Agnes M. Azimzadeh

Fish oil, but not flaxseed oil, decreases inflammation and prevents pressure overload-induced cardiac dysfunction

AIMS Clinical studies suggest that intake of omega-3 polyunsaturated fatty acids (omega-3 PUFA) may lower the incidence of heart failure. Dietary supplementation with omega-3 PUFA exerts metabolic and anti-inflammatory effects that could prevent left ventricle (LV) pathology; however, it is unclear whether these effects occur at clinically relevant doses and whether there are differences between omega-3 PUFA from fish [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and vegetable sources [alpha-linolenic acid (ALA)]. METHODS AND RESULTS We assessed the development of LV remodelling and pathology in rats subjected to aortic banding treated with omega-3 PUFA over a dose range that spanned the intake of humans taking omega-3 PUFA supplements. Rats were fed a standard food or diets supplemented with EPA+DHA or ALA at 0.7, 2.3, or 7% of energy intake. Without supplementation, aortic banding increased LV mass and end-systolic and -diastolic volumes. ALA supplementation had little effect on LV remodelling and dysfunction. In contrast, EPA+DHA dose-dependently increased EPA and DHA, decreased arachidonic acid in cardiac membrane phospholipids, and prevented the increase in LV end-diastolic and -systolic volumes. EPA+DHA resulted in a dose-dependent increase in the anti-inflammatory adipokine adiponectin, and there was a strong correlation between the prevention of LV chamber enlargement and plasma levels of adiponectin (r = -0.78). Supplementation with EPA+DHA had anti-aggregatory and anti-inflammatory effects as evidenced by decreases in urinary thromboxane B(2) and serum tumour necrosis factor-alpha. CONCLUSION Dietary supplementation with omega-3 PUFA derived from fish, but not from vegetable sources, increased plasma adiponectin, suppressed inflammation, and prevented cardiac remodelling and dysfunction under pressure overload conditions.

Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62Lhigh) and effector memory (TEM; CD62Llow) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-γ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-γ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease.

Inducing CTLA-4–Dependent Immune Regulation by Selective CD28 Blockade Promotes Regulatory T Cells in Organ Transplantation

An improved method of immunosuppression allows better immune function and prolongs the survival of transplanted organs in nonhuman primates. Increasing Tolerance with Less Toxicity According to Chinese legend, 2500 years ago the physician Pien Ch’iao exchanged the hearts of two warriors, one of strong spirit and weak will and the other of weak spirit and strong will, with the aim of achieving balance in their characters. Of course, without modern surgical techniques and the ability to suppress organ rejection by the recipient’s immune system, such transplants would have been impossible. It was not until the discovery of the powerful immunosuppressant drug cyclosporin that organ transplants between unrelated individuals became routinely successful, beginning in the 1980s. Cyclosporin and some newer similar drugs, however, have toxic side effects, and none of them can stop eventual rejection of the organ, so researchers are seeking other options. Now, Vanhove and colleagues test an alternative immunosuppressive strategy. T cells are a major culprit in the immune response directed against transplanted organs, and therefore a number of immunosuppressive drugs target T cells. Cyclosporin, for example, inhibits calcineurin, a protein phosphatase that under normal conditions increases the expression of interleukin 2, which—among other functions—promotes the proliferation of activated T cells. Certain newer approaches to immunosuppression target the process by which T cells become activated. In general, this process is induced by antigen binding to the T cell receptor and reinforced by costimulatory molecules—CD80 and CD86—binding to the T cell CD28 receptor. Additionally, CD80/86 can bind to the CTLA-4 receptor, resulting in T cell inhibition. CTLA-4 is also required for the function of regulatory T cells (Tregs), which suppress aberrant immune responses and are important for inducing tolerance toward transplanted tissue. Several drugs that block CD80/86 have been developed as a nontoxic alternative to calcineurin inhibitors, but in addition to affecting the CD28-mediated pathway of T cell stimulation, these reagents also inhibit the CTLA-4 signals required for Treg function. To avoid inhibiting CTLA-4–dependent regulation of the immune system while suppressing T cell activation, the Vanhove, Blancho, and Pierson labs aimed to block CD28 function directly in experiments on kidney and heart transplantation in baboons and monkeys. Previous work showed that down-regulation of CD28 activity with an anti-CD28 monoclonal antibody inhibited rejection of organ transplants in rodents. For the new nonhuman primate studies, the researchers used a fragment of a human CD28-specific monoclonal antibody fused to a carrier molecule to increase its half-life in vivo. This fusion antibody, sc28AT, recognizes CD28 from monkeys and baboons as well as humans, and it competes with CD80/86 for binding to CD28. sc28AT increased the number of functional Tregs and reduced chronic rejection of the transplanted tissue in the primates when used together with a calcineurin inhibitor. This is good news because it suggests that the dose of such inhibitors (and their toxic side effects) could be reduced in patients when given with a molecule targeting CD28. It remains to be determined whether this approach offers practical advantages over straight CD80/86 blockade when tested in the clinic. Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4–dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4–dependent immune regulation and promotes allograft survival.

THE INNATE IMMUNE RESPONSE AND ACTIVATION OF COAGULATION IN α1,3-GALACTOSYLTRANSFERASE GENE-KNOCKOUT XENOGRAFT RECIPIENTS

Background. The role of the innate immune system in the development of thrombotic microangiopathy (TM) after α1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain. Methods. Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out. Results. Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft. Conclusions. Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.

