Monitoring B cell alloresponses in rats.

Antibody-mediated rejection is a major cause of graft failure in organ transplantation. For this reason, B cell responses are of particular interest to transplantation research. Rats are important model organisms for transplant studies, but B cell alloimmune assays and B cell subset markers are poorly established in rats. We alloimmunized rats by donor blood injection using the high responder rat strain combination Brown Norway (donor) and Lewis (recipient) rats. Using splenocytes from alloimmunized and control rats, we established assays to assess allospecific B cell proliferation and the capacity to generate allospecific B memory cells and alloantibody-secreting cells after antigenic rechallenge in vitro using a mixed lymphocyte reaction. Furthermore, we defined a simple gating and sorting strategy for pre- and post-germinal center follicular B cells, as well as non-switched and switched plasmablasts. Our protocols for assessing B cell alloresponses and B cell subsets in rats may help to accelerate research into the role of B cells and manipulation of humoral alloresponses in transplant research.

Circulating anti-human leukocyte antigen IgM antibodies as a potential early predictor of allograft rejection and a negative clinical outcome after lung transplantation.

PURPOSE: Anti-human leukocyte antigen (HLA) immunoglobulin (Ig) M production stimulated by an alloantigen is sensitive, making IgM a novel potential marker of allorejection after organ transplantation. This study examined the relationship between the serum levels of anti-HLA IgM early after clinical lung transplantation (LTx) and the post-transplant outcomes. METHODS: Thirty-one consecutive patients who underwent deceased LTx were included. Immunoreactivity against HLA was retrospectively analyzed by measuring the anti-HLA IgM levels in the serum sampled for the first 14 days after LTx. The flow panel reactive antibody technique was used. The ratio of the anti-class I IgM level at each day to baseline was obtained, and the peak IgM level was determined for each case. The correlation between the peak IgM level and subsequent development of acute rejection (AR), chronic lung allograft dysfunction (CLAD), and survival outcomes were examined. RESULTS: The peak IgM level was a significant risk factor for AR within 90 days in univariate and multivariate analyses. In the long term, the patients with positive IgM (peak level > 1.8) tended to have a poorer CLAD-free and overall survival than those with negative IgM. CONCLUSION: Elevation of anti-HLA IgM levels early after LTx may be correlated with a higher incidence of rejection and negative clinical outcomes.

Acute Atherosis Lesions at the Fetal-Maternal Border: Current Knowledge and Implications for Maternal Cardiovascular Health.

Decidua basalis, the endometrium of pregnancy, is an important interface between maternal and fetal tissues, made up of both maternal and fetal cells. Acute atherosis is a uteroplacental spiral artery lesion. These patchy arterial wall lesions containing foam cells are predominantly found in the decidua basalis, at the tips of the maternal arteries, where they feed into the placental intervillous space. Acute atherosis is prevalent in preeclampsia and other obstetric syndromes such as fetal growth restriction. Causal factors and effects of acute atherosis remain uncertain. This is in part because decidua basalis is challenging to sample systematically and in large amounts following delivery. We summarize our decidua basalis vacuum suction method, which facilitates tissue-based studies of acute atherosis. We also describe our evidence-based research definition of acute atherosis. Here, we comprehensively review the existing literature on acute atherosis, its underlying mechanisms and possible short- and long-term effects. We propose that multiple pathways leading to decidual vascular inflammation may promote acute atherosis formation, with or without poor spiral artery remodeling and/or preeclampsia. These include maternal alloreactivity, ischemia-reperfusion injury, preexisting systemic inflammation, and microbial infection. The concept of acute atherosis as an inflammatory lesion is not novel. The lesions themselves have an inflammatory phenotype and resemble other arterial lesions of more extensively studied etiology. We discuss findings of concurrently dysregulated proteins involved in immune regulation and cardiovascular function in women with acute atherosis. We also propose a novel hypothesis linking cellular fetal microchimerism, which is prevalent in women with preeclampsia, with acute atherosis in pregnancy and future cardiovascular and neurovascular disease. Finally, women with a history of preeclampsia have an increased risk of premature cardiovascular disease. We review whether presence of acute atherosis may identify women at especially high risk for premature cardiovascular disease.

The Impact of Inflammation on the Immune Responses to Transplantation: Tolerance or Rejection?

