Objectives. We compared the common pathway components C3a, C5a and membrane attack complex (MAC), also known as C5b-9, and the alternative pathway components factor B and properdin in patients with ANCA-associated vasculitis (AAV) and healthy controls, and conducted a meta-analysis of the available clinical evidence for the role of complement activation in the pathogenesis of AAV. Methods. Complement components were evaluated in 59 patients with newly diagnosed or relapsing granulomatosis with polyangiitis or microscopic polyangiitis and 36 healthy volunteers. In 28 patients, testing was repeated in remission. Next, we performed a meta-analysis by searching databases to identify studies comparing complement levels in AAV patients and controls. A random-effects model was used for statistical analyses. Results. The median concentrations of MAC, C5a, C3a, and factor B were higher in active AAV patients (p<0.001). Achievement of remission was associated with reductions in C3a (p=0.005), C5a (p=0.035), and factor B levels (p=0.045), whereas MAC and properdin levels did not change. In active AAV, there were no effects of ANCA specificity, disease phenotype, previous immunosuppression, or disease severity on complement levels. A total of 1122 articles were screened, and five studies, including this report, were entered in the meta-analysis. Plasma MAC, C5a, and factor B in patients with active AAV were increased compared to patients in remission (excluding factor B) and controls. Changes in C3a were of borderline significance. Conclusion. Our findings and the results of the meta-analysis support activation of the complement system predominantly via the alternative pathway in AAV patients.

Allergic sensitization is commonly assessed in patients by performing the skin prick test (SPT) or determining specific IgE levels in blood samples with the ImmunoCAP assay, which measures each allergen and sample separately. This paper explores the possibility to investigate respiratory allergies with a high throughput method, the Meso Scale Discovery (MSD) multiplex immunoassay, measuring IgE levels in low volumes of blood. The MSD multiplex immunoassay, developed and optimized with standards and allergens from Radim, was validated against the SPT and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients and 3 controls), blood collection and the SPT were performed within the same hour. Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay and the ImmunoCAP assay, except for house dust mite. The sensitivity of the MSD multiplexed assay was ≥75% for most allergens compared to the SPT and ImmunoCAP assay. Additionally, the specificity of the MSD multiplex immunoassay was ≥80% - the majority showing 100% specificity. Only the rye allergen had a low specificity when compared to the SPT, probably due to cross-reactivity. The reproducibility of the MSD multiplex immunoassay, assessed as intra- and inter-assay reproducibility and biological variability between different sampling moments, showed significantly high correlations (r=0.943-1) for all tested subjects (apart from subject 13; r=0.65-0.99). The MSD multiplex immunoassay is a reliable method to detect specific IgE levels against respiratory allergens in a multiplexed and high throughput way, using blood samples as small as from a finger prick.

Decreasing graft rejection and increasing graft and patient survival are great challenges facing liver transplantation (LT). Different T-cell subsets participates in the acute cellular rejection (ACR) of the allograft. Cell-mediated immunity markers of the recipient could help to understand the mechanisms underlying acute rejection. This study aimed to analyse CD4+CD154+ and CD8+CD154+ T-cells in a cohort of adult liver patients undergoing LT to determine the influence on ACR using multiparametric flow cytometry functional assay. Thiry patients were immunologically monitored at baseline and during 1 year post-transplant. Two groups were established, with (ACR) and without (NACR) acute cellular rejection. Leukocyte, total lymphocyte, percentages of CD4+CD154+ and CD8+CD154+ T-cells, HLA mismatch between recipient-donor and their relation with ACR as well as the acute rejection frequencies were analysed. T-cells were stimulated with concanavalin A (Con-A) and surface antigens were analysed by FACS analysis. A high percentage of CD4+CD154+ T-cells (p=0.001) and a low percentage of CD8+CD154+ T-cells (p=0.002) at baseline were statistically significant in ACR. A receiver operating characteristic analysis determined the cut-off values capable to stratify patients at high risk of ACR with high sensitivity and specificity for CD4+CD154+ (p=0.001) and CD8+CD154+ T-cells (p=0.002). In logistic regression analysis, CD4+CD154+, CD8+CD154+ and HLA mismatch were confirmed as independent risk factors to ACR. Post-transplant percentages of both T-cell subsets were significantly higher in ACR, despite variations compare to pre-transplant. These findings support the selection of candidates for LT based on the pre-transplant percentages of CD4+CD154+ and CD8+CD154+ T-cells in parallel with other transplant factors

