As vaccine-elicited antibodies have now been associated with HIV protective efficacy, a thorough understanding of mucosal and systemic B-cell development and maturation is needed. We phenotyped mucosal memory B-cells, investigated isotype expression and homing patterns, and defined plasmablasts and plasma cells at three mucosal sites (duodenum, jejunum and rectum) in rhesus macaques, the commonly used animal model for pre-clinical vaccine studies. Unlike humans, macaque mucosal memory B-cells lacked CD27 expression; only two sub-populations were present: naïve (CD21+CD27−) and tissue-like (CD21−CD27−) memory. Similar to humans, IgA was the dominant isotype expressed. The homing markers CXCR4, CCR6, CCR9 and α4β7 were differentially expressed between naïve and tissue-like memory B-cells. Mucosal plasmablasts were identified as CD19+CD20+/−HLA-DR+Ki-67+IRF4+CD138+/− and mucosal plasma cells as CD19+CD20−HLA-DR−Ki-67−IRF4+CD138+. Both populations were CD39+/−CD27−. Plasma cell phenotype was confirmed by spontaneous IgA secretion by ELISpot of positively-selected cells and J-chain expression by real-time PCR. Duodenal, jejunal and rectal samples were similar in B-cell memory phenotype, isotype expression, homing receptors and plasmablast/plasma cell distribution among the three tissues. Thus rectal biopsies adequately monitor B-cell dynamics in the gut mucosa, and provide a critical view of mucosal B-cell events associated with development of vaccine-elicited protective immune responses and SIV/SHIV pathogenesis and disease control.
SIV/SHIV rhesus macaque model; mucosal memory B cell phenotypes and distribution; homing markers; plasmablasts/plasma cells
Necrosis, an inflammatory form of cell death, has been considered to be an accidental death and/or cell death due to injury. However, the literature in the last decade has established that necrosis is a regulated form of cell death, and that inhibition of specific molecular pathways leading to necrosis can block it and reduce inflammation. Since necrotic lesions are observed in several immune mediated human pathologies, in this review we will discuss the impact that this form of programmed cellular demise has in the pathology of immune mediated nephropathies.
Nephritis; Autoimmunity; Cell Death; Necrosis
Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune
system might improve protective efficacy. We compared SIV-specific humoral immunity
induced in rhesus macaques by five vaccine regimens. Systemic regimens included
ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization;
and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal
replication-competent Ad-env priming with systemic Env boosting. A
Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by
MVA-env and Env boosts. Serum antibodies mediating neutralizing,
phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env
vaccines. Memory B cells and plasma cells were maintained in bone marrow. RepAd/Env
vaccination induced early SIV-specific IgA in rectal secretions before Env boosting,
although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env,
RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest combined RepAd
priming with ALVAC/Env or DNA&Env regimen boosting might induce potent,
functional, long-lasting systemic and mucosal SIV-specific antibodies.
Simian Immunodeficiency Virus; poxvirus-, adenovirus-, and DNA-based vaccines; mucosal and systemic humoral immunity; memory B cells; functional antibody activities
Sjogren’s syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17 Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.
Plasmacytoid dendritic cells (pDCs) are bone marrow-derived immune cells with the ability to express copious amounts of type I and III interferon (IFN) and can differentiate into antigen-presenting dendritic cells as a result of stimulation by pathogen-derived nucleic acid. These powerful combined functionalities allow pDCs to bridge the innate and adaptive immune systems resulting in a concerted pathogen response. The contribution of pDCs to gastrointestinal immunity is only now being elucidated and is proving to be a critical component in systemic immunity. This review will explore the immunology of pDCs and will discuss their involvement in human disease and tolerance with an emphasis on those in the gastrointestinal lymphoid tissue.
GALT; HIV; interferon; pDC; gut; tolerance; autoimmunity
Noninvasive carotid measurements have independent value in the estimation of future cardiovascular (CV) outcomes in systemic lupus erythematosus (SLE). Natural IgM-antibodies to phosphorylcholine (PC) epitopes can enhance apoptotic-cell clearance and induce anti-inflammatory pathways. Herein, we show that subclinical CV disease, as detected by carotid ultrasound, in a cross-sectional SLE cohort was associated with lower levels of IgM anti-PC, as well as lower levels of the ratio of IgM anti-PC/total IgM, compared to patients without plaque (p=0.004 and p=0.02, respectively). The IgM anti-PC/total IgM association remained significant after adjusting for age, cholesterol and hypertension. Adiponectin and sE-selectin were significantly elevated in patients with plaque, and statistical models showed that combining adiponectin, sE-selectin and IgM anti-PC/total IgM was better for predicting plaque than either test alone.
