Characterization of ABH-subtype donor-specific antibodies in ABO-A-incompatible kidney transplantation.

ABO-incompatible (ABOi) transplantation requires preemptive antibody reduction; however, the relationship between antibody-mediated rejection (AMR) and ABO-antibodies, quantified by hemagglutination (HA), is inconsistent, possibly reflecting variable graft resistance to AMR or HA assay limitations. Using an ABH-glycan microarray, we quantified ABO-A antigen-subtype (A-subtype)-specific IgM and IgG in 53 ABO-O recipients of ABO-A kidneys, before and after antibody removal (therapeutic plasma exchange [TPE] or ABO-A-trisaccharide immunoadsorption [IA]) and 1-year posttransplant. IgM binding to all A-subtypes correlated highly (R2  ≥ .90) and A-subtype antibody specificities was reduced equally by IA versus TPE. IgG binding to the A-subtypes (II-IV) expressed in kidney correlated poorly (.27 ≤ R2  ≤ .69). Reduction of IgG specific to A-subtype-II was equivalent for IA and TPE, whereas IgG specific to A-subtypes-III/IV was not as greatly reduced by IA (p < .005). One-year posttransplant, IgG specific to A-II remained the most reduced antibody. Immunostaining revealed only A-II on vascular endothelium but A-subtypes II-III/IV on tubular epithelium. These results show that ABO-A-trisaccharide is sufficient for IgM binding to all A-subtypes; this is true for IgG binding to A-II, but not subtypes-III/IV, which exhibits varying degrees of specificity. We identify A-II as the major, but importantly not the sole, antigen relevant to treatment and immune modulation in adult ABO-A-incompatible kidney transplantation.

Immunoglobulin isotype compositions of ABO specific antibodies are dependent on the individual patient blood group and blood group specificity: Results from a healthy donor cohort.

Antibodies specific for the blood group ABO system antigens are of clinical significance and immunological interest. Routine clinical methods typically employ direct or indirect haemagglutination methods to measure IgM and IgG, respectively. We have developed a simple, single tube method to quantify IgM, IgG, and IgA specific for A and B antigens in order to improve accuracy and reproducibility, and to investigate the relationships between ABO group antibody type, and antibody level. Plasma samples from 300 healthy blood donors were studied. Levels of IgM and IgG binding to reagent group A and B red cells were measure by agglutination (HA) and multi-colour flow cytometry (MC-FC). IgA was also measured by MC-FC. Our FC method was found to be significantly more reproducible than HA for the measurement of blood group A and B specific antibodies. We found statistically significant correlations between antibodies measured by GC-HA and MC-FC, but sufficient differences to indicate that these methods are not equivalent. By MC-FC, IgM, IgG and IgA levels and isotope profiles were found to be dependent on both the donor ABO type and the specificity of the antibody. This study demonstrated heterogeneity in the immunoglobulin class profiles of ABO-blood group specific antibodies within the healthy population. Differences in isotype profiles of ABO-blood group specific antibodies may indicate fundamental differences in the immune mechanisms that generate these antibodies. This is likely to be relevant to the clinical situations where management or diagnosis depend on ABO-specific antibody detection and measurement.

PAF-R on activated T cells: Role in the IL-23/Th17 pathway and relevance to multiple sclerosis.

IL-23 is a potent stimulus for Th17 cells. These cells have a distinct developmental pathway from Th1 cells induced by IL-12 and are implicated in autoimmune and inflammatory disorders including multiple sclerosis (MS). TGF-ß, IL-6, and IL-1, the transcriptional regulator RORγt (RORC) and IL-23 are implicated in Th17 development and maintenance. In human polyclonally activated T cells, IL-23 enhances IL-17 production. The aims of our study were: 1). To validate microarray results showing preferential expression of platelet activating factor receptor (PAF-R) on IL-23 stimulated T cells. 2). To determine whether PAF-R on activated T cells is functional, whether it is co-regulated with Th17-associated molecules, and whether it is implicated in Th17 function. 3). To determine PAF-R expression in MS. We show that PAF-R is expressed on activated T cells, and is inducible by IL-23 and IL-17, which in turn are induced by PAF binding to PAF-R. PAF-R is co-expressed with IL-17 and regulated similarly with Th17 markers IL-17A, IL-17F, IL-22 and RORC. PAF-R is upregulated on PBMC and T cells of MS patients, and levels correlate with IL-17 and with MS disability scores. Our results show that PAF-R on T cells is associated with the Th17 phenotype and function. Clinical Implications Targeting PAF-R may interfere with Th17 function and offer therapeutic intervention in Th17-associated conditions, including MS.

