD45+lymphocytes, CD19+ B lymphocytes, CD3+ T lymphocytes, CD4+ helper T cells, CD8+ cytotoxic T cells and CD56+ NK cells. We also measured the levels of the NK cytotoxicity receptors NKp30 and NKp46 on the NK cell population.ResultsOne hundred and nine patients were recruited to the study and there was a wound complication rate of 13.4%. Absolute numbers of CD3+ T lymphocytes, CD4+ helper T cells, CD8+ cytotoxic T cells and CD56+ NK cells were significantly lower 4 hours post-operatively compared to pre-operative levels (p&lt;0.05), although levels had typically recovered after 24 hours. However, NKp30 expression remained significantly reduced at 24 hours (p&lt;0.05). Mastectomy patients had a significantly greater fall in T lymphocyte numbers than those having breast conserving surgery (p&lt;0.05). Patients who went on to develop wound complications post-operatively had a significantly greater fall in their CD4+ helper T cells at 4 hours post-operatively, than those patients who did not go on to develop wound complications (p&lt;0.05).ConclusionsBreast cancer surgery results in severe disruption to the immune system, with dramatic changes in levels of immune regulatory blood cells populations. Changes are predominantly immuno-suppressive. The greater the immune disruption as a result of surgery, the more likely the patient is to develop a wound complication. We believe that this peri-operative immuno-suppression may also provide a window of opportunity for the successful dissemination of tumour cells post-operatively thereby increasing the risk of future metastases; we are maintaining follow up on this patient cohort in order to test correlations between peri-operative immuno-suppression and systemic recurrences. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4132.

Preformed donor-relevant antibodies are a contraindication to successful renal transplantation, causing irreversible damage to the endothelial cells on the donor organ that leads to vessel obstruction and swift rejection. Such sensitised patients represent a challenge to successful renal transplantation and individual treatment plans have become necessary in order to obtain a negative cross-match. Current methods used to treat the recipient before transplant are based on the removal of donor-relevant antibodies and include the use of high-dose intravenous Ig, immunoadsorption and plasmapheresis. All methods of reducing antibody levels require immunosuppression therapies in order to halt antibody re-synthesis. Whilst significant efforts are made to control the recipient's immune system, scant regard is given to direct modulation of the donor antigens present on the transplanted organ.In the present study a strategy was explored in which pre-conditioning of the donor, in a live donor situation, could lead to a transient reduction of human leucocyte antigen (HLA) expression at the time of transplant, thus reducing organ antigenicity. Several studies have shown that specific compounds can act as immunomodulatory agents. Whilst the use of prescription drugs in a healthy donor would be obviously inappropriate, n-3 fish oil (a widely-used food supplement) has been shown to have a direct effect on peripheral monocytes by inhibiting the expression of surface molecules involved in antigen presentation (1) . Although controversial, the relatively innocuous nature of fish oils indicates their potential as an acceptable immune-response modifier for use in the diet of the organ donor before transplant.In order to quickly screen a number of potential compounds, work was undertaken to establish an in vitro screen for HLA class II expression and other relevant cell-surface molecules on human monocytic cell lines. This procedure will be used to test potential compounds (health food or dietary supplements) to assess their ability to down regulate molecules involved in antigen presentation. The pre-monocytic cell line THP-1 was analysed by two-colour flow cytometry to measure baseline expression of CD14/HLA-DR. Cells were subsequently stimulated with lipopolysaccharide (LPS) for 24 h or 48 h and analysed for CD14/HLA-DR up-regulation. A representative example showing CD14 (FL-1)/HLA-DR(FL-2) expression from unstimulated and LPS-stimulated THP-1 cells is shown in the Figure.