Although increasing evidence demonstrates the association between intestinal dysbiosis and pancreatic diseases, such as chronic pancreatitis and pancreatic cancer, it remains largely unknown whether intestinal dysbiosis is involved in the immunopathogenesis of autoimmune pancreatitis (AIP). Recently, we found that intestinal dysbiosis mediates experimental AIP via the activation of plasmacytoid dendritic cells (pDCs), which can produce IFN-α and IL-33. However, candidate pathobionts for type 1 AIP have not been identified. In this study, we tried to identify pathobionts associated with type 1 AIP. Fecal samples were obtained from type 1 AIP patients before and after prednisolone (PSL) treatment and subjected to 16S ribosomal RNA sequencing to evaluate the composition of intestinal bacteria. Induction of remission by PSL was associated with the complete disappearance of Klebsiella species from feces, in two of the three analyzed patients with type 1 AIP. To assess the pathogenicity of Klebsiella species, mild experimental AIP was induced in MRL/MpJ mice by repeated injections of 10 μg of polyinosinic-polycytidylic acid (poly (I:C)) in combination with oral administration of heat-killed Klebsiella pneumoniae. The AIP pathology score was significantly higher in MRL/MpJ mice that received both oral administration of heat-killed K. pneumoniae and intraperitoneal injections of poly (I:C) than in those administered with either agent alone. Pancreatic accumulation of pDCs capable of producing large amounts of IFN-α and IL-33 was also significantly higher in mice that received both treatments. These data suggest that intestinal colonization by K. pneumoniae may play a pathogenic role in AIP.

Abstract In recent years, immune checkpoint therapy to reverse NK and T cell exhaustion has emerged as a promising treatment in various cancers. Recently, the FDA has approved anti-PD-1 pembrolizumab for patients with recurrent or metastatic cervical cancer. Other checkpoint molecules, such as TIGIT and Tim-3 have yet to be fully explored in this disease. Here, we found that PD-1, TIGIT, and Tim-3 are over-expressed on some peripheral blood CD56dim and CD56bright NK cells and T cells in cervical cancer patients and women with premalignant lesions. However, we observed stronger significance and separation between groups when these three molecules were examined together. These cells, with an apparently “exhausted” phenotype, were significantly augmented in patients. Different PD-1 levels (PD1low, PD1int, PD-1hi) on T cells were used to further define checkpoint positive populations. Soluble PD-L1 was observed to be increased in cervical cancer. Within the cancer group, the highest levels of sPD-L1 and triple positive or double positive cells and tumor stage were found similarly within tumors of different stages. Our results might show an overview of what is happening in patients with precancerous lesions and cervical cancer, and may give an early clue as to whom to administer monoclonal checkpoint blocking therapies.

The aim of this multicenter retrospective study was to evaluate the incidence of hyperprogressive disease after treatment with pembrolizumab as a second-line treatment in patients (n=167) with non-small-cell lung cancer (NSCLC) with metastatic disease whose tumors expressed programmed death-ligand-1 in ≥1% and to search for factors associated with its development. All patients received platinum-containing chemotherapy as a first-line treatment. The neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and their derivations were retrospectively analyzed. The psoas major muscle area (PMMA) was calculated at the L3 position on computed tomography before chemotherapy and immunotherapy. Patients with ∆PMMA≥10% were considered to have sarcopenia (low muscle mass). We also performed multinomial logistic regression to estimate the effects of hematological biomarkers and ∆PMMA on the response to immunotherapy. Hyperprogressors (HPs) had significantly higher NLRs, PLRs and ∆PMMA levels than the other patients. Moreover, in multivariate regression analysis, higher levels of ∆PMMA were associated with a decreased likelihood of being a progressor (P) (OR, 0.81; 95% CI, 0.65-0.99; p=0.047) or a nonprogressor (NP) (OR, 0.76; 95% CI, 0.62-0.94; p=0.012) vs an HP. In multivariate analysis, higher NLRs tended to decrease the likelihood of being a P vs an HP (OR, 0.66; 95% CI, 0.42-1.06; p=0.09) and significantly decrease the likelihood of being an NP vs an HP (OR, 0.44; 95% CI, 0.28-0.69; p<0.0001). Our data suggest that a high pre-immunotherapy NLR and the presence of sarcopenia are potential risk factors for the development of hyperprogressive disease.