Changes in natural killer cells and exhausted memory regulatory T Cells with corticosteroid therapy in acute autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436).

Clinical Potential of Regulatory T Cell Therapy in Liver Diseases: An Overview and Current Perspectives.

The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients.

Clinical outcomes with ABO antibody titer variability in a multicenter study of ABO-incompatible kidney transplantation in the United Kingdom.

BACKGROUND: ABO blood group-incompatible kidney transplantation (ABOiKTx) outcomes are good, but complications are more common than in conventional transplantation. Regimens that use extracorporeal antibody removal therapy (EART) and enhanced immunosuppression are guided by titration of ABO blood group antibodies (using hemagglutination [HA] dilution assays), and these assays vary significantly in performance between centers. This study aims to describe the differences in titer measurement and the effect on clinical practice and outcomes. STUDY DESIGN AND METHODS: This multicentre, prospective cohort study of 100 ABOiKTx recipients assessed treatment and outcome data, including HA assay results measured retrospectively in a single central laboratory. RESULTS: Patient and allograft survival at 1 year was 99% and 94%, respectively. There were significant differences in the number of pretransplantation EART sessions in centers undertaking plasma exchange (PEx), compared with immunoadsorption (IA) (median, 6 vs. 4 sessions; p = 0.007). The pre-EART HA titer in both groups was the same when centrally assayed. The local HA assay used to guide treatment yielded significantly higher titers in centers undertaking PEx compared with IA (median, 128 vs. 32; p < 0.005). Patients undergoing PEx rather than IA were significantly more likely to suffer postoperative hematoma (12.9% vs. 1.8%; p = 0.05) or any perioperative collection requiring drainage (19.4% vs. 3.6%; p = 0.02). CONCLUSION: The colinearity of HA assay sensitivity with the receipt of PEx and EART limits some conclusions regarding the likely direction of causation. However, the association of differences in clinical practice with recognized perioperative complications of ABOiKTx identifies targets for further investigation and quality improvement.

Human intrahepatic regulatory T cells are functional, require IL-2 from effector cells for survival, and are susceptible to Fas ligand-mediated apoptosis.

UNLABELLED: Regulatory T cells (Treg ) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal-endothelium (postendothelial migrated Treg [PEM Treg ]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg . Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)-2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL-2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL-2 in the inflamed liver. Liver-infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL-2 or blockade of CD95. CONCLUSION: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL-2 leads to impaired function and increased susceptibility of Treg to epithelial cell-induced Fas-mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL-2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138-150).

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1.

BACKGROUND & AIMS: Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. METHODS: The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. RESULTS: Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEß7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4ß7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. CONCLUSIONS: Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future.

Helicobacter pylori infection reduces disease severity in an experimental model of multiple sclerosis.

Recent research has demonstrated that infection with the bacterial pathogen Helicobacter pylori is less common amongst patients with multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). We aimed to compare the prevalence of H. pylori amongst MS patients and healthy controls, and also investigated the impact of this infection on an animal model for MS, experimental autoimmune encephalomyelitis (EAE). The H. pylori status of 71 MS patients and 42 healthy controls was determined by serology. Groups of C57BL/6 mice were infected with H. pylori, or given diluent alone as a placebo, prior to inducing EAE. Clinical scores were assessed for all mice, and spleens and spinal cord tissue were harvested. CD4(+) T cell subsets were quantified by flow cytometry, and T cell proliferation assays were performed. In MS patients the seroprevalence of H. pylori was half that of healthy controls (p = 0.018). Over three independent experiments, prior H. pylori infection had a moderate effect in reducing the severity of EAE (p = 0.012). In line with this, the antigen-specific T cell proliferative responses of infected animals were significantly reduced (p = 0.001), and there was a fourfold reduction in the number of CD4(+) cells in the CNS. CD4(+) populations in both the CNS and the spleens of infected mice also contained greatly reduced proportions of IFNγ(+), IL-17(+), T-bet(+), and RORγt(+) cells, but the proportions of Foxp3(+) cells were equivalent. There were no differences in the frequency of splenic CD4(+)cells expressing markers of apoptosis between infected and uninfected animals. H. pylori was less prevalent amongst MS patients. In mice, the infection exerted some protection against EAE, inhibiting both Th1 and Th17 responses. This could not be explained by the presence of increased numbers of Foxp3(+) regulatory T cells, or T cell apoptosis. This is the first direct experimental evidence showing that H. pylori may provide protection against inflammatory demyelination in the CNS.

