Anti-major Histocompatibility Complex Class Research Articles

Targeting mast cell activation alleviates anti-MHC I antibody and LPS-induced TRALI in mice by pharmacologically blocking the TLR3 and MAPK pathway

Transfusion-related lung injury (TRALI) poses a significant risk following blood transfusion and remains the primary cause of transfusion-related morbidity and mortality, primarily driven by the activation of immune cells through anti-major histocompatibility complex class I (anti-MHC I) antibody. However, it remains to be defined how immune microenvironmental cue contributes to TRALI. Here, we uncover that activated mast cells within the immune microenvironment promote lung inflammation and injury in antibody-mediated TRALI, both in vitro and in vivo. This was demonstrated by co-culturing lipopolysaccharide (LPS)-pretreated mast cell line with anti-MHC I antibody and establishing a “two-hit” TRALI mouse model through intratracheal injection of LPS followed by tail-vein injection of anti-MHC I antibody. Importantly, mast cell-deficient KitW-sh/W-sh mice exhibited markedly reduced lung inflammation and injury responses in antibody-mediated TRALI compared with wild-type mice. Mechanistically, activation of toll-like receptor 3 (TLR3)/mitogen-activated protein kinase (MAPK) signaling pathway in mast cells contributes to the enhanced production of proinflammatory factors. These excessive proinflammatory factors produced by activated mast cells contribute to lung inflammation and injury in antibody-mediated TRALI. Pharmacologically targeting the TLR3/MAPK pathway to inhibit mast cell activation normalizes the proinflammatory microenvironment and alleviates lung inflammation and injury in the preclinical TRALI mouse model. Overall, we find that activation of mast cells via the TLR3/MAPK pathway contributes to lung inflammation and injury in antibody-mediated TRALI, providing novel insights into its underlying mechanisms. Furthermore, targeting activated mast cells and the associated pathway offers potential therapeutic strategies for antibody-mediated TRALI.

Outcome-based Risk Assessment of Non-HLA Antibodies in Heart Transplantation: A Systematic Review

Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, subclinical, or EMB-negative rejection events. While non-human leukocyte antigen (non-HLA) antibodies have emerged as important risk factors for antibody-mediated rejection after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has the potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included the number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and donor-specific anti-human leukocyte antigen antibody (HLA-DSA) responses. A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support versus against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-major histocompatibility complex class I chain-related gene A (MICA) and anti-angiotensin II type I receptor antibodies (AT1R) in HT rejection, evidence for their involvement was equivocal. Conversely, the strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs

AbstractTransfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti–major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S–mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

Cancer stem-like cells evade CD8+CD103+ tumor-resident memory T (TRM) lymphocytes by initiating an epithelial-to-mesenchymal transition program in a human lung tumor model

BackgroundCancer stem cells (CSC) define a population of rare malignant cells endowed with ‘stemness’ properties, such as self-renewing, multipotency and tumorigenicity. They are responsible for tumor initiation and progression, and...

Platelet FcγRIIA-induced serotonin release exacerbates the severity of transfusion-related acute lung injury in mice

AbstractTransfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti–major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A+ mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A+ animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A+ mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A+ mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A+ mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients.

A decline in club cell secretory proteins in lung transplantation is associated with release of natural killer cells exosomes leading to chronic rejection

In human lung transplant recipients, a decline in club cell secretory protein (CCSP) in bronchoalveolar lavage fluid has been associated with chronic lung allograft dysfunction (CLAD) as well as the induction of exosomes and immune responses that lead to CLAD. However, the mechanisms by which CCSP decline contributes to CLAD remain unknown. To define the mechanisms leading to CCSP decline and chronic rejection, we employed two mouse models: 1) chronic rejection after orthotopic single lung transplantation and 2) anti-major histocompatibility complex (MHC) class I-induced obliterative airway disease. In the chronic rejection mouse model, we detected circulating exosomes with donor MHC (H2b) and lung self-antigens and also development of antibodies to H2b and lung self-antigens and then a decline in CCSP. Furthermore, DBA2 mice that received injections of these exosomes developed antibodies to donor MHC and lung self-antigens. In the chronic rejection mouse model, natural killer (NK) and CD8 T cells were the predominant graft-infiltrating cells on day 14 of rejection followed by exosomes containing NK cell-associated and cytotoxic molecules on day 14 and 28. When NK cells were depleted, exosomes with NK cell-associated and cytotoxic molecules as well as fibrosis decreased. Induction of exosomes led to immune responses to donor MHC and lung self-antigens, resulting in CCSP decline, leading to NK cell infiltration and release of exosomes from NK cells. These results suggest a novel role for exosomes derived from NK cells in the pathogenesis of chronic lung allograft rejection.