Background. Anti-citrullinated protein/peptide antibodies (ACPA) play important roles in the pathogenesis of rheumatoid arthritis (RA). ACPA-positive (ACPA+) and ACPA-negative (ACPA-) RA were suggested to be different disease subsets with distinct differences in genetic variation and clinical outcomes. The aims of the present study were to compare gene expression profiles in ACPA+ and ACPA- RA and identify novel candidate gene signatures that might serve as therapeutic targets. Methods. Comprehensive transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from ACPA+ and ACPA- RA patients, and healthy controls was performed via RNA sequencing. A validation cohort was used to further investigate differentially expressed genes via PCR and ELISA. Spearman’s correlation test was used to evaluate the correlation of differentially expressed genes and the clinical and laboratory data of the patients. The role of differentially expressed genes in osteoclastogenesis was further investigated. Results. Expression of C-X-C motif chemokine ligand 2 (CXCL2) was significantly increased in ACPA+ RA than in ACPA- RA, which was validated in PBMCs and serum. CXCL2 promoted the migration of CD14+ monocytes and increased osteoclastogenesis in RA patients. RAW264.7 macrophages were used to investigate specific mechanisms, and the results suggested that CXCL2 stimulated osteoclastogenesis via ERK MAPK and NFκB pathways. Conclusion. CXCL2 was highly expressed in ACPA+ RA than in ACPA- RA. CXCL2 promoted osteoclastogenesis and was related to bone erosion in RA, which suggest that the blockade of CXCL2 might be a novel strategy for the treatment of RA.

Tuberculosis (TB) is one of the top 10 causes of mortality worldwide from a single infectious agent and has significant implications for global health. In 2018, 1.5 million people died from TB worldwide and 440,000 of those were from India. The WHO End-TB strategy aims to reduce TB deaths by 95% and new TB cases by 90% by 2035, with a call for more basic research on TB pathogenesis and immunity. A major hurdle in the development of effective TB vaccines and therapies is the absence of defined immune-correlates of protection. In this context, the role of regulatory T cells (Treg), which are essential for maintaining immune homeostasis, is even less understood. This review aims to address this knowledge gap by providing an overview of the emerging patterns of Treg function in TB. The review also provides a comprehensive critical analysis of the key features of Treg cells in TB; highlights the importance of a balanced immune response as being important in TB and discusses the importance of probing not just Treg frequency but also qualitative aspects of Treg function as part of a comprehensive search for novel TB treatments.

Growing evidence shows that a homozygous 6.7-kb deletion of the novel anti-inflammatory molecule leukocyte immunoglobulin like receptor A3 (LILRA3) is associated with many autoimmune disorders. However, its effects on pathogenesis of inflammatory bowel disease (IBD) have not been clarified yet. LILRA3 is mainly expressed in monocytes, whereas its effects on biological behaviors of monocytes have not been systematically reported. To investigate the association between LILRA3 polymorphism and IBD susceptibility, LILRA3 polymorphism was assessed in 378 IBD patients and 509 healthy controls in our study, quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC) were employed to detect the LILRA3 expression in IBD patient blood and intestinal samples. Despite no association of the polymorphism with IBD development was found, LILRA3 expression was markedly increased in IBD patients compared with healthy controls. Human U937 monocyte cell line was employed to establish LILRA3-overexpressing cells and the effects of LILRA3 on the biological behaviors of U937 cells were systematically explored. We found that overexpression of LILRA3 in monocytes led to significant decreases in secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Additionally, LILRA3 abated monocyte migration by reducing the expression of several chemokines and enhanced monocyte phagocytosis by increasing CD36 expression. Furthermore, LILRA3 promoted monocyte proliferation through a combination of Akt and MEK/Erk signaling pathways. We report for the first time that LILRA3 is related to IBD and functions as an anti-inflammatory modulator in U937 cells.

Peroxiredoxins (PRXs) are intracellular antioxidative enzymes but work as inflammatory amplifiers under the extracellular condition. To date, the function of PRXs in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) is not fully understood. The aim of this study was to investigate whether PRXs play a role in the pathogenesis of MS and NMOSD. We analyzed levels of PRXs (PRX1, PRX5, and PRX6) in the CSF and serum of 16 patients with MS, 16 patients with NMOSD, and 15 patients with other neurological disorders (ONDs). We identified potential correlations between significantly elevated PRXs levels and the clinical variables in patients with MS and NMOSD. Additionally, pathological analyses of PRXs (PRX1-6) in the central nervous system were performed using the experimental autoimmune encephalomyelitis (EAE), animal model of MS. We found that serum levels of PRX5 and PRX6 in patients with MS and NMOSD were higher compared with those in patients with ONDs (p < 0.05). Furthermore, high levels of PRX5 and PRX6 were partly associated with blood–brain barrier dysfunction and disease duration in NMOSD patients. No significant elevation was found in CSF PRXs levels of MS and NMOSD. Spinal cords from EAE mice showed remarkable PRX5 staining, especially in CD45+ infiltrating cells. In conclusion, PRX5 and PRX6 may play a role in the pathogeneses of MS and NMOSD.