Reciprocal Regulation of Substance P and IL-12/IL-23 and the Associated Cytokines, IFNγ/IL-17: A Perspective on the Relevance of This Interaction to Multiple Sclerosis.

UNLABELLED: The neuropeptide substance P (SP) exhibits cytokine-like properties and exerts different effects in autoimmune inflammation. Various immune cells express SP and its neurokinin-1 receptor (NK1R) isoforms. A role for SP has been demonstrated in a number of autoimmune conditions, including multiple sclerosis (MS). In this work, we studied the role of SP and NK1R in human immune cells with a focus on their relationship with IL-12/IL-23 family cytokines and the associated IFN-γ/IL-17. AIMS: (1) To determine the role of SP mediated effects on induction of various inflammatory cytokines in peripheral blood mononuclear cells (PBMC); (2) to investigate the expression of SP and its receptor in T cells and the effects of stimulation with IL-12 and IL-23. Quantitative real-time PCR, flow cytometry, ELISA, promoter studies on PBMC and primary T cells from healthy volunteers, and Jurkat cell line. Treatment with SP significantly increased the expression of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA in human PBMC. Expression of NK1R and SP in T cells was upregulated by IL-23 but a trend was observed with IL-12. The IL-23 effect likely involves IL-17 production that additionally mediates IL-23 effects. Mutual interactions exist with SP enhancing the cytokines IL-23 and IL-12, and SP and NK1R expression being differentially but potentially synergistically regulated by these cytokines. These findings suggest a proinflammatory role for SP in autoimmune inflammation. We propose a model whereby immunocyte derived SP stimulates Th1 and Th17 autoreactive cells migrating to the central nervous system (CNS), enhances their crossing the blood brain barrier and perpetuates inflammation in the CNS by being released from damaged nerves and activating both resident glia and infiltrating immune cells. SP may be a therapeutic target in MS.

IL-12 inhibits glucocorticoid-induced T cell apoptosis by inducing GMEB1 and activating PI3K/Akt pathway.

Interleukin (IL)-12 is an important pro-inflammatory cytokine that has been shown to play a role in T cell survival, at least in part by activating the PI3K/Akt pathway. Glucocorticoid modulatory element binding protein (GMEB)1 and 2 are closely related proteins that modify the glucocorticoid receptor binding locus and thus modulate glucocorticoid-mediated gene induction effects, including apoptosis. GMEB1 associates with caspases and prevents apoptosis of cells in the nervous system. We have observed, in preliminary studies, that IL-12 up-regulates GMEB mRNA in human T cells, and postulated that this may contribute to the anti-apoptotic effect of IL-12 on T cells, in particular with regard to glucocorticoid induced apoptosis. Here, we confirm that IL-12 rescue of dexamethasone induced T cell apoptosis involves the PI3K/Akt pathway and that IL-12 induces GMEB1 and GMEB2. A siRNA knockdown of GMEB1 reverses the protective effect of IL-12 on dexamethasone induced T cell apoptosis. Thus, IL-12 protects T cells from glucocorticoid induced apoptosis via PI3K/Akt pathway and via induction of GMEB1, which is likely to reduce transactivation of the glucocorticoid receptor and induction of apoptotic genes. As glucocorticoid induced apoptosis occurs both in physiological and pathological/therapeutic situations, and IL-12 is actively involved in a variety of inflammatory and immune responses, the ability of IL-12 to inhibit steroid responses and increase T cell survival through GMEB1 has wide ranging implications. Manipulating GMEB may be used therapeutically to enhance the resistance or the sensitivity to steroids.

Orexin A (hypocretin-1) levels are not reduced while cocaine/amphetamine regulated transcript levels are increased in the cerebrospinal fluid of patients with multiple sclerosis: no correlation with fatigue and sleepiness.

BACKGROUND: Fatigue and sleep disturbance are common features of multiple sclerosis (MS). Our objectives were to determine cerebrospinal fluid levels of orexin A (hypocretin-1), a hypothalamic peptide involved in sleep, in patients with MS, and correlate them with fatigue, sleepiness, and levels of cocaine and amphetamine regulated transcript (CART) another neuropeptide regulating metabolism with wider nervous system distribution. METHODS: Consecutive patients with MS (n=34), other inflammatory (n=24) or non-inflammatory (n=42) neurological diseases, undergoing lumbar puncture were investigated. Orexin and CART were measured by RIA by investigators unaware of the patients' diagnosis. RESULTS: Orexin A was slightly decreased in the cerebrospinal fluid of patients with inflammatory disease. There was no evidence of orexin A deficiency in MS, although there was a non-significant trend toward a decrease compared to non-inflammatory neurological diseases (p=0.06). CART levels were increased in MS compared to the non-inflammatory disease group (p=0.03). There were no significant correlations between CSF levels of orexin A and CART, fatigue, and hypersomnolence. CONCLUSIONS: Cerebrospinal fluid orexin A is decreased in CNS inflammatory diseases other than MS, where it shows a trend toward reduction, but does not correlate significantly with CART or with measures of fatigue and hypersomnolence.