Pulmonary coagulation and fibrinolysis abnormalities that favor fibrin deposition in the lungs of mouse antibody-mediated transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a life-threatening disease caused by blood transfusion. However, its pathogenesis is poorly understood and specific therapies are not available. Experimental and clinical studies have indicated that alveolar fibrin deposition serves a pathological role in acute lung injuries. The present study investigated whether pulmonary fibrin deposition occurs in a TRALI mouse model and the possible mechanisms underlying this deposition. The TRALI model was established by priming male Balb/c mice with lipopolysaccharide (LPS) 18 h prior to injection of an anti-major histocompatibility complex class I (MHC-I) antibody. Untreated mice and mice administered LPS plus isotype antibody served as controls. At 2 h after TRALI induction, blood and lung tissue were collected. Disease characteristics were assessed based on lung tissue histology, inflammatory responses and alterations in the alveolar-capillary barrier. Immunofluorescence staining was used to detect pulmonary fibrin deposition, platelets and fibrin-platelet interactions. Levels of plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TATc), tissue factor pathway inhibitor (TFPI), coagulation factor activity and fibrin degradation product (FDP) in lung tissue homogenates were measured. Severe lung injury, increased inflammatory responses and a damaged alveolar-capillary barrier in the LPS-primed, anti-MHC-I antibody-administered mice indicated that the TRALI model was successfully established. Fibrin deposition, fibrin-platelet interactions and platelets accumulation in the lungs of mouse models were clearly promoted. Additionally, levels of TATc, coagulation factor V (FV), TFPI and PAI-1 were elevated, whereas FDP level was decreased in TRALI mice. In conclusion, both impaired fibrinolysis and enhanced coagulation, which might be induced by boosted FV activity, increased pulmonary platelets accumulation and enhanced fibrin-platelet interactions and contributed to pulmonary fibrin deposition in TRALI mice. The results provided a therapeutic rationale to target abnormalities in either coagulation or fibrinolysis pathways for antibody-mediated TRALI.

Platelet depletion limits the severity but does not prevent the occurrence of experimental transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI. In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction. Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation. This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology.

Inhibition of the CD40/CD40L complex protects mice against ALI-induced pancreas degradation.

Acute lung injury (ALI) is a severe complication of transfusion. In a previous study, we saw that inhibition of the CD40/CD40L complex allowed restoration of ALI lesions in an experimental mouse model. This study focused on pancreas-associated injury development during experimental ALI pathogenesis and its limitation through CD40/CD40L complex inhibition. An ALI mouse model was established through intraperitoneal lipopolysaccharide and intravenous anti-major histocompatibility complex class I monoclonal antibody injection. Preemption of lesions was achieved with intravenous injection of neutralizing anti-CD40L monoclonal antibody 30 minutes before the trigger, that is, anti-major histocompatibility complex class I monoclonal antibody administration. Histology and immunoassay analyses were used to evaluate pancreatic lesions. ALI development induced significant degradation of the lungs and pancreas and was associated with pancreatic lesions. Different scores were established showing more severe injury to the pancreas in ALI conditions; however, injury was significantly reduced through CD40/CD40L complex inhibition. This study supports the idea that several organs are exposed during ALI development, and particularly when such experimental ALI aims at mimicking transfusion-associated ALI; nevertheless, preventive treatment inhibiting CD40/CD40L (sCD40L) complex formation provides protection from lung disease as well as disease of other organs.