Anti-CD20 treatment depletes B-cells in blood and lymphatic tissue of cynomolgus monkeys

INTRODUCTION Macaque species offer a valuable model for translational allo-transplantation and tolerance studies. Cardiac allograft vasculopathy in Macaca fascicularis is associated with elaboration of anti-donor antibodies. Since T-independent pathways of B cell activation have been described, and anti-B cell strategies have proven to be a fruitful tolerogenic adjunct in rodent and xenogenic models, here we investigate whether an anti-CD20 antibody (rituximab) would be useful to deplete B-cells in a pre-clinical allo-transplantation setting in macaques. METHODS Three cynomolgus macaques which had previously rejected a cardiac allograft and one with concurrent subacute vascular rejection were treated weekly with rituximab 20 mg/kg i.v. for 4 and 2 weeks, respectively. B-cell levels (CD19+ cells) were measured by flow cytometry in peripheral blood, spleen, lymph node and bone marrow cells at various intervals after initiation of treatment. B-cells and plasma cells were also analyzed by immunohistochemistry at necropsy in spleen, lymph node, tonsil and thymus tissue sections. Anti-donor antibody titers were measured by flow cytometry. RESULTS B-cells expressing CD19 were not detectable in the peripheral blood in any animal within 24 h after initial treatment, or over the ensuing month. At necropsy, the germinal centers in spleen and lymph node were completely depleted of CD20+ B-cells in 2 animals, leaving a hypocellular trabecular pattern around preserved plasma cell follicles. Substantial but incomplete depletion of B-cells was demonstrated in the other 2 animals, in each instance immunohistochemical findings in spleen and lymph node exhibiting higher sensitivity for residual B-cells compared to FACS. Anti-donor antibody titers exhibited kinetics similar to untreated animals over this short follow-up. COMMENT Treatment with anti-CD20 very efficiently depletes peripheral and tissue B-cells but not plasma cells in this macaque species. Biopsy of lymph node is necessary and may be sufficient to assess B-cell clearance in secondary lymphoid organs in this model.

B‐Cell Depletion Extends the Survival of GTKO.hCD46Tg Pig Heart Xenografts in Baboons for up to 8 Months

Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Rejection in Gal knockout (GTKO) pigs due to elicited non‐Gal antibody response required further genetic modifications of donor pigs and better control of the B‐cell response to xenoantigens. We report significant prolongation of heterotopic alpha Galactosyl transferase “knock‐out” and human CD46 transgenic (GTKO.hCD46Tg) pig cardiac xenografts survival in specific pathogen free baboons. Peritransplant B‐cell depletion using 4 weekly doses of anti‐CD20 antibody in the context of an established ATG, anti‐CD154 and MMF‐based immunosuppressive regimen prolonged GTKO.hCD46Tg graft survival for up to 236 days (n = 9, median survival 71 days and mean survival 94 days). B‐cell depletion persisted for over 2 months, and elicited anti‐non‐Gal antibody production remained suppressed for the duration of graft follow‐up. This result identifies a critical role for B cells in the mechanisms of elicited anti‐non‐Gal antibody and delayed xenograft rejection. Model‐related morbidity due to variety of causes was seen in these experiments, suggesting that further therapeutic interventions, including candidate genetic modifications of donor pigs, may be necessary to reduce late morbidity in this model to a clinically manageable level.

CTLA4 Expression Is an Indicator and Regulator of Steady-State CD4+FoxP3+ T Cell Homeostasis

The presence of FoxP3+ regulatory T cells (Tregs) is necessary for control of deleterious immune responses in the steady state; however, mechanisms for maintaining the frequency and quality of endogenous Tregs are not well defined. In this study, we used in vivo modulators of the CD28 and CTLA4 pathways administered to intact mice to reveal mechanisms controlling the homeostasis and phenotype of endogenous Tregs. We demonstrate that expression of the negative costimulatory regulator CTLA4 on FoxP3+ Tregs in vivo is a direct consequence of their rapid, perpetual homeostasis. Up-regulation of CTLA4 expression occurs only on FoxP3+ Tregs undergoing extensive proliferation and can be abrogated by inhibiting the CD28 pathway, coinciding with a reduction in FoxP3+ Treg proliferation and frequency. We further demonstrate that CTLA4 negatively regulates steady-state Treg homeostasis, given that inhibiting CTLA4 signaling with an anti-CTLA4 blocking Ab greatly enhances Treg proliferation and overall Treg frequency. Our findings provide new insight into the origin and role of CTLA4 expression on natural FoxP3+ Tregs and reveal opposing effects of costimulation modulators on the steady-state level and quality of Tregs, with implications regarding their effects on endogenous Tregs in patients receiving immunotherapy.

Selective CD28 blockade attenuates acute and chronic rejection of murine cardiac allografts in a CTLA-4-dependent manner.

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single‐chain Fv‐based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA‐4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154‐blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA‐4 during α28scFv‐based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)‐1 and 2,3‐indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA‐4 is thus a promising approach to inhibit pathogenic alloimmunity.

CCR5 Blockade Modulates Inflammation and Alloimmunity in Primates

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Δ32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck’s compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.

Preemptive CD20+ B cell depletion attenuates cardiac allograft vasculopathy in cyclosporine-treated monkeys

Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (alphaCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with alphaCD20 or CsA alone. In animals treated with both alphaCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic.