B cell repertoire in children with skin barrier dysfunction supports altered IgE maturation associated with allergic food sensitization.

The skin is a major immune organ and skin barrier dysfunction is a major risk factor for the development of the inappropriate immune response seen in allergic disease. Skin barrier disruption alters the landscape of antigens experienced by the immune system and the downstream impacts on the antibody repertoire remain poorly characterized, particularly for the IgE isotype responsible for allergic specificity and in early life, when allergic disease is developing. In this study, we sequenced antibody gene repertoires from a large and well-characterized cohort of children with atopic dermatitis and found that food sensitization was associated with lower mutation frequencies in the IgE compartment. This trend was abrogated in children living with pets during the first year of life. These results elucidate potential molecular mechanisms underlying the protective effects of pet ownership and non-antiseptic environs reported for allergic disease, and the hygiene hypothesis more broadly. We also observed increased IgE diversity and increased isotype-switching to the IgE isotype, suggesting that B cell development, particularly isotype-switching, is heavily altered in the those with food allergen sensitizations relative to those without food allergen sensitizations. Unlike for food antigens, aeroallergen sensitization exhibited no effect on IgE mutation or diversity. Consistent patterns of antibody rearrangement were associated with food allergen sensitization in subjects with atopic dermatitis. Thus, we propose the Immune Repertoire in Atopic Disease (IRAD) score, to quantify this repertoire shift and to aid clinically in patient diagnosis and risk stratification.

B cell clonal expansion within immune infiltrates in human cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV) is associated with intragraft B cell infiltrates. Here, we studied the clonal composition of B cell infiltrates using 4 graft specimens with CAV. Using deep sequencing, we analyzed the immunoglobulin heavy chain variable region repertoire in both graft and blood. Results showed robust B cell clonal expansion in the graft but not in the blood for all cases. Several expanded B cell clones, characterized by their uniquely rearranged complementarity-determining region 3, were detected in different locations in the graft. Sequences from intragraft B cells also showed elevated levels of mutated rearrangements in the graft compared to blood B cells. The number of somatic mutations per rearrangement was also higher in the graft than in the blood, suggesting that B cells continued maturing in situ. Overall, our studies demonstrated B cell clonal expansion in human cardiac allografts with CAV. This local B cell response may contribute to the pathophysiology of CAV through a mechanism that needs to be identified.

A Comparative Study of Single and Dual Perfusion During End-ischemic Subnormothermic Liver Machine Preservation.

BACKGROUND: It remains controversial if arterial perfusion in addition to portal vein perfusion during machine preservation improves liver graft quality. Comparative studies using both techniques are lacking. We studied the impact of using single or dual machine perfusion of donation after circulatory death rat livers. In addition, we analyzed the effect of pulsatile versus continuous arterial flow. METHODS: Donation after circulatory death rat livers (n = 18) were preserved by 6 hours cold storage, followed by 1 hour subnormothermic machine perfusion (20°C, pressure of 40/5 mm Hg) and 2 hours ex vivo warm reperfusion (37°C, pressure of 80/11 mm Hg, 9% whole blood). Machine preservation was either through single portal vein perfusion (SP), dual pulsatile (DPP), or dual continuous perfusion (DCP) of the portal vein and hepatic artery. Hydrodynamics, liver function tests, histopathology, and expression of endothelial specific genes were assessed during 2 hours warm reperfusion. RESULTS: At the end of reperfusion, arterial flow in DPP livers tended to be higher compared to DCP and SP grafts. However, this difference was not significant nor was better flow associated with better outcome. No differences in bile production or alanine aminotransferase levels were observed. SP livers had significantly lower lactate compared to DCP, but not DPP livers. Levels of Caspase-3 and tumor necrosis factor-α were similar between the groups. Expression of endothelial genes Krüppel-like-factor 2 and endothelial nitric oxide synthase tended to be higher in dual perfused livers, but no histological evidence of better preservation of the biliary endothelium or vasculature of the hepatic artery was observed. CONCLUSIONS: This study shows comparable outcomes after using a dual or single perfusion approach during end-ischemic subnormothermic liver machine preservation.

Prevalence of polyreactive innate clones among graft--infiltrating B cells in human cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) has been associated with graft-infiltrating B cells, although their characteristics are still unclear. In this study we examined the frequency, localization and reactivity profile of graft-infiltrating B cells to determine their contribution to the pathophysiology of CAV. METHODS: B cells, plasma cells and macrophages were examined by immunohistochemistry in 56 allografts with CAV, 49 native failed hearts and 25 autopsy specimens. A total of 102 B-cell clones were immortalized directly from the infiltrates of 3 fresh cardiac samples with CAV. Their secreted antibodies were assessed using enzyme-linked immunoassay and flow cytometry. RESULTS: B-cell infiltration was observed around coronary arteries in 93% of allograft explants with CAV. Comparatively, intragraft B cells were less frequent and less dense in the intraventricular myocardium from where routine biopsies are obtained. Plasma cells and macrophages were also detected in 85% and 95% of explants, respectively. Remarkably, B-cell infiltrates were not associated with circulating donor-specific antibodies (DSA) or prior episodes of antibody-mediated rejection (AMR). Among all B-cell clones generated from 3 explants with CAV, a majority secreted natural antibodies reactive to multiple autoantigens and apoptotic cells, a characteristic of innate B cells. CONCLUSIONS: Our study reveals a high frequency of infiltrating B cells around the coronary arteries of allografts with CAV, independent of DSA or AMR. These cells are enriched for innate B cells with a polyreactive profile. The findings shift the focus from conventional DSA-producing B cells to the potentially pathogenic polyreactive B cells in the development of clinical CAV.