Osteopontin Mediates Murine Transfusion-Related Acute Lung Injury through Stimulation of Pulmonary Neutrophil Accumulation

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress which develops within 6 hours of blood transfusion. It is the leading cause of transfusion-related deaths and the pathogenesis is complex and incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI.Osteopontin (OPN) is an extracellular matrix protein with multiple biological functions. OPN is involved in normal physiological processes, such as cell migration and adhesion, but has also been implicated in a wide range of disease states, including cancer, atherosclerosis, glomerulonephritis, and several chronic inflammatory diseases. Interestingly, OPN is upregulated at sites of inflammation and tissue remodeling. As inflammation is an important risk factor for TRALI development, and as neutrophils (PMNs) are known effector cells in the pathogenesis of TRALI which migrate and accumulate in the lungs during TRALI development, we investigated the potential contribution of OPN in the onset of antibody-mediated TRALI.We utilized a previously established murine TRALI model (Kapur et al, Blood 2017, Blood Advances 2018) in which C57BL/6 mice were first primed with a low dose of lipopolysaccharide (LPS) and depleted of their CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex (MHC) class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes by analysis of pulmonary edema (lung wet-to-dry weight ratios, W/Ds) and the levels of pulmonary neutrophils. Wildtype (WT) mice suffered from antibody-mediated TRALI compared to untreated naïve mice, as was shown by their significantly increased lung W/Ds (4.72 vs 4.50, respectively, P<0.0001). This also corresponded to significantly increased levels of pulmonary PMNs compared to untreated naïve mice (34% vs 5%, P<0.0001). In contrast, C57BL/6 OPN knock-out mice were resistant to antibody-mediated TRALI induction as they did not display any significant increase in lung W/Ds levels or pulmonary PMNs compared to untreated naïve OPN knock-out mice (lung W/Ds 4.83 vs 4.75, and pulmonary PMNs 18% vs 16%, respectively). Strikingly, administration of purified recombinant OPN during TRALI induction in C57BL/6 OPN knock-out mice, significantly induced a TRALI reaction (lung W/Ds 5.12 vs 4.75 as compared to untreated naïve OPN knock-out mice, P<0.05). Mechanistically, this TRALI inducing effect of OPN administration to OPN knock-out mice was associated with increased levels of pulmonary PMNs (38% vs 16%, as compared to untreated naïve OPN knock-out mice, P<0.0001). In vivo blocking of OPN in WT mice with an anti-OPN antibody demonstrated decreased lung W/Ds as compared to treatment with an isotype antibody (4.48 vs 4.69, respectively, P<0.05). The OPN blocking response during TRALI was associated with a decreased level of pulmonary PMN accumulation as compared to treatment with an isotype antibody (14% vs 34%, respectively, P<0.0001). As the PMN-chemoattractant macrophage inflammatory protein (MIP)-2 has previously been described to be upregulated in murine antibody-mediated TRALI, we investigated if the OPN-associated pulmonary PMN accumulation and TRALI induction could be related to the levels of MIP-2. We found that plasma MIP-2 levels were increased in mice that were infused with anti-MHC class I antibodies as compared to naïve controls, but that addition or blocking of OPN did not affect these MIP-2 levels. This indicates that the OPN-related PMN responses in TRALI are independent of plasma MIP-2 levels.Collectively, these data indicate OPN as a novel pathogenic factor which enhances antibody-mediated murine TRALI through stimulation of PMN migration towards the lungs, independent of MIP-2. This may suggest that blocking OPN (using an anti-OPN antibody) may prevent TRALI by impairing pulmonary PMN accumulation and could be a therapeutic avenue to explore in combatting this serious adverse complication of blood transfusion. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of therapeutic plasma exchange on angiotensin II type‐1 receptor antibodies on two kidney transplant recipients

Angiotensin II type-1 receptor antibody (AT1RAb) has been reported to cause antibody mediated rejection (AMR) in kidney transplant recipients possibly by contraction of renal arteries. We here report 2 kidney transplant recipients with elevated AT1RAbs and negative HLA donor specific antibodies (DSA) and anti-major histocompatibility complex class I chain-related gene A (MICA) Abs who received therapeutic plasma exchange (TPE) treatment followed by IVIG. CASE 1: Thirty-eight-year-old patient received second kidney transplant for end stage renal disease (ESRD) with chronic rejection. Three years post-transplant, she developed AMR with AT1RAb level >40 U/mL. She received 5 TPE and AT1RAb decreased by 20%, and biopsy showed improvement of AMR. She received another 3 TPE and AT1RAb decreased by 60%. Her creatinine (Cr) was stabilized at around 1.4 mg/dL. CASE 2: Twenty-four-year-old patient received kidney transplant for ESRD with unclear etiology. Two weeks post-transplant, her Cr rose with AT1RAb level at 18 U/mL and biopsy showed possible AMR. She received 6 TPE treatments and AT1RAb decreased by 55% and biopsy showed improvement of AMR. She received weekly TPE for subsequently rising AT1RAb but TPE was discontinued because of unsuccessful decrease of AT1RAb. Her Cr was stabilized at around 1.7 mL/dL. We reported 2 patients who received TPE treatments to decrease AT1RAbs. A course of TPE treatment successfully decreased AT1RAb. Histological improvement was observed quickly and Cr was also stabilized following the TPE treatment. Further study is necessary to determine the optimal use of TPE in renal transplant recipients with AT1RAbs.