Background: Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier suggesting a potential effect on CNS resident cells. Objective: We examined, if CdA modifies the phenotype and function of naïve and activated primary mouse microglia, when applied in different concentrations including 0.1-1 µM that putatively overlaps human CSF concentrations. Methods: Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory IL-4 were co-treated with different concentrations of CdA for 24 hours. Viability was assessed by MTT assay. Phagocytotic ability and morphology were examined by flow cytometry, and random migration by IncuCyte Zoom and TrackMate. Change in gene expression was examined by qPCR, and protein secretion by Meso Scale Discovery. Results: LPS and IL-4 upregulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0.1-1 µM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 µM increased the IL-4-induced gene expression of Arg1 and LPS-induced expression of IL-1beta, TNF, iNOS, and Arg1, but protein secretion remained unaffected. CdA 10 µM potentiated the increased expression of anti-inflammatory TNFR2 but not TNFR1 induced by LPS. Conclusion: Microglia acquire a less activated phenotype when treated with 0.1–1 µM CdA that putatively overlaps human CSF concentrations. This may be related to the upregulated gene expression of DCK upon activation and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

Properdin is the only one positive regulator of the complement system. In this study, we characterize the prevalence, functional consequences and disease associations of autoantibodies against properdin in a cohort of patients with autoimmune disease systemic lupus erythematosus (SLE), suffering from lupus nephritis (LN). We detected autoantibodies against properdin in plasma of 22.5% of the LN patients (16/71) by ELISA. The binding of these autoantibodies to properdin was dose-dependent and was validated by surface plasmon resonance. Higher levels of anti-properdin were related to high levels of anti-dsDNA and ANA and to low concentrations of C3 and C4 in patients and also with histological signs of LN activity and chronicity. The high negative predictive value (NPV) of anti-properdin and anti-dsDNA combination suggested that patients who are both negative for anti-properdin and anti-dsDNA will not have severe nephritis. IgG from anti- properdin positive patients’ plasma increased the C3b deposition on late apoptotic cells by flow cytometry. Nevertheless, these IgGs did not modify substantially the binding of properdin to C3b, the C3 convertase C3bBb and the pro-convertase C3bB, evaluated by surface plasmon resonance. In conclusion, anti- properdin autoantibodies exist in LN patients. They have weak but relevant functional consequences, which could have pathological significance.

The aim of this study was to investigate the pathogenesis of combination ipilimumab and nivolumab-associated colitis (IN-COL) by measuring gut-derived and peripheral blood mononuclear cell (GMNC; PBMC) profiles. We studied GMNC and PBMC from patients with IN-COL, IN-treated with no adverse-events (IN-NAE), ulcerative colitis (UC) and healthy volunteers by flow cytometry. In the gastrointestinal-derived cells we found high levels of activated CD8+ T cells and mucosal-associated invariant T (MAIT) cells in IN-COL, changes that were not evident in IN-NAE or UC. UC but not IN-C was associated with a high proportion of regulatory T cells (Treg). We sought to determine if local tissue responses could be measured in peripheral blood. Peripherally, checkpoint-inhibition instigated a rise in activated memory CD4+ and CD8+ T cells, regardless of colitis. Low circulating MAIT cells at baseline was associated with IN-COL patients, compared with IN-NAE in one of two cohorts. UC but not IN-COL was associated with high levels of circulating plasmablasts. In summary, the alterations in T cell subsets measured in IN-COL-affected tissue, characterised by high levels of activated CD8+ T cells and MAIT cells and a low proportion of Treg, reflected a pathology distinct from UC. These tissue changes differed from the periphery, where T cell activation was a widespread on-treatment effect, and circulating MAIT cell count was low but not reliably predictive of colitis (Figure1).

Background: Pulmonary sarcoidosis is characterized by an exaggerated CD4+ T-cell response and formation of non-necrotizing granulomas. Tumour necrosis factor α (TNF-α) is regarded as crucial for granuloma formation and TNF-α inhibitors offer a 3rd line treatment option for patients not responding to conventional treatment. However, not all patients benefit from treatment, and an optimal dose and treatment duration have not been established. Insight into the influence of TNF-α inhibitors on lung immune cells may provide clues to what drives inflammation in sarcoidosis and improve our understanding of treatment outcomes. Objectives: To evaluate effects of treatment with the TNF- α inhibitor infliximab on lung immune cells and clinical features of the patients. Methods: Thirteen patients with sarcoidosis refractory to conventional treatment were assessed with bronchoalveolar lavage (BAL), spirometry and CT scan in close adjacent to start of infliximab treatment. These investigations were repeated after six months of treatment. Results: Treatment with TNF- α inhibitor infliximab was well tolerated with no adverse events, except for one patient who developed a probable adverse event with liver toxicity. Ten patients were classified as responders, having a reduced CD4/CD8 ratio, a decreased percentage of CD4+ T-cells expressing the activation marker CD69 and number of mast cells (p<0.05 for all). The percentage of T regulatory cells (Tregs), defined as FoxP3+ CD4+ T-cells decreased in most patients. Conclusions: Six months of infliximab treatment in patients with sarcoidosis led to signs of decreased CD4+ T-cell alveolitis and decreased mastocytosis in the lungs of responders.

