Hedgehog costimulation during ischemia-reperfusion injury potentiates cytokine and homing responses of CD4+ T cells.

Introduction: Ischemia reperfusion injury (IRI) confers worsened outcomes and is an increasing clinical problem in solid organ transplantation. Previously, we identified a "PtchHi" T-cell subset that selectively received costimulatory signals from endothelial cell-derived Hedgehog (Hh) morphogens to mediate IRI-induced vascular inflammation. Methods: Here, we used multi-omics approaches and developed a humanized mouse model to resolve functional and migratory heterogeneity within the PtchHi population. Results: Hh-mediated costimulation induced oligoclonal and polyclonal expansion of clones within the PtchHi population, and we visualized three distinct subsets within inflamed, IRI-treated human skin xenografts exhibiting polyfunctional cytokine responses. One of these PtchHi subsets displayed features resembling recently described T peripheral helper cells, including elaboration of IFN-y and IL-21, expression of ICOS and PD-1, and upregulation of positioning molecules conferring recruitment and retention within peripheral but not lymphoid tissues. PtchHi T cells selectively homed to IRI-treated human skin xenografts to cause accelerated allograft loss, and Hh signaling was sufficient for this process to occur. Discussion: Our studies define functional heterogeneity among a PtchHi T-cell population implicated in IRI.

The CD8α-PILRα interaction maintains CD8+ T cell quiescence.

T cell quiescence is essential for maintaining a broad repertoire against a large pool of diverse antigens from microbes and tumors, but the underlying molecular mechanisms remain largely unknown. We show here that CD8α is critical for the maintenance of CD8+ T cells in a physiologically quiescent state in peripheral lymphoid organs. Upon inducible deletion of CD8α, both naïve and memory CD8+ T cells spontaneously acquired activation phenotypes and subsequently died without exposure to specific antigens. PILRα was identified as a ligand for CD8α in both mice and humans, and disruption of this interaction was able to break CD8+ T cell quiescence. Thus, peripheral T cell pool size is actively maintained by the CD8α-PILRα interaction in the absence of antigen exposure.

Smooth Muscle Cell Reprogramming in Aortic Aneurysms.

The etiology of aortic aneurysms is poorly understood, but it is associated with atherosclerosis, hypercholesterolemia, and abnormal transforming growth factor ß (TGF-ß) signaling in smooth muscle. Here, we investigated the interactions between these different factors in aortic aneurysm development and identified a key role for smooth muscle cell (SMC) reprogramming into a mesenchymal stem cell (MSC)-like state. SMC-specific ablation of TGF-ß signaling in Apoe-/- mice on a hypercholesterolemic diet led to development of aortic aneurysms exhibiting all the features of human disease, which was associated with transdifferentiation of a subset of contractile SMCs into an MSC-like intermediate state that generated osteoblasts, chondrocytes, adipocytes, and macrophages. This combination of medial SMC loss with marked increases in non-SMC aortic cell mass induced exuberant growth and dilation of the aorta, calcification and ossification of the aortic wall, and inflammation, resulting in aneurysm development.

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.

The DNA Methylcytosine Dioxygenase Tet2 Sustains Immunosuppressive Function of Tumor-Infiltrating Myeloid Cells to Promote Melanoma Progression.

Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.

Interferon-γ-mediated allograft rejection exacerbates cardiovascular disease of hyperlipidemic murine transplant recipients.

RATIONALE: Transplantation, the most effective therapy for end-stage organ failure, is markedly limited by early-onset cardiovascular disease (CVD) and premature death of the host. The mechanistic basis of this increased CVD is not fully explained by known risk factors. OBJECTIVE: To investigate the role of alloimmune responses in promoting CVD of organ transplant recipients. METHODS AND RESULTS: We established an animal model of graft-exacerbated host CVD by combining murine models of atherosclerosis (apolipoprotein E-deficient recipients on standard diet) and of intra-abdominal graft rejection (heterotopic cardiac transplantation without immunosuppression). CVD was absent in normolipidemic hosts receiving allogeneic grafts and varied in severity among hyperlipidemic grafted hosts according to recipient-donor genetic disparities, most strikingly across an isolated major histocompatibility complex class II antigen barrier. Host disease manifested as increased atherosclerosis of the aorta that also involved the native coronary arteries and new findings of decreased cardiac contractility, ventricular dilatation, and diminished aortic compliance. Exacerbated CVD was accompanied by greater levels of circulating cytokines, especially interferon-γ and other Th1-type cytokines, and showed both systemic and intralesional activation of leukocytes, particularly T-helper cells. Serological neutralization of interferon-γ after allotransplantation prevented graft-related atherosclerosis, cardiomyopathy, and aortic stiffening in the host. CONCLUSIONS: Our study reveals that sustained activation of the immune system because of chronic allorecognition exacerbates the atherogenic diathesis of hyperlipidemia and results in de novo cardiovascular dysfunction in organ transplant recipients.