Gastrointestinal Flora Dictates the Biological Response in Murine Transfusion Related Acute Lung Injury (TRALI)

Transfusion related acute lung injury (TRALI) is a syndrome of respiratory distress which occurs within 6 hours of blood transfusion. It is the leading cause of transfusion-related fatalities and the pathogenesis is incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Recently, using novel murine models of TRALI, CD4+T cells were found to be important protector cells against antibody-mediated TRALI and administration of interleukin (IL)-10 was demonstrated to be a successful treatment strategy for TRALI, rescuing mice therapeutically from pulmonary edema, the hallmark of acute lung injury (Kapur et al, Blood 2017, 129(18):2557-2569). Whether the gut microbiome plays any role in the development of TRALI is currently unknown. For that purpose, we compared the biological TRALI response in mice housed in a barrier-free (BF) setting versus mice housed in a specific pathogen-free (SPF) environment. We utilized our TRALI model in which C57BL/6 mice were first depleted of CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes for several parameters including pulmonary edema (lung wet-to-dry weight ratios, W/Ds), rectal temperatures (indicative of systemic shock), plasma levels of macrophage inflammatory protein (MIP)-2 (murine homologue of IL-8, a neutrophil chemoattractant), levels of pulmonary neutrophils (major effector cells in TRALI) and lung histology. We observed that after one week of housing, baseline rectal temperatures of BF mice significantly increased from 38.7o C to 39.5° C (p<0.001) while SPF mice remained stable around 38.3 °C. When comparing BF versus SPF mice, the baseline temperature of BF mice was significantly higher than SPF mice after one week of housing (P<0.0001). The gut flora was subsequently depleted in BF mice via administration of broad-spectrum antibiotics through the drinking water. When both aerobic and anaerobic gut microbes were efficiently depleted after a week of antibiotic treatment, rectal temperatures of the treated BF mice significantly dropped again to 38.7 °C (P<0.05). When inducing TRALI by depleting CD4+ T cells and injecting TRALI-antibodies, the rectal temperatures of the untreated BF mice remained low while the rectal temperatures of SPF mice and gut flora depleted-BF mice normalized again from 60 minutes onwards (34.9 versus 36.8 versus 37.8 °C, respectively). Moreover, the untreated BF mice suffered from TRALI while the SPF mice and the gut flora depleted-BF mice were protected from TRALI development (lung W/Ds 5.77 versus 4.63 versus 4.49, respectively) which was also evident from lung histology-analyses. The prevention of TRALI in SPF mice and gut flora depleted BF mice was paralleled by decreased plasma MIP-2 levels compared to the untreated BF mice which suffered from TRALI (MIP-2: 0.09 versus 0.08 versus 82.94 pg/ml, respectively) which also corresponded to pulmonary neutrophil numbers. As previously shown, low IL-10 levels were associated with TRALI development and IL-10 KO mice were found to be susceptible to antibody-mediated murine TRALI (without prior in vivo cell-depletion). However, when IL-10 KO mice were housed under SPF conditions and injected with TRALI-inducing antibodies, they did not display increased lung W/Ds levels compared to naïve SPF mice indicating that the gut flora precedes and dictates the biological TRALI response. These data collectively link the gut flora to the development of antibody-mediated TRALI in mice and antibiotic treatment prevents TRALI and may perhaps prevent the onset of human TRALI which may help in TRALI risk-assessment prior to transfusion. DisclosuresSemple:Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy; UCB: Consultancy.