These results support the hypothesis that IgM-natural autoantibodies may inhibit atherogenesis, and confirms the utility of IgM anti-PC levels as a biomarker for subclinical CV disease.
IgM; phosphorylcholine; systemic lupus erythematosus; atherosclerosis; adiponectin; E-selectin; IMT
To prepare a novel Bispecific Antibody (BsAb) as a potential targeted therapy for T1D, we produced a “functionally inert” monoclonal antibody (mAb) against Glucose transporter-2 (GLUT-2) expressed on β-cells to serve as an anchoring antibody. The therapeutic arm is an agonistic mAb against Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), a negative regulator of T-cell activation expressed on activated CD4+ T-cells. A BsAb was prepared by chemically coupling an anti-GLUT2 mAb to an agonistic anti-CTLA-4 mAb. This BsAb was able to bind to GLUT2 and CTLA-4 in vitro, and to pancreatic islets, both in vitro and in vivo. We tested the safety and efficacy of this BsAb by treating Non-Obese Diabetes (NOD) mice and found that it could delay the onset of diabetes with no apparent undesirable side effects. Thus, engagement of CTLA-4 on activated T cells from target tissue can be an effective way to treat type-1 diabetes.
anti-CTLA-4; anti-Glut2; dendritic cells; regulatory T cells; diabetes; tolerance
Ischemia, lack of blood flow, and reperfusion, return of blood flow, is a common phenomenon affecting millions of Americans each year. Roughly 30,000 Americans per year experience intestinal ischemia-reperfusion (IR), which is associated with a high mortality rate. Previous studies of the intestine established a role for neutrophils, eicosanoids, the complement system and naturally occurring antibodies in IR-induced pathology. Furthermore, data indicate involvement of a lipid or lipid-like moiety in mediating IR-induced damage. It has been proposed that exposure of neo-antigens are recognized by antibodies, triggering action of the complement cascade. While it is evident that the pathophysiology of IR-induced injury is complex and multi-factorial, we focus this review on the involvement of eicosanoids, phospholipids and neo-antigens in the early pathogenesis. Lipid changes occurring in response to IR, neo-antigens exposed and the role of a phospholipid transporter, phospholipid scramblase 1 will be discussed.
ischemia; reperfusion; lipids; neo-antigen
The autosomal recessive form of the Hyper IgE syndrome (AR-HIES) with dedicator of cytokinesis 8 (DOCK8) deficiency is associated with difficult to treat persistent viral skin infections, including papilloma virus infection. Type I interferons play an important role in the defense against viruses. We examined the effect of therapy with IFN–α 2b in an 11-year old boy with DOCK8 deficiency due to a homozygous splice donor site mutation in DOCK8 intron 40. His unremitting warts showed dramatic response to IFN–α 2b therapy. Immunological studies revealed decreased circulating plasmacytoid dendritic cells (pDCs) and profound deficiency of IFN-α production by his peripheral blood mononuclear cells in response to treatment with CpG oligonucleotides. These findings indicate that underlying pDC deficiency and impaired IFN-α production may predispose to chronic viral infections in DOCk8 deficiency. IFN–α 2b therapy maybe useful in controlling recalcitrant viral infections in these patients.
Combined Immunodeficiency; Dedicator of Cytokinesis 8; DOCK8; Hyper Immunoglobulin E syndrome; interferon–α 2b; Warts; Papilloma Virus
Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.
SLE; anti-DNA antibody; antigenicity; spermine; polyamines
Natural killer T (NKT) cells are known to play a role against certain microbial infections, including malaria and HIV, two major global infectious diseases. Strategies that acn harness and amplify the immunotherapeutic potential of NKT cells can serve as powerful tools in the fight against such diseases. 7DW8-5, a novel glycolipid, may be one such tool. The interaction of 7DW8-5 with CD1d molecules induces activation of NKT cells, thereby activating various immune-competent cells including dendritic cells (DCs) to provide a significant adjuvant effect for several vaccines. This review discusses the discovery and characterization of 7DW8-5 and the practical considerations of its preclinical and clinical development as a potential glycolipid adjuvant for candidate malaria and HIV vaccines.