PPARγ negatively regulates T cell activation to prevent follicular helper T cells and germinal center formation.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates lipid and glucose metabolism. Although studies of PPARγ ligands have demonstrated its regulatory functions in inflammation and adaptive immunity, its intrinsic role in T cells and autoimmunity has yet to be fully elucidated. Here we used CD4-PPARγKO mice to investigate PPARγ-deficient T cells, which were hyper-reactive to produce higher levels of cytokines and exhibited greater proliferation than wild type T cells with increased ERK and AKT phosphorylation. Diminished expression of IκBα, Sirt1, and Foxo1, which are inhibitors of NF-κB, was observed in PPARγ-deficient T cells that were prone to produce all the signature cytokines under Th1, Th2, Th17, and Th9 skewing condition. Interestingly, 1-year-old CD4-PPARγKO mice spontaneously developed moderate autoimmune phenotype by increased activated T cells, follicular helper T cells (TFH cells) and germinal center B cells with glomerular inflammation and enhanced autoantibody production. Sheep red blood cell immunization more induced TFH cells and germinal centers in CD4-PPARγKO mice and the T cells showed increased of Bcl-6 and IL-21 expression suggesting its regulatory role in germinal center reaction. Collectively, these results suggest that PPARγ has a regulatory role for TFH cells and germinal center reaction to prevent autoimmunity.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

OBJECTIVE: Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. METHODS AND RESULTS: Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. CONCLUSIONS: We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Risperidone-related improvement of irritability in children with autism is not associated with changes in serum of epidermal growth factor and interleukin-13.

Risperidone has been shown to improve serious behavioral problems in children with autism. Here we asked whether risperidone-associated improvement was related to changes in concentrations of inflammatory molecules in the serum of these subjects. Seven molecules were identified as worthy of further assessment by performing a pilot analysis of 31 inflammatory markers in 21 medication-free subjects with autism versus 15 healthy controls: epidermal growth factor (EGF), interferon-γ (IFN-γ), interleukin (IL)-13, IL-17, monocyte chemoattractant protein-1 (MCP-1), IL-1 and IL-1-receptor antagonist. Serum concentrations of these markers were then established in a different set of subjects that participated in a double-blind, clinical trial and an expanded group of healthy subjects. In the first analysis, samples obtained from subjects with autism at baseline visits were compared to visits after 8-week treatment with placebo (n=37) or risperidone (n=40). The cytokine concentrations remained stable over the 8-week period for both risperidone and placebo groups. In the second analysis, we explored further the differences between medication-free subjects with autism (n=77) and healthy controls (recruited independently; n=19). Serum levels of EGF were elevated in subjects with autism (median=103 pg/mL, n=75) in comparison to healthy controls (75 pg/mL, n=19; p<0.05), and levels of IL-13 were decreased in autism (median=0.8 pg/mL, n=77) in comparison to controls (9.8 pg/mL, n=19; p=0.0003). These changes did not correlate with standardized measures used for a diagnosis of autism. In summary, risperidone-induced clinical improvement in subjects with autism was not associated with changes in the serum inflammatory markers measured. Whether altered levels of EGF and IL-13 play a role in the pathogenesis or phenotype of autism requires further investigation.

Peroxisome proliferator-activated receptor-γ agonists prevent in vivo remodeling of human artery induced by alloreactive T cells.

BACKGROUND: Ligands activating the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) have antiinflammatory effects. Vascular rejection induced by allogeneic T cells can be responsible for acute and chronic graft loss. Studies in rodents suggest that PPARγ agonists may inhibit graft vascular rejection, but human T-cell responses to allogeneic vascular cells differ from those in rodents, and the effects of PPARγ in human transplantation are unknown. METHODS AND RESULTS: We tested the effects of PPARγ agonists on human vascular graft rejection using a model in which human artery is interposed into the abdominal aorta of immunodeficient mice, followed by adoptive transfer of allogeneic (to the artery donor) human peripheral blood mononuclear cells. Interferon-γ-dependent rejection ensues within 4