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10

AbstractTransfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti–major histocompatibility complex class I antibodies, we found CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD11c+ dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.

Role of dendritic cell maturation factors produced by human invariant NKT cells in immune tolerance.

In this study, we used the culture supernatant of iNKT cells to identify human myeloid DC maturation factors produced by human CD4+ iNKT cells. S100A8 had a strong maturation effect. Notably, the recombinant S100A8 protein displayed properties of DC maturation functioning, and the induction of DC differentiation by both the purified and the recombinant protein were blocked by anti-S100A8 and anti-TLR-4 mAbs. DC differentiation induced by anti-major histocompatibility complex class II/CD1d Ab, S100A8, or both was qualitatively indistinguishable from that induced by the coculture of DCs and iNKT cells or via culture supplementation with supernatants from activated CD4+ iNKT cells. S100A8 also induced CD4+/CD25+/Foxp3+ Treg cells from naïve T cells. S100A8 may contribute to DC differentiation by elevating transcription factors or activating transcription factor-2, heat shock factor-1, or both, in mature DCs. S100A8 is a novel candidate iNKT cell-dependent DC maturation factor.

Elevation of C-reactive protein levels in patients with transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within six hours following blood transfusion. In most cases, donor antibodies are suggested to be involved, however, the pathogenesis is poorly understood. A two-hit model is generally assumed to underlie TRALI pathogenesis where the first hit consists of a patient predisposing factor such as inflammation and the second hit is due to donor antibodies present in the transfused blood. We recently demonstrated that the acute phase protein C-reactive protein (CRP) could enhance murine anti-major histocompatibility complex (MHC) class I-mediated TRALI. Whether CRP is increased in human TRALI patients which would support its role as a risk factor for human TRALI, is currently unknown. For that purpose, we measured CRP levels in the plasma of human TRALI patients and found CRP levels to be significantly elevated compared to transfused control patients. These data support the notion that CRP may be a novel first hit risk factor in human TRALI and that modulation of CRP levels could be an effective therapeutic strategy for this serious adverse event of transfusion.

Interferon Gamma and Contact-dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell-Mediated Antibody-dependent Chronic Rejection.

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor-specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody-mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN-γ) versus the contact-dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune-deficient C57BL/6 rag-/- γc-/- recipients, who also received monoclonal anti-major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild-type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN-γ-deficient NK cells or host IFN-γ neutralization led to amelioration of these lesions. Use of either perforin-deficient NK cells or CD95 (Fas)-deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell-derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN-γ production and contact-dependent cytotoxic activity are rate-limiting effector pathways that contribute to this form of antibody-induced chronic allograft vasculopathy.

Serum miR-338-5p, Soluble B-Cell–Activating Factor, Allo-Antibodies, and Renal Transplantation

BackgroundThe objective of the study was to explore the expression features of serum miR-338-5p and soluble B-cell–activating factor (sBAFF) in renal transplant recipients. MethodsFollow-up renal transplant recipients (n = 49) were enrolled in this study (male/female: 38/11). Healthy volunteers were controlled; 2 mL of peripheral blood from each subject was collected. Total RNA was extracted from serum by use of the miRNeasy Serum/Plasma Kit (QIAGEN), and miR-338-5p was amplified by means of quantitative real-time reverse transcriptase–polymerase chain reaction. sBAFF was detected by means of enzyme-linked immunoassay. LABScreen Mix (LSM12) (One Lambda) was used to test the level of anti–human leukocyte antigen (HLA) I antibody (Ab), anti–HLA II Ab, and anti–major histocompatibility complex class I chain-related A (MICA) Ab. All data are shown as mean ± SD and were analyzed by use of SPSS software 17.0. ResultsCompared with healthy volunteers, serum miR-338-5p in recipients was statistically downregulated (2.79 ± 2.5 versus 0.09 ± 0.12, P < .001); sBAFF in recipients was significantly upregulated (1321 ± 950 pg/mL versus 534 ± 327 pg/mL, P < .01); serum anti–HLAII Ab, anti–MICA Ab, and anti–HLA+MICA Abs all statistically increased in recipients (P < .05). Spearman correlation analysis showed that miR-338-5p was significantly negatively correlated with sBAFF (r = −0.51, P < .001) and anti–HLA II antibody with mean fluorescence intensity value >1000 (r = −0.322, P < .05). Analysis results also suggested that sBAFF was significantly negatively correlated with anti–MICA Ab, with mean fluorescence intensity value >1000 (r = −0.579, P < .05). ConclusionsmiR-338-5p is closely correlated with the procedure of renal allograft antibody-mediated rejection.

Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD.

Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients’ prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10−5). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6).

Harmful Effect of Preformed Anti-MICA Antibodies on Renal Allograft Evolution in Early Posttransplantation Period

Pretransplantation anti-major histocompatibility complex class I chain-related molecule A (MICA) sensitization is an uncommon event and its role on kidney graft evolution is not completely defined. A retrospective study of patients transplanted between 2005 and 2011 in our center (n=727) was performed. Recipients were classified in four groups, according either to multiplexed flow cytometry-recorded anti-human leukocyte antigen (HLA) and anti-MICA antibodies or to percent panel-reactive antibody (PRA; by complement-dependent cytotoxicity) and anti-MICA antibodies. In the total cohort, 52 (7.15%) patients had preformed anti-MICA antibodies, and these were not related with anti-HLA, previous transplantations, or recipient female sex (potential pregnancies). Kaplan-Meier curves showed global allograft survival differences (P=0.042) mostly due to pronounced decrease in PRA+MICA+ group early after transplantation. Biopsy-proven allograft rejection rate increased after month 12 in PRA+MICA- group and was higher early after transplantation in PRA+MICA+ group (P=0.033). In paired comparisons, rejection incidence was superior in PRA+MICA- versus PRA-MICA- patients (17% vs. 7%; P=0.007) at 24 months, confirming the widely reported deleterious effect of PRA+ status, but at 3 months rejection was higher in PRA+MICA+ versus PRA-MICA- patients (14% vs. 2%; P=0.009). Among patients categorized according anti-HLA and anti-MICA antibodies, the most striking difference in rejection was observed at 3 months (8% in HLA-MICA+ vs. 2% in HLA-MICA- patients; P=0.032). In the multivariate analysis, HLA-MICA+ status at 3 months independently conferred the highest risk for rejection (odds ratio, 5.07; P=0.049). Pretransplantation sensitization against MICA and HLA are independent events. Preformed anti-MICA antibodies independently increase risk for kidney rejection and enhance the deleterious effect of PRA+ status early after transplantation.

The Effect of Anti-Human Leukocyte Antigen, Anti-Major Histocompatibility Complex Class 1 Chain-Related Antigen A, and Anti-Glutathione Transferase-T1 Antibodies on the Long-Term Survival of Renal Allograft

BackgroundOne of the most important mechanisms of allograft rejection is the production of donor-specific antibodies (DSA). Anti-major histocompatibility complex class-I chain-related antigen A (MICA) and anti-glutathione S transferase-T1 (GSTT1) antibodies cause graft dysfunction and reduce graft survival. The aim of this study was to examine the effects of anti-human leukocyte antigen class I–II, anti-MICA, and anti-GSTT1 antibodies in development of antibody-mediated rejection. MethodsAmong the 32 renal transplant patients included in this study 65% experienced antibody-mediated rejection (AMR; chronic active AMR [CAMR], n = 17; acute AMR [AAMR], n = 4) and 35%, ACR. The anti-HLA class I–II and anti-MICA antibodies were determined by using LUMINEX, anti-GSTT1 antibodies by enzyme-linked immunosorbent assay. GSTT1 genotyping of patients and donors was performed by polymerase chain reaction. ResultsAntibody was detected in 19 of 21 patients undergoing antibody-mediated rejection (90%). We detected anti-GSTT1 in 4, anti-MICA in 8, anti-HLA class I in 5, and anti-HLA class II in 9 patients with CAMR (P = .007). If the patients were divided into 2 groups according to being C4d+ and C4d− both anti-HLA class I and class II antibodies were found significantly more frequently among the C4d+ group (P = .019, P = .024). No difference was determined between AMR and ACR groups in terms of anti-GSTT1 and anti-MICA antibodies. ConclusionsIn this study, we observed the role of anti-HLA class II antibodies in the development of CAMR and in long-term allograft survival. It is observed that anti-MICA and anti-GSTT1 antibodies showed no effect on rejection mechanisms.