Alexandru Schiopu

In vivo prevention of transplant arteriosclerosis by ex vivo-expanded human regulatory T cells

Transplant arteriosclerosis is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent transplant arteriosclerosis. Therefore, we designed this study to test the hypothesis that human regulatory T cells (Treg cells) expanded ex vivo can prevent transplant arteriosclerosis. Here we show the comparative capacity of Treg cells, sorted via two separate strategies, to prevent transplant arteriosclerosis in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of transplant arteriosclerosis in human arteries was prevented by treatment of ex vivo–expanded human Treg cells. Additionally, we show that Treg cells sorted on the basis of low expression of CD127 provide a more potent therapy to conventional Treg cells. Our results demonstrate that human Treg cells can inhibit transplant arteriosclerosis by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting transplant arteriosclerosis in both allograft transplantation and other immune-mediated causes of vasculopathy.

Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis

Background—Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. Methods and Results—Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE−/− mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. Conclusions—These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.

S100A8 and S100A9: DAMPs at the Crossroads between Innate Immunity, Traditional Risk Factors, and Cardiovascular Disease

Amplification of innate immune responses by endogenous danger-associated molecular patterns (DAMPs) promotes inflammation. The involvement of S100A8 and S100A9, DAMPs belonging to the S100 calgranulin family, in the pathogenesis of cardiovascular disease is attracting an increasing amount of interest. S100A8 and S100A9 (also termed MRP8 and MRP14) preferentially form the S100A8/A9 heterodimer (MRP8/14 or calprotectin) and are constitutively expressed in myeloid cells. The levels of circulating S100A8/A9 in humans strongly correlate to blood neutrophil counts and are increased by traditional cardiovascular risk factors such as smoking, obesity, hyperglycemia, and dyslipidemia. S100A8/A9 is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE) and has been shown to promote atherogenesis in mice. In humans, S100A8/A9 correlates with the extent of coronary and carotid atherosclerosis and with a vulnerable plaque phenotype. S100A8/A9 is locally released following myocardial infarction and amplifies the inflammatory responses associated with myocardial ischemia/reperfusion injury. Elevated plasma levels of S100A8/A9 are associated with increased risk of future coronary events in healthy individuals and in myocardial infarction survivors. Thus, S100A8/A9 might represent a useful biomarker and therapeutic target in cardiovascular disease. Importantly, S100A8/A9 blockers have been developed and are approved for clinical testing.

Role of T cells in graft rejection and transplantation tolerance

Transplantation is the most effective treatment for end-stage organ failure, but organ survival is limited by immune rejection and the side effects of immunosuppressive regimens. T cells are central to the process of transplant rejection through allorecognition of foreign antigens leading to their activation, and the orchestration of an effector response that results in organ damage. Long-term transplant acceptance in the absence of immunosuppressive therapy remains the ultimate goal in the field of transplantation and many studies are exploring potential therapies. One promising cellular therapy is the use of regulatory T cells to induce a state of donor-specific tolerance to the transplant. This article first discusses the role of T cells in transplant rejection, with a focus on the mechanisms of allorecognition and the alloresponse. This is followed by a detailed review of the current progress in the field of regulatory T-cell therapy in transplantation and the translation of this therapy to the clinical setting.

Functional Regulatory T Cells Produced by Inhibiting Cyclic Nucleotide Phosphodiesterase Type 3 Prevent Allograft Rejection

A clinically approved agent can generate ex vivo graft-reactive, functional mouse and human regulatory T cells. Regulatory T cells Go cAMPing As summer approaches, campers yearn to leave their homes and sleep out under the stars. However, camping isn’t as blissful at it seems from the comfort of a couch in an air-conditioned home: Bugs, heat, and lack of facilities all take their toll on the sensitive adventurer. Transplanted organs face similar challenges in a new host. Not only must they acclimatize to a new environment, but they have to avoid attack from the recipient’s immune system. One way to dodge damage is with the help of immunosuppressive regulatory T cells (Tregs). Feng et al. now find that stimulating these Tregs in the presence of PDE (phosphodiesterase 3) inhibition enriches functional allospecific Tregs, which then can prevent transplant rejection. Adenosine 3′,5′-monophosphate (cAMP) not only directly inhibits effector T cells, it also promotes production of Tregs. Feng et al. hypothesize that inhibiting PDEs, which break down cAMP, would increase cAMP concentrations in Tregs in culture and promote expansion of these cells in the presence of allogeneic dendritic cells. In the mouse PDE3 inhibition with cilostamide in conjunction with allostimulation enriched for functional alloreactive Tregs; these cells inhibited effector T cell function in vitro and prevented graft rejection. PDE3 inhibition also generates human Tregs and these inhibit T cell proliferation and prevent the rejection of human vessel transplants in a humanized mouse transplant model. The ability to selectively enhance allospecific Treg proliferation using cilostamide, a clinically approved agent, should decrease the need for general immunosuppression in transplant recipients. Allospecific Tregs expanded with cilostamide may provide a transplanted organ with an environment that is less like the great outdoors and instead has most of the comforts of home. Regulatory T cells (Tregs) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring Tregs, an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive Tregs. Here we demonstrate that stimulation of mouse CD4+ T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3+ T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4+ effectors or donor-reactive CD8+ T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3+ CD4+ T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies.

Association Between IgM Against an Aldehyde-Modified Peptide in Apolipoprotein B-100 and Progression of Carotid Disease

Background and Purpose— Autoantibodies against antigens in oxidized low-density lipoprotein are common in people; experimental studies suggest that these immune responses have a functional role in the disease process. The aim of this study was to evaluate the relationship between the immune response against one defined oxidized low-density lipoprotein antigen, the aldehyde-modified peptide corresponding amino acids 3136 and 3155 (MDA-p210) in apolipoprotein (apo) B-100, and progression of carotid intima media thickness (IMT). Methods— IgM and IgG against MDA-p210 were determined by enzyme-linked immunosorbent assay at baseline and after 12 months of treatment with placebo, metoprolol, fluvastatin, or metoprolol/fluvastatin in 751 individuals participating in the BCAPS. Carotid IMT was assessed by ultrasonography at baseline and after 18 and 36 months of treatment. Results— Antibody levels did not change in response to treatment, but high baseline MDA-p210 IgM levels were associated with a more rapid progression of carotid disease both at 18 (r=0.09, P<0.05) and 36 months (r=0.12, P<0.005). At 36 months, the difference in IMT progression rate per year between those with high MDA-p210 IgM levels and those with low was 0.011 mm (95% CI=0.005 to 0.018 mm, P<0.0001). Treatment with fluvastatin markedly decreased the progression of IMT among subjects with high but not with low MDA-p210 IgM levels. There was no association between MDA-p210 IgG and carotid IMT progression. Conclusions— IgM against the aldehyde-modified peptide corresponding amino acids 3136 and 3155 in apo B-100 is common in subjects with asymptomatic carotid disease, and high levels are associated with a more rapid progression of carotid IMT. The observation that the effect of fluvastatin was restricted to subjects with high MDA-p210 IgM levels may reflect the increased rate of disease progression in this group.

Oxidized LDL Antibodies in Treatment and Risk Assessment of Atherosclerosis and Associated Cardiovascular Disease

Immune responses against oxidized forms of LDL play a critical role in activation and regulation of the inflammatory process that characterizes all stages of atherosclerosis. In humans oxidized LDL is targeted by both IgM and IgG autoantibodies. Immunization of hypercholesterolemic animals with oxidized LDL has been shown to inhibit atherosclerosis demonstrating that at least some of these immune responses have a protective effect. The identification of the structures in oxidized LDL that are responsible for activation of immunity has made it possible to develop novel therapeutic approaches for treatment of atherosclerosis based on active (vaccines) and passive (antibodies) immunization. Studies performed in atherosclerosis-prone mice demonstrate that both peptide-based vaccines and recombinant IgG targeting epitopes in oxidized LDL significantly reduce atherosclerosis. There is also evidence antibodies against oxidized LDL could also be used for imaging atherosclerosis.

Plasma S100A8/A9 Correlates With Blood Neutrophil Counts, Traditional Risk Factors, and Cardiovascular Disease in Middle-Aged Healthy Individuals

Objective— The S100 alarmins A8, A9, and A8/A9, secreted by activated neutrophils and monocytes/macrophages, are involved in the pathogenesis of various inflammatory diseases. S100A8/A9 has previously been linked to atherogenesis and cardiovascular (CV) disease. We investigated whether S100A8, A9, and A8/A9 correlate with carotid artery disease and CV risk in apparently healthy individuals. Approach and Results— We measured baseline S100A8, A9, and A8/A9 in 664 individuals aged 63 to 68 years, with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer population cohort. We examined the correlations between S100 proteins and circulating cell populations, plasma cytokines, carotid artery disease, and incidence of CV events during a median follow-up period of 16.2 years. We found that plasma S100A8/A9 concentration is positively influenced by circulating neutrophil numbers, smoking, body mass index, glycosylated hemoglobin A1c, and low-density lipoprotein. High-density lipoprotein was negatively associated with S100A8/A9. S100A8/A9 and the neutrophil counts were positively correlated with intima-media area in the common carotid artery, independently of age, sex, and CV risk factors. S100A8/A9 and circulating neutrophils presented similar associations with the incidence of coronary events (hazard ratio [95% confidence interval] per 1 SD: 1.28 [1.03–1.59] and 1.26 [1.04–1.53], respectively) and CV death (1.34 [1.06–1.71] and 1.59 [1.33–1.90], respectively). These relationships were mainly supported by strong associations in women, which were independent of traditional risk factors. There were no independent relationships between S100A8 and S100A9, and CV disease. Conclusions— Our study supports the value of S100A8/A9 as a potentially important link between neutrophils, traditional CV risk factors, and CV disease.

Regulatory T cells: hypes and limitations.

PURPOSE OF REVIEW In recent years there has been increased interest in understanding the physiology and function of regulatory T cells. In this review we focus on the characterization of regulatory T-cell subsets and their potential therapeutic use in organ transplantation. RECENT FINDINGS Regulatory T cells can play an instrumental role in the establishment of operational tolerance to allografts. The level of expression and the extent of posttranslational acetylation of the regulatory T-cell specific transcription factor Foxp3 are important modulators of their suppressive activity. Low expression of CD127 can be used as a novel marker to define pure regulatory T-cell populations and the expression of CD45RA on CD4CD25 regulatory T cells characterizes a population with a more stable phenotype upon expansion in vitro. Interleukin-35 is a recently discovered immunosuppressive cytokine secreted by CD4CD25 regulatory T cells. Although the presence of allospecific memory T cells in the pretransplant period and the use of immunosuppressants might interfere with the effectiveness of regulatory T-cell-based therapies, encouraging results indicate that the immunosuppressive drug rapamycin does not affect the expansion and function of regulatory T cells and could be included in a combined therapy. SUMMARY Important advances have been made towards establishing regulatory T cells as a viable therapy in transplantation and the first clinical trials using human regulatory T cells are currently underway. There are, however, important limitations and safety issues that have to be addressed before this therapy can be fully translated into the clinic.

A high quality diet is associated with reduced systemic inflammation in middle-aged individuals

OBJECTIVE To examine if overall diet quality is associated with cellular and soluble biomarkers of systemic inflammation in middle-aged individuals. METHODS A group of 667 individuals, aged 63-68 years, selected from the cardiovascular arm of the Malmö Diet and Cancer cohort, participated in this study. Baseline examinations consisted of an extensive socio-demographic questionnaire, anthropometric measurements, blood sampling and detailed dietary data. Mononuclear leukocytes frozen at baseline were thawed and analysed with flow cytometry to quantify monocyte subsets based on CD14 and CD16 expression. Plasma cytokines were measured using multiplexed immune assays. A diet quality index consisting of six components (saturated fatty acids, polyunsaturated fatty acids, fish and shellfish, dietary fibre, fruit and vegetables, and sucrose) was constructed to measure adherence to the Swedish Nutrition Recommendations/Dietary Guidelines. General linear models were used to investigate associations between index scores and several biomarkers of inflammation. RESULTS A higher percentage of women reported adherence to the nutritional recommendations and had better overall diet quality than men. Participants with higher diet quality were more likely to have a healthier lifestyle. The levels of high-sensitive CRP, S100A8/A9, TNF-α, white blood cells, neutrophils, lymphocytes and CD14(+)CD16(++) were lower in participants with higher index scores. The associations remained significant after adjustment for potential confounders. CONCLUSION In this cross-sectional study, we found that a high diet quality is associated with lower systemic inflammation. As the incidence of cardiovascular disease and cancer is directly correlated with the levels of inflammation, our findings might indicate a protective role of high-quality diet.