Leveraging the tolerogenic potential of TNF-α and regulatory B cells in organ transplantation.

A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-ß, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance.

Suppression of allograft rejection by regulatory B cells induced via TLR signaling.

B lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and Ab production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to downregulate unwanted immune responses, such as transplant rejection. In this study, we found that PMA- and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through TLR4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-ß and not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced Tregs in a TGF-ß-dependent manner. RNA-Seq analyses corroborated the involvement of TGF-ß pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested targets to foster Breg regulation.

Interleukin-27 in liver xenotransplantation: A rational target to mitigate ischemia reperfusion injury and increase xenograft survival.

Transplantation of xenogeneic organs is an attractive solution to the existing organ shortage dilemma, thus, securing a clinically acceptable prolongation of xenograft survival is an important goal. In preclinical transplantation models, recipients of liver, kidney, heart, or lung xenotransplants demonstrate significant graft damages through the release of pro-inflammatory molecules, including the C-reactive protein, cytokines, and histone-DNA complexes that all foster graft rejection. Recent studies have demonstrated that mitigation of ischemia reperfusion injury (IRI) greatly improves xenograft survival. Organ IRI develops primarily on a complex network of cytokines and chemokines responding to molecular cues from the graft milieu. Among these, interleukin 27 (IL-27) plays an immunomodulatory role in IRI onset due to graft environment-dependent pro- and anti- inflammatory activities. This review focuses on the impact of IL-27 on IRI of liver xenotransplants and provides insights on the function of IL-27 that could potentially guide genetic engineering strategies of donor pigs and/or conditioning of organs prior to transplantation.

An Unbiased Machine Learning Exploration Reveals Gene Sets Predictive of Allograft Tolerance After Kidney Transplantation.

Efforts at finding potential biomarkers of tolerance after kidney transplantation have been hindered by limited sample size, as well as the complicated mechanisms underlying tolerance and the potential risk of rejection after immunosuppressant withdrawal. In this work, three different publicly available genome-wide expression data sets of peripheral blood lymphocyte (PBL) from 63 tolerant patients were used to compare 14 different machine learning models for their ability to predict spontaneous kidney graft tolerance. We found that the Best Subset Selection (BSS) regression approach was the most powerful with a sensitivity of 91.7% and a specificity of 93.8% in the test group, and a specificity of 86.1% and a sensitivity of 80% in the validation group. A feature set with five genes (HLA-DOA, TCL1A, EBF1, CD79B, and PNOC) was identified using the BSS model. EBF1 downregulation was also an independent factor predictive of graft rejection and graft loss. An AUC value of 84.4% was achieved using the two-gene signature (EBF1 and HLA-DOA) as an input to our classifier. Overall, our systematic machine learning exploration suggests novel biological targets that might affect tolerance to renal allografts, and provides clinical insights that can potentially guide patient selection for immunosuppressant withdrawal.

Properties of regulatory B cells regulating B cell targets.

Regulatory B cells (Bregs) have shown promise as anti-rejection therapy applied to organ transplantation. However, less is known about their effect on other B cell populations that are involved in chronic graft rejection. We recently uncovered that naïve B cells, stimulated by TLR ligand agonists, converted into B cells with regulatory properties (Bregs-TLR) that prevented allograft rejection. Here, we examine the granular phenotype and regulatory properties of Breg-TLR cells suppressing B cells. Cocultures of Bregs-TLR with LPS-activated B cells showed a dose-dependent suppression of targeted B cell proliferation. Adoptive transfers of Bregs-TLR induced a decline in antibody responses to antigenically disparate skin grafts. The role of Breg BCR specificity in regulation was assessed using B cell-deficient mice replenished with transgenic BCR (OB1) and TCR (OT-II) lymphocytes of matching antigenic specificity. Results indicated that proliferation of OB1 B cells, mediated through help from CD4+ OT-II cells, was suppressed by OB1 Bregs of similar specificity. Transcriptomic analyses indicated that Bregs-TLR suppression is associated with a block in targeted B cell differentiation controlled by PRDM1 (Blimp1). This work uncovered the regulatory properties of a new brand of Breg cells and provided mechanistic insights into potential applications of Breg therapy in transplantation.