NKT cell; Glycolipid; CD1d; 7DW8-5; HIV; Malaria; Vaccine; Adjuvant
Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.
Microvesicle; anti-DNA; lupus; autoimmunity; apoptosis
This study was undertaken to test the hypothesis that Sjogren’s syndrome Antigen A (SSA)/Ro60-reactive T cells are activated by peptides originating from oral and gut bacteria. T cell hybridomas generated from HLA-DR3 transgenic mice recognized 3 regions on Ro60, with core epitopes mapped to amino acids 228-238, 246-256 and 371-381. BLAST analysis identified several mimicry peptides, originating from human oral, intestinal, skin and vaginal bacteria, as well as environmental bacteria. Amongst these, a peptide from the von Willebrand factor type A domain protein (vWFA) from the oral microbe Capnocytphaga ochracea was the most potent activator. Further, Ro60-reactive T cells were activated by recombinant vWFA protein and whole E. coli expressing this protein. These results demonstrate that peptides derived from normal human microbiota can activate Ro60-reactive T cells. Thus, immune responses to commensal microbiota and opportunistic pathogens should be explored as potential triggers for initiating autoimmunity in SLE and Sjögren’s syndrome.
Sjögren’s syndrome; SLE; Ro60/SSA; Molecular Mimicry; T Epitopes; Microbiota
Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults. Elevations in CD8+CD146+ T cells are found in as Behcet’s disease and birdshot retinochoroidopathy, which have been reported to have HLA class I associations. Sarcoidosis does not have a class I association and displays an increase in CD4+ CD146+ T cells but not in CD8+CD146+ T cells. CD146 on these cells may facilitate their ability to bind to, and migrate through, endothelium, as has been reported for CD4+CD146+ T cells.
CD146/MCAM; Tc17; inflammation; class I antigen
Individuals carrying DRB1*0401 who smoke cigarettes are at an increased risk of developing severe seropositive RA. To determine how cigarette smoke (CS) interacts with host genetic factors in the induction of RA-associated autoimmunity, we used transgenic mice carrying the RA-susceptible HLA genes DR4 and DQ8, but lacking all endogenous murine class II molecules. Cigarette smoke exposure augmented peptidylarginine deiminase (PAD) enzyme expression, and enhanced immune responses to citrullinated collagen and vimentin. Here we show for the first time that DQ molecules can present citrullinated peptides much more efficiently than native peptides. Interestingly, CS exposure suppressed collagen-induced arthritis (CIA) in DRB1*0401 mice although innate immune response was enhanced. On the other hand, CS exposure exacerbated CIA in DQ8 mice, which was accompanied by an increased expression of Th17 gene transcripts in lungs. These observations suggest that cigarette smoke promotes antigen-specific autoimmunity that is profoundly influenced by host genetic factors.
Smoking; Rheumatoid arthritis; HLA transgenic mice; Citrullination; Innate immune response
IL-15 is involved in regulating host defense and inflammation. Monocytes produce the biologically active cell surface IL-15 in response to IFN-γ. Although aging can alter the immune system, little is known about whether and how aging affects IFN-γ-mediated IL-15 production in human monocytes. We showed that monocytes of healthy older adults (age ≥ 65) had increased cell surface IL-15 expression in response to IFN-γ compared to those of healthy young adults (age ≤ 40). This finding stems in part from increased IFN-γ receptor (R)1/2 expression on monocytes in older adults, leading to enhanced STAT1 activation and interferon regulatory factor 1 synthesis with increased IL15 gene expression. Our study suggests that with aging the IFN-γ-mediated IL-15 production pathway in human monocytes is uncompromised, but rather augmented, and could be considered as a therapeutic target point to modulate host defense and inflammation in older adults.
IL-15; IFN-γ; human; monocytes; aging
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27−, CD45RO−) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.
Multiple Sclerosis; CD8; T cells; Regulatory; IL12
Infection by Staphylococcus aureus is on the rise, and there is need for a better understanding of host immune responses that combat S. aureus. Here we use DNA barcoding to enable deep sequencing of the paired heavy- and light-chain immunoglobulin genes expressed by individual plasmablasts derived from S. aureus-infected humans. Bioinformatic analysis of the antibody repertoires revealed clonal families of heavy-chain sequences and enabled rational selection of antibodies for recombinant expression. Of the ten recombinant antibodies produced, seven bound to S. aureus, of which four promoted opsonophagocytosis of S. aureus. Five of the antibodies bound to known S. aureus cell-surface antigens, including fibronectin binding protein A. Fibronectin binding protein A-specific antibodies were isolated from two independent S. aureus infected patients and mediated neutrophil killing of S. aureus in in vitro assays. Thus, our DNA barcoding approach enabled efficient identification of antibodies involved in protective host antibody responses against S. aureus.
Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms’ tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity.
T cells; Graft versus host disease; Cdk2; T regulatory cells
To explore the possibility of using a mini-array of multiple tumor-associated antigens (TAAs) as an approach to the diagnosis of hepatocellular carcinoma (HCC), 14 TAAs were selected to examine autoantibodies in sera from patients with chronic hepatitis, liver cirrhosis and HCC by immunoassays. Antibody frequency to any individual TAA in HCC varied from 6.6% to 21.1%. With the successive addition of TAAs to the panel of TAAs, there was a stepwise increase of positive antibody reactions. The sensitivity and specificity of 14 TAAs for immunodiagnosis of HCC was 69.7% and 83.0%, respectively. This TAAs mini-array also identified 43.8% of HCC patients who had normal alpha-fetoprotein (AFP) levels in serum. In summary, this study further supports the hypothesis that a customized TAA array used for detecting anti-TAA autoantibodies can constitute a promising and powerful tool for immunodiagnosis of HCC and may be especially useful in patients with normal AFP levels.
Autoantibodies; Tumor-associated antigens (TAAs); Cancer immunodiagnosis; Hepatocellular carcinoma
Hepatitis C virus (HCV) induces persistent infection and causes chronic liver disease in most infected patients. Vigorous HCV-specific CD4+ and CD8+ T cell responses against HCV multiple epitopes are necessary for spontaneous viral clearance during the acute phase, but the virus appears to have multiple strategies to evade these defenses. There are relatively few studies on the role of immune responses during the chronic phase of infection. CD4+ T cell responses appear to protect against liver injury and may be important to clearance during interferon and ribavirin based therapy. Classic cytotoxic T cells (CTL) may primarily damage the liver in chronic HCV, but there may be subpopulations of T cells that protect against liver inflammation. Resolution of these outstanding questions is important to the development of a prophylactic vaccine as well as improving therapeutic options for those with chronic infection.
Hepatitis C virus; CD4 T cells; CD8+ T cells; chronic infection; liver injury; persistence; immune escape; immunopathogenesis
Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that recruits monocytes into the subendothelial cell layer in atherosclerotic lesions. Elevated homocysteine (hyper-homocysteinemia), which is usually associated with low-folate status, is a known risk factor for many pathologies with inflammatory etiologies. The present study was undertaken to examine whether there are associations between MCP-1 concentrations and folate/Hcy phenotype or methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype in healthy young adults. In females, MCP-1 concentrations were positively correlated with Hcy and negatively correlated with both serum and red blood cell folate; female smokers and MTHFR 677T carriers had particularly elevated MCP-1 concentrations. Similar relationships were not seen in males. These findings may have implications for understanding the female predominance observed for a range of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.
MCP-1; Folate; Homocysteine; Autoimmune
We sought to determine if a specific class I and II HDAC inhibitor (ITF2357) was able to decrease disease in lupus-prone NZB/W mice through regulation of T cell profiles. From 22 - 38 weeks-of-age, NZB/W and non-lupus NZW mice were treated with ITF2357 (5 mg/kg or 10 mg/kg), or vehicle control. Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA and cytokine levels were measured every 4 weeks. Kidney disease was determined by sera IgG levels, immune complex deposition, and renal pathology. T lymphocyte profiles were assessed using flow cytometric analyses. Our results showed NZB/W mice treated with the high-dose of ITF2357 had decreased renal disease and inflammatory cytokines in the sera. Treatment with ITF2357 decreased the Th17 phenotype while increasing the percentage of Tregs as well as Foxp3 acetylation. These results suggest that specific HDAC inhibition may decrease disease by altering T cell differentiation and acetylation.
Systemic Lupus Erythematosus; histone deacetylase; regulatory T cells
We used a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of expressed antibodies. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy chain VJ and light chain VJ sequence usage, do so most effectively. Vaccine-induced heavy chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years’ seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years’ seasonal influenza, suggesting that ‘original antigenic sin’ shapes the antibody response to influenza vaccination.