The role of osteopontin in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS).

Osteopontin (OPN) is an extracelluar matrix protein with chemokine, cytokine and intergrin properties. It has multiple immunological functions and is secreted by activated macrophages, leukocytes and activated T lymphocytes. It is present in extracellular fluids and is up-regulated at sites of inflammation. OPN has intracellular and secreted isoforms. It has been shown to be involved in inflammation and autoimmune disorders, including multiple sclerosis. Multiple sclerosis (MS) is an immune mediated inflammatory disease of the central nervous system (CNS) in which autoreactive T cells attack the myelin-oligodendrocyte complex. Experimental autoimmune encephalomyelitis (EAE) is a widely used experimental model for MS. This review presents updated evidence for the role of OPN in MS and EAE, starting with the data provided by microarray analysis showing elevated levels of OPN transcripts in MS brain lesions and spinal cords of rats with EAE. This plausible target has since been validated in EAE, by showing that OPN knockout mice are protected from severe EAE. Increased levels of OPN were reported in plasma and CSF of MS patients in comparison to healthy controls. Potential mechanisms of OPN involvement in inflammatory demyelination are discussed. The involvement of OPN, in part via non-immune effects, in remyelination and its neuroprotective potential need to balanced against its pro-inflammatory properties.

Expression of activity-dependent neuroprotective protein in the immune system: possible functions and relevance to multiple sclerosis.

BACKGROUND: Activity-dependent neuroprotector (ADNP) is a neuroprotective molecule containing an 8-amino acid peptide, NAPVSIPQ (NAP), that is sufficient for its neuroprotective effects. OBJECTIVE: To assess the expression of ADNP in the human immune system in normal subjects and multiple sclerosis patients. MaterialsandMethods: ADNP expression was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and multiple sclerosis (MS) patients using staining with anti-ADNP (NAP) antibodies and markers for T cells, B cells, monocytes and natural killer cells. ADNP mRNA was determined in peripheral blood from MS patients (n = 24) and matched controls (n = 21). Expression of activation markers CD69 and CD154 and of IFN-gamma was assessed by flow cytometry in stimulated PBMCs. Effects of NAP on immune cell proliferation was assessed by tritiated thymidine incorporation. RESULTS: Monocytes, B cells and T cells, but not regulatory (CD4+CD25+) T cells expressed ADNP. NAP peptide decreased the expression of CD69, CD154 and IFN-gamma in PBMC and caused suppressed anti-CD3-/anti-CD28-stimulated PBMC proliferation. ADNP mRNA was reduced in MS compared to control peripheral blood. CONCLUSION: ADNP is expressed in many immune system cells. ADNP mRNA is reduced in PBMCs in MS. The peptide NAP, which plays an important role in neuroprotection, has potential immunomodulatory properties.

Increased osteopontin levels in the cerebrospinal fluid of patients with multiple sclerosis.

OBJECTIVE: To determine cerebrospinal fluid levels of osteopontin (OPN), a proinflammatory cytokine that was found to be overexpressed in multiple sclerosis lesions and increased in plasma during relapses and in secondary progressive multiple sclerosis. DESIGN: Case series. Osteopontin, interleukin 12p40 (IL-12p40), IL-10, and matrix metalloproteinase 9 were measured by enzyme-linked immunosorbent assay by an investigator unaware of the patients' diagnoses. PATIENTS: Consecutive patients with multiple sclerosis (n = 27), or other inflammatory (n = 11) or non-inflammatory (n = 23) neurological diseases, undergoing lumbar puncture, were investigated. RESULTS: Osteopontin was significantly elevated in the cerebrospinal fluid of patients with multiple sclerosis (mean [SD], 415 [186] ng/mL) and other inflammatory diseases (563 [411] ng/mL) compared with those with noninflammatory neurological diseases (286 [150] ng/mL). Cerebrospinal fluid OPN levels were slightly higher than plasma OPN levels. Cerebrospinal fluid OPN levels positively correlated with the ability to detect cerebrospinal fluid IL-12p40. CONCLUSION: Osteopontin in the cerebrospinal fluid may be, in part, of central nervous system origin, and may play an important role in central nervous system inflammation.