Transplantation (Tx) remains the optimal therapy for end-stage disease (ESD) of various solid organs. Although alloimmune events remain the leading cause of long-term allograft loss, many patients develop innate and adaptive immune responses leading to graft tolerance. The focus of this review is to provide an overview of selected aspects of the effects of inflammation on this delicate balance following solid organ transplantation. Initially, we discuss the inflammatory mediators detectable in an ESD patient. Then, the specific inflammatory mediators found post-Tx are elucidated. We examine the reciprocal relationship between donor-derived passenger leukocytes (PLs) and those of the recipient, with additional emphasis on extracellular vesicles, specifically exosomes, and we examine their role in determining the balance between tolerance and rejection. The concept of recipient antigen-presenting cell "cross-dressing" by donor exosomes is detailed. Immunological consequences of the changes undergone by cell surface antigens, including HLA molecules in donor and host immune cells activated by proinflammatory cytokines, are examined. Inflammation-mediated donor endothelial cell (EC) activation is discussed along with the effect of donor-recipient EC chimerism. Finally, as an example of a specific inflammatory mediator, a detailed analysis is provided on the dynamic role of Interleukin-6 (IL-6) and its receptor post-Tx, especially given the potential for therapeutic interdiction of this axis with monoclonal antibodies. We aim to provide a holistic as well as a reductionist perspective of the inflammation-impacted immune events that precede and follow Tx. The objective is to differentiate tolerogenic inflammation from that enhancing rejection, for potential therapeutic modifications. (Words 247).

Lymphatic Reconstruction in Kidney Allograft Aggravates Chronic Rejection by Promoting Alloantigen Presentation.

Chronic rejection of the renal allograft remains a major cause of graft loss. Here, we demonstrated that the remodeling of lymphatic vessels (LVs) after their broken during transplantation contributes to the antigen presenting and lymph nodes activating. Our studies observed a rebuilt of interrupted lymph draining one week after mouse kidney transplantation, involving preexisting lymphatic endothelial cells (LECs) from both the donor and recipient. These expanding LVs also release C-C chemokine ligand 21 (CCL21) and recruit CCR7+ cells, mainly dendritic cells (DCs), toward lymph nodes and spleen, evoking the adaptive response. This rejection could be relieved by LYVE-1 specific LVs knockout or CCR7 migration inhibition in mouse model. Moreover, in retrospective analysis, posttransplant patients exhibiting higher area density of LVs presented with lower eGFR, severe serum creatinine and proteinuria, and greater interstitial fibrosis. These results reveal a rebuilt pathway for alloantigen trafficking and lymphocytes activation, providing strategies to alleviate chronic transplantation rejection.

Heterologous Immunity of Virus-Specific T Cells Leading to Alloreactivity: Possible Implications for Solid Organ Transplantation.

Exposure of the adaptive immune system to a pathogen can result in the activation and expansion of T cells capable of recognizing not only the specific antigen but also different unrelated antigens, a process which is commonly referred to as heterologous immunity. While such cross-reactivity is favourable in amplifying protective immune responses to pathogens, induction of T cell-mediated heterologous immune responses to allo-antigens in the setting of solid organ transplantation can potentially lead to allograft rejection. In this review, we provide an overview of murine and human studies investigating the incidence and functional properties of virus-specific memory T cells cross-reacting with allo-antigens and discuss their potential relevance in the context of solid organ transplantation.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines.

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV- Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-ß), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

Mouse Models of Antigen Presentation in Hematopoietic Stem Cell Transplantation.

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for hematopoietic malignancies. The therapeutic effect relies on donor T cells and NK cells to recognize and eliminate malignant cells, known as the graft-versus-leukemia (GVL) effect. However, off target immune pathology, known as graft-versus-host disease (GVHD) remains a major complication of alloSCT that limits the broad application of this therapy. The presentation of recipient-origin alloantigen to donor T cells is the primary process initiating GVHD and GVL. Therefore, the understanding of spatial and temporal characteristics of alloantigen presentation is pivotal to attempts to separate beneficial GVL effects from detrimental GVHD. In this review, we discuss mouse models and the tools therein, that permit the quantification of alloantigen presentation after alloSCT.

A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells.

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

Memory B Cells in Pregnancy Sensitization.

Memory B cells play an important role in immunity to pathogens as these cells are poised to rapidly differentiate into antibody-secreting cells upon antigen re-encounter. Memory B cells also develop over the course of HLA-sensitization during pregnancy and transplantation. In this review, we discuss the potential contribution of memory B cells to pregnancy sensitization as well as the impact of these cells on transplant candidacy and outcomes. We start by summarizing how B cell subsets are altered in pregnancy and discuss what is known about HLA-specific B cell responses given our current understanding of fetal antigen availability in maternal secondary lymphoid tissues. We then review the molecular mechanisms governing the generation and maintenance of memory B cells during infection - including the role of T follicular helper cells - and discuss the experimental evidence for the development of these cells during pregnancy. Finally, we discuss how memory B cells impact access to transplantation and transplant outcomes for a range of transplant recipients.

The lysophospholipid-binding molecule CD1D is not required for the alloimmunization response to fresh or stored RBCs in mice despite RBC storage driving alterations in lysophospholipids.

BACKGROUND: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. STUDY DESIGN AND METHODS: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. RESULTS: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG. CONCLUSION: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency.

Immunological Consequences of In Utero Exposure to Foreign Antigens.