Dynamics of B Cell Recovery In Kidney/Bone Marrow Transplant Recipients.

BACKGROUND: Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation. METHODS: Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients' serum reactivity to HLA was assessed by Luminex. RESULTS: B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24CD38 transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA. CONCLUSIONS: Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.

The human thymus perivascular space is a functional niche for viral-specific plasma cells.

The human thymus is susceptible to viral infections that can severely alter thymopoiesis and compromise the mechanisms of acquired tolerance to self-antigens. In humans, plasma cells residing primarily in the bone marrow confer long-lasting protection to common viruses by secreting antigen-specific antibodies. Since the thymus also houses B cells, we examined the phenotypic complexity of these thymic resident cells and their possible protective role against viral infections. Using tissue specimens collected from subjects ranging in age from 5 days to 71 years, we found that starting during the first year of life, CD138+ plasma cells (PC) begin accumulating in the thymic perivascular space (PVS) where they constitutively produce IgG without the need for additional stimulation. These, thymic PC secrete almost exclusively IgG1 and IgG3, the two main complement-fixing effector IgG subclasses. Moreover, using antigen-specific ELISpot assays, we demonstrated that thymic PC include a high frequency of cells reactive to common viral proteins. Our study reveals an unrecognized role of the PVS as a functional niche for viral-specific PCs. The PVS is located between the thymic epithelial areas and the circulation. PCs located in this compartment may therefore provide internal protection against pathogen infections and preserve the integrity and function of the organ.

Evidence to Support a Contribution of Polyreactive Antibodies to HLA Serum Reactivity.

BACKGROUND: Assessing the serum reactivity to HLA is essential for the evaluation of transplant candidates and the follow-up of allograft recipients. In this study, we look for evidence at the clonal level that polyreactive antibodies cross-reactive to apoptotic cells and multiple autoantigens can also react to HLA and contribute to the overall serum reactivity. METHODS: We immortalized B cell clones from the blood of 2 kidney transplant recipients and characterized their reactivity to self-antigens, apoptotic cells as well as native, denatured, and cryptic HLA determinants using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, flow cytometry and Luminex assays. We also assessed the reactivity of 300 pretransplant serum specimens to HLA and apoptotic cells. RESULTS: We report here 4 distinct B cell clones cross-reactive to self and HLA class I. All 4 clones reacted to numerous HLA class I alleles but did not appear to target canonical "shared" epitopes. In parallel experiments, we observed a strong correlation between IgG reactivity to HLA and apoptotic cells in pretransplant serum samples collected from 300 kidney transplant recipients. Further analysis revealed that samples with higher reactivity to apoptotic cells displayed significantly higher class I percent panel-reactive antibodies compared to samples with low reactivity to apoptotic cells. CONCLUSIONS: We provide here (1) proof of principle at the clonal level that human polyreactive antibodies can cross-react to HLA, multiple self-antigens and apoptotic cells and (2) supportive evidence that polyreactive antibodies contribute to overall HLA reactivity in the serum of patients awaiting kidney transplant.

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-ß. In this study, we show that the co-culture of naive T cells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-ß, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-ß, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Expansion and somatic hypermutation of B-cell clones in rejected human kidney grafts.

BACKGROUND: B-cell infiltrates are common in rejected kidney allografts, yet their composition is still unclear. The aim of our study was to characterize the clonal composition of B-cell infiltrates of rejected human kidney grafts. METHODS: We used a molecular approach to characterize the partial B-cell repertoires of 5 failed human kidney grafts with detectable B-cell infiltrates. A comparison between the intragraft and blood repertoire was also conducted for 1 case. RESULTS: Redundant sequences were observed in both blood and graft, although the level of clonal amplification was significantly higher for the graft. Somatic hypermutations (SHMs) were also more frequent in sequences found in the graft compared to the blood. The rate of nonsilent mutations was significantly higher in complementarity determining regions (CDRs) compared to framework regions in blood sequences as well as in graft sequences found at low frequency. In contrast, this preferential distribution was lost in sequences found at high frequency in the graft, suggesting a lack of affinity maturation in situ. Lastly, follicular dendritic cells were undetectable in CD20 infiltrates in all samples examined. CONCLUSIONS: We provide here evidence that B-cell clones expand and undergo SHMs in situ. However, the even distribution of nonsilent SHM in high-frequency graft sequences together with the absence of follicular dendritic cells do not support the view that infiltrating B cells are part of functional germinal centers.