Backgroud: A long-term hepatic inflammatory response is a risk factor for liver cancer initiation and progression. Interleukin (IL)-35 is the newest member of the IL-12 cytokine family, and has been reported to play an essential role in the immunosuppressive liver microenvironment. Herein we focus on the expression profiles of IL-35 in hepatocellular carcinoma (HCC) and the effect on local immune status. Methods: HCC transcriptome array data were downloaded from Gene Expression Omnibus. The bioinformatics analysis was performed by the BRB array tools and online Ingenuity Pathway Analysis software. The serum IL-35 level was detected by AimPlet bead-based immunoassay. In situ IL-35 expression detection was performed by immunohistochemical staining and western blot. Results: Our results showed that there were large amounts of IL-35 expressed in HCC serum and tumor tissues. IL-35 expression affects the transcript of thousands of genes, most of which correlated with T-cell immunity. This study proved that enhancement of regulatory T cells (Tregs) and impairment of cytolytic T cells are prominent effects of IL-35. Conclusions: Elevated IL-35 played critical roles in HCC patients through affecting the balance between Tregs and cytotoxic T cells. Dissection of the precise targets and the underlying molecular mechanisms will lead to alternative treatments for HCC patients.

Though the pathogenesis of acute myeloid leukemia (AML) is still unknown, accumulating evidence has revealed that immune response plays a vital part in the pathogenesis. Here, we investigated the involvement of 24 single-nucleotide polymorphisms (SNPs) of immuno-related genes, including cytokines (IL2, IL4, IL9, IL-12A, IL-22, IFNG, and TGFB1), transcriptional regulatory genes (TBX21, STAT1, STAT3, STAT5B, STAT6, GATA3, FOXP3, and IRF4), and others (IL2RA IL6R NFKBIA), in 269 AML inpatients and 200 healthy controls. Furthermore, we analyzed the relationship between the SNPs and clinical characteristics. Immuno-related SNP genotyping was performed on the Sequenom MassARRAY iPLEX platform. All the SNPs in healthy controls were consistent with Hardy–Weinberg equilibrium. All final p values were adjusted by Bonferroni multiple testing. Our results showed that IL-22 (rs2227491) was significantly associated with the white blood cell (WBC) counts. STAT5B (rs6503691) showed a close relationship with the recurrent genetic abnormalities in patients with AML. We verified the negatively independent effect of age and risk of cytogenetics on overall survival (OS). More importantly, the GG genotype of IL-12A (rs6887695) showed a negative impact on AML prognosis independently. Furthermore, the relative expression of IL-12 was decreased in GG genotype, no matter under codominant or recessive model. However, no correlation was observed between the SNPs mentioned above and disease susceptibility, risk stratification, and survival. Our findings suggest that immuno-related gene polymorphisms are associated with prognosis in AML, which may perform as novel inspection targets for AML patients.

Background NOD-like receptor pyrin 7 (NLRP7) has been identified as the major gene responsible for the recurrent hydatidiform mole (RHM). The immunological role of NLRP7 mutation in HM patients has not been conclusively demonstrated. Hence, we aim to demonstrate this role in our study. Methods We followed 12 new patients with NLRP7 nonsynonymous variations (NSVs) from date to date. Peripheral blood mononuclear cells (PBMCs) were collected from patients with and without NLRP7 mutation, separately. Supernatant IL-1β secretion, intracellular pro-IL-1β and mature-IL-1β expressions were measured after 24h lipopolysaccharide (LPS) stimulation. Plasmids with corresponding NSVs were generated to evaluate the ability of processing pro-IL-1β into mature-IL-1β in vitro. Results Homozygous or compound heterozygous NLRP7 mutation secreted less IL-1β in root of abnormal intracellular pro-IL-1β or mature-IL-1β according to different domain defective. Plasmids with NSVs could also affect processing or/and trafficking together with caspase-1 and apoptosis-associated speck-like protein (ASC). Conclusion Inflammasome related NLRP7 mutation is a potential mechanism of RHM.