Immunologic tolerance refers to a state of immune nonreactivity specific to particular antigens as an important issue in the field of transplantation and the management of autoimmune diseases. Tolerance conceptually originated from Owen's observation of blood cell sharing in twin calves. Owen's conceptual framework subsequently constituted the backbone of Medawar's "actively acquired tolerance" as the major tenet of modern immunology. Based upon this knowledge, the delivery of genetically distinct hematopoietic stem cells into pre-immune fetuses represented a novel and unique approach to their engraftment without the requirement of myeloablation or immunosuppression. It might also make fetal recipients commit donor alloantigens to memory of their patterns as "self" so as to create a state of donor-specific tolerance. Over the years, the effort made experimentally or clinically toward in utero marrow transplantation could not reliably yield sufficient hematopoietic chimerism for curing candidate diseases as anticipated, nor did allogeneic graft tolerance universally develop as envisaged by Medawar following in utero exposure to various forms of alloantigens from exosomes, lymphocytes or marrow cells. Enduring graft tolerance was only conditional on a state of significant hematopoietic chimerism conferred by marrow inocula. Notably, fetal exposure to ovalbumin, oncoprotein and microbial antigens did not elicit immune tolerance, but instead triggered an event of sensitization to the antigens inoculated. These fetal immunogenic events might be clinically relevant to prenatal imprinting of atopy, immune surveillance against developmental tumorigenesis, and prenatal immunization against infectious diseases. Briefly, the immunological consequences of fetal exposure to foreign antigens could be tolerogenic or immunogenic, relying upon the type or nature of antigens introduced. Thus, the classical school of "actively acquired tolerance" might oversimplify the interactions between developing fetal immune system and antigens. Such interactions might rely upon fetal macrophages, which showed up earlier than lymphocytes and were competent to phagocytose foreign antigens so as to bridge toward antigen-specific adaptive immunity later on in life. Thus, innate fetal macrophages may be the potential basis for exploring how the immunological outcome of fetal exposure to foreign antigens is determined to improve the likelihood and reliability of manipulating fetal immune system toward tolerization or immunization to antigens.

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis.

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.

Marginal zone B cells mediate a CD4 T-cell-dependent extrafollicular antibody response following RBC transfusion in mice.

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell-deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell-mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Characterization of CD177-reactive iso- and auto-antibodies.

BACKGROUND: CD177 is a surface protein on neutrophils and a main mediator for the surface expression of proteinase 3 (PR3). Its functions are largely unknown. At least three types of antibodies have been described to target CD177: isoantibodies, which are formed in CD177-null individuals as a result of an immune reaction following transfusion or pregnancy; autoantibodies present in sera from patients with autoimmune neutropenia; and antineutrophil cytoplasmic antibodies in sera from patients with glomerulonephritis with polyangiitis. In this study, we aimed to compare the binding characteristics of auto- and iso-antibodies to optimize their detectability in the neutrophil serology laboratory. STUDY DESIGN AND METHODS: The reactivity of iso- and auto-antibodies against CD177 was studied using granulocytes, "native" CD177/PR3 complex, and recombinant CD177 or PR3. RESULTS: All iso- and auto-antibodies were reactive with CD177/PR3 when immobilized with monoclonal antibody (moab) 7D8. Seventy-five percent of autoantibodies, but none of the isoantibodies, did not react with CD177/PR3 immobilized with moab MEM166. The majority of autoantibodies did not react with recombinant CD177, whereas most isoantibodies tested positive. DISCUSSION: Our results suggest that iso- and auto-antibodies against CD177 target different epitopes. Isoantibodies mainly target CD177 alone, while the majority of autoantibodies target a native epitope present on the neutrophil surface, but absent from recombinant CD177 which lacks PR3. Moab MEM166 binds to the native epitope and hinders the binding of CD177 autoantibodies. The results may help to design diagnostic strategies, especially for the identification of autoantibodies.

Triggering Receptor Expressed on Myeloid Cells-1 Agonist Regulates Intestinal Inflammation via Cd177+ Neutrophils.

Triggering receptor expressed on myeloid cell-1 (TREM-1) signaling is expressed on neutrophils and monocytes that is necessary for the successful antimicrobial response and resolution of inflammation in the gut. In this study, we determined the effect of an anti-TREM-1 agonistic antibody (α-TREM-1) on colitis and identify its underlying mechanism of action. Administration of α-TREM-1 alleviated colitis in mice and resolved dysbiosis, which required TLR4/Myd88 signaling. α-TREM-1 increased the production of neutrophil extracellular traps and interleukin-22 by CD177+ neutrophils, which led to pathogen clearance and protection of the intestinal barrier. TREM-1 activation using an α-TREM-1 antibody protects against colitis by rebalancing the microbiota and protecting the epithelium against the immune response as well as modulates the function of neutrophils and macrophages. These results highlight the importance of the TREM-1 pathway in intestinal homeostasis and suggest that α-TREM-1 treatment may be an effective therapeutic strategy for inflammatory bowel disease.