Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.

Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs in vitro and explored the fine specificity of their suppressive function and their mechanism of action in vitro and in vivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function in vivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

BACKGROUND: To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. METHODS: We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). RESULTS: ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. CONCLUSION: The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

The contribution of the donor vascularised hand and face allograft in transplant rejection: An immunological perspective.

Overcoming immunological rejection remains a barrier to the safe adoption of Vascularised Composite Allotransplantation (VCA). To mitigate this risk, clinical protocols have been derived from solid organ transplantation, targeting recipient immunomodulation, yet VCA is unique. Face and hand composite allografts are composed of multiple different tissues, each with their own immunological properties. Experimental work suggests that allografts carry variable numbers and populations of donor leukocytes in an organ specific manner. Ordinarily, these passenger leukocytes are transferred from the donor graft into the recipient circulation after transplantation. Whether alloantigen presentation manifests as acute allograft rejection or transplant tolerance is unknown. This review aims to characterise the immunological properties of the constituent parts of the donor face and hand, the potential fate of donor leukocytes and to consider theoretical graft specific interventions to mitigate early rejection.

Low-affinity CD8+ T cells provide interclonal help to high-affinity CD8+ T cells to augment alloimmunity.

Following solid organ transplantation, small precursor populations of polyclonal CD8+ T cells specific for any graft-expressed antigen preferentially expand their high-affinity clones. This phenomenon, termed "avidity maturation," results in a larger population of CD8+ T cells with increased sensitivity to alloantigen, posing a greater risk for graft rejection. Using a mouse model of minor-mismatched skin transplantation, coupled with the tracking of 2 skin graft-reactive CD8+ T cell receptor-transgenic tracer populations with high and low affinity for the same peptide-major histocompatibility complex, we explored the conventional paradigm that CD8+ T cell avidity maturation occurs through T cell receptor affinity-based competition for cognate antigen. Our data revealed "interclonal CD8-CD8 help," whereby lower/intermediate affinity clones help drive the preferential expansion of their higher affinity counterparts in an interleukin-2/CD25-dependent manner. Consequently, the CD8-helped high-affinity clones exhibit greater expansion and develop augmented effector functions in the presence of their low-affinity counterparts, correlating with more severe graft damage. Finally, interclonal CD8-CD8 help was suppressed by costimulation blockade treatment. Thus, high-affinity CD8+ T cells can leverage help from low-affinity CD8+ T cells of identical specificity to promote graft rejection. Suppressing provision of interclonal CD8-CD8 help may be important to improve transplant outcomes.

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.

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).

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.

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.

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.

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.

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.

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.