Cyclooxygenase-2 deficiency enhances Th2 immune responses and impairs neutrophil recruitment in hepatic ischemia/reperfusion injury.

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2(-/-) to elaborate COX-2 products favored a Th2-type response in these mice. COX-2(-/-) livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-x(L) were normally expressed in COX-2(-/-) livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2(-/-) livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.

Fibronectin-alpha4beta1 integrin interactions regulate metalloproteinase-9 expression in steatotic liver ischemia and reperfusion injury.

Ischemia/reperfusion injury is a major cause of the highly dysfunctional rate observed in marginal steatotic orthotopic liver transplantation. In this study, we document that the interactions between fibronectin, a key extracellular matrix protein, and its integrin receptor alpha4beta1, expressed on leukocytes, specifically up-regulated the expression and activation of metalloproteinase-9 (MMP-9, gelatinase B) in a well-established steatotic rat liver model of ex vivo ice-cold ischemia followed by isotransplantation. The presence of the active form of MMP-9 was accompanied by massive intragraft leukocyte infiltration, high levels of proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, and impaired liver function. Interestingly, MMP-9 activity in steatotic liver grafts was, to a certain extent, independent of the expression of its natural inhibitor, the tissue inhibitor of metalloproteinases-1. Moreover, the blockade of fibronectin-alpha4beta1-integrin interactions inhibited the expression/activation of MMP-9 in steatotic orthotopic liver transplantations without significantly affecting the expression of metalloproteinase-2 (MMP-2, gelatinase A). Finally, we identified T lymphocytes and monocytes/macrophages as major sources of MMP-9 in steatotic liver grafts. Hence, these findings reveal a novel aspect of the function of fibronectin-alpha4beta1 integrin interactions that holds significance for the successful use of marginal steatotic livers in transplantation.

Fibronectin-alpha 4 beta 1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

We tested a hypothesis that interactions between fibronectin (FN), the major extracellular matrix component, and its integrin alpha 4 beta 1 receptor is important in the development of ischemia/reperfusion injury of steatotic liver transplants. We examined the effect of connecting segment-1 (CS1) peptide-facilitated blockade of FN-alpha 4 beta 1 interaction in a well-established steatotic rat liver model of ex vivo cold ischemia followed by iso-transplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers before and after cold ischemic storage. Lean Zucker recipients of fatty liver transplants received an additional 3-day course of CS1 peptides after transplant. CS1 peptide therapy significantly inhibited the recruitment of T lymphocytes, neutrophil activation/infiltration, and repressed the expression of proinflammatory tumor necrosis factor-alpha and interferon-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase expression, peroxynitrite formation, and hepatic necrosis. Importantly, CS1 peptide therapy improved function/histological preservation of steatotic liver grafts, and extended their 14-day survival in lean recipients from 40% in untreated to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-alpha 4 beta 1 interaction protects against severe ischemia/reperfusion injury experienced otherwise by steatotic OLTs. These novel findings document the potential of targeting FN-alpha 4 beta 1 in vivo interaction to increase the transplant donor pool through modulation of marginal steatotic livers.

Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury.

BACKGROUND: Ischemia/reperfusion (I/R) injury is a critical factor in the dysfunction of steatotic orthotopic liver transplants. Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic I/R injury. METHODS: We used adenovirus (Ad)-based HO-1 gene transfer to analyze the effects of HO-1 overexpression in a well-established fatty Zucker rat model of I/R followed by orthotopic liver transplantation. RESULTS: Ad-HO-1 gene therapy increased recipient survival (80% vs. 40-50% in controls) and significantly diminished hepatocyte injury, as compared with untreated and Ad-beta-galactosidase (Ad-beta-Gal)-treated livers. Orthotopic liver transplants in the Ad-HO-1 group exhibited less macrophage infiltration in the portal areas, as compared with controls. Unlike untreated and Ad-beta-Gal-treated orthotopic liver transplant controls, which showed elevated levels of inducible nitric oxide synthase by infiltrating macrophages, inducible nitric oxide synthase expression in the Ad-HO-1 group was almost absent. In contrast, endothelial nitric oxide synthase was comparable in Ad-HO-1- and Ad-beta-Gal-transduced fatty orthotopic liver transplants. Intragraft expression of antiapoptotic Bcl-2 and Bag-1 was increased in Ad-HO-1-treated orthotopic liver transplants, as compared with Ad-beta-Gal controls. Moreover, increased HO enzymatic activity was accompanied by inhibition of caspase-3 protein expression. CONCLUSIONS: HO-1 gene transfer significantly prolongs survival of steatotic orthotopic liver transplants, depresses macrophage infiltration, suppresses local expression of inducible nitric oxide synthase, and modulates pro- and antiapoptotic pathways.