Alzheimer's disease brain contains tau fractions with differential prion-like activities.

Neurofibrillary tangles (NFTs) made of abnormally hyperphosphorylated tau are a hallmark of Alzheimer's disease (AD) and related tauopathies. Regional distribution of NFTs is associated with the progression of the disease and has been proposed to be a result of prion-like propagation of misfolded tau. Tau in AD brain is heterogenous and presents in various forms. In the present study, we prepared different tau fractions by sedimentation combined with sarkosyl solubility from AD brains and analyzed their biochemical and pathological properties. We found that tau in oligomeric fraction (O-tau), sarkosyl-insoluble fractions 1 and 2 (SI1-tau and SI2-tau) and monomeric heat-stable fraction (HS-tau) showed differences in truncation, hyperphosphorylation, and resistance to proteinase K. O-tau, SI1-tau, and SI2-tau, but not HS-tau, were hyperphosphorylated at multiple sites and contained SDS- and ß-mercaptoethanol-resistant high molecular weight aggregates, which lacked the N-terminal portion of tau. O-tau and SI2-tau displayed more truncation and less hyperphosphorylation than SI1-tau. Resistance to proteinase K was increased from O-tau to SI1-tau to SI2-tau. O-tau and SI1-tau, but not SI2-tau or HS-tau, captured tau from cell lysates and seeded tau aggregation in cultured cells. Heat treatment could not kill the prion-like activity of O-tau to capture normal tau. Hippocampal injection of O-tau into 18-month-old FVB mice induced significant tau aggregation in both ipsilateral and contralateral hippocampi, but SI1-tau only induced tau pathology in the ipsilateral hippocampus, and SI2-tau and HS-tau failed to induce any detectable tau aggregation. These findings suggest that O-tau and SI1-tau have prion-like activities and may serve as seeds to recruit tau and template tau to aggregate, resulting in the propagation of tau pathology. Heterogeneity of tau pathology within AD brain results in different fractions with different biological and prion-like properties, which may pose a major challenge in targeting tau for development of effective therapeutic treatments.

Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells.

Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.

Regulatory B cells require antigen recognition for effective allograft tolerance induction.

Through multiple mechanisms, regulatory B cells (Breg) have been shown to play an important role in the development of allograft tolerance. However, a careful understanding of the role of antigen-specificity in Breg-mediated allograft tolerance has remained elusive. In experimental models of islet and cardiac transplantation, it has been established that Bregs can be induced in vivo by anti-CD45RB ± anti-TIM1antibody treatment, resulting in prolonged, Breg-dependent allograft tolerance. The importance of Breg antigen recognition has been suggested but not confirmed through adoptive transfer experiments, using tolerant WT C57BL/6 animals challenged with either BALB/c or C3H grafts. However, the importance of receptor-specificity has not been formally tested. Here, we utilize the novel ovalbumin-specific B cell receptor transnuclear (OBI) mice in multiple primary tolerance and adoptive transfer experiments to establish that Breg-dependent allograft tolerance relies on antigen recognition by B cells. Additionally, we identify that this Breg-dependent tolerance relies on the function of transforming growth factor-ß. Together, these experiments mark important progress toward understanding how best to improve Breg-mediated allograft tolerance.

Rap GTPase Interactor: A Potential Marker for Cancer Prognosis Following Kidney Transplantation.

Post-transplant (post-Tx) kidney cancer has become the second-highest cause of death in kidney recipients. Late diagnosis and treatment are the main reasons for high mortality. Further research into early diagnosis and potential treatment is therefore required. In this current study, through genome-wide RNA-Seq profile analysis of post-Tx malignant blood samples and post-Tx non-malignant control blood samples (CTRL-Tx), we found Rap GTPase Interactor (RADIL) and Aprataxin (APTX) to be the most meaningful markers for cancer diagnosis. Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) of the RADIL-APTX signature model was 0.92 (P < 0.0001). Similarly, the AUC of RADIL alone was 0.91 (P < 0.0001) and that of APTX was 0.81 (P = 0.001). Additionally, using a semi-supervised method, we found that RADIL alone could better predict malignancies in kidney transplantation recipients than APTX alone. Kaplan-Meier analysis indicated that RADIL was expressed significantly higher in the early stages (I and II) of kidney, liver, stomach, and pancreatic cancer, suggesting the potential use of RADIL in early diagnosis. Multivariable Cox regression analysis found that RADIL together with other factors (including age, stage III, stage IV and CD8+ T cells) play a key role in kidney cancer development. Among those factors, RADIL could promote kidney cancer development (HR > 1, P < 0.05) while CD8+ T cells could inhibit kidney cancer development (HR < 1, P < 0.05). RADIL may be a new immunotherapy target for kidney cancer post kidney transplantation.

Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation.

It remains unclear whether plasma phospholipid transfer protein (PLTP) is involved in hyper-coagulation or hypo-coagulation. This study investigated the direct effect of PLTP on platelet aggregation and the underlying mechanism. Washed platelets from humans or mice and mouse platelet-rich plasma and human recombinant PLTP were isolated. PLTP is present in human platelets. We assessed adenosine diphosphate (ADP)-, collagen- and thrombin-induced platelet aggregation, phosphatidylserine externalization and photothrombosis-induced cerebral infarction in mice. PLTP over-expression increased platelet aggregation, while PLTP deficiency had the opposing reaction. Human recombinant PLTP increased both mouse and human platelet aggregation in a dose-dependent manner. Phosphatidylserine externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild-type controls, platelets from PLTP transgenic mice had significantly more phosphatidylserine on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less phosphatidylserine on the surface, thus PLTP influences fibrinogen binding on the plasma membrane. Moreover, recombinant PLTP together with ADP significantly increased phosphatidylserine exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. PLTP over-expression significantly accelerated the incidence of photothrombosis-induced infarction in mice, whereas PLTP deficiency significantly reduced the frequency of infarction. We concluded that PLTP promotes phosphatidylserine externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. This effect plays an important role in the initiation of thrombin generation and platelet aggregation under sheer stress conditions. Thus, PLTP is involved in hyper-coagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.

Chronic intermittent hyperoxia alters the development of the hypoxic ventilatory response in neonatal rats.

Chronic exposure to sustained hyperoxia alters the development of the respiratory control system, but the respiratory effects of chronic intermittent hyperoxia have rarely been investigated. We exposed newborn rats to short, repeated bouts of 30% O2 or 60% O2 (5 bouts h(-1)) for 4-15 days and then assessed their hypoxic ventilatory response (HVR; 10 min at 12% O2) by plethysmography. The HVR tended to be enhanced by intermittent hyperoxia at P4 (early phase of the HVR), but it was significantly reduced at P14-15 (primarily late phase of the HVR) compared to age-matched controls; the HVR recovered when individuals were returned to room air and re-studied as adults. To investigate the role of carotid body function in this plasticity, single-unit carotid chemoafferent activity was recorded in vitro. Intermittent hyperoxia tended to decrease spontaneous action potential frequency under normoxic conditions but, contrary to expectations, hypoxic responses were only reduced at P4 (not at P14) and only in rats exposed to higher O2 levels (i.e., intermittent 60% O2). Rats exposed to intermittent hyperoxia had smaller carotid bodies, and this morphological change may contribute to the blunted HVR. In contrast to rats exposed to intermittent hyperoxia beginning at birth, two weeks of intermittent 60% O2 had no effect on the HVR or carotid body size of rats exposed beginning at P28; therefore, intermittent hyperoxia-induced respiratory plasticity appears to be unique to development. Although both intermittent and sustained hyperoxia alter carotid body development and the HVR of rats, the specific effects and time course of this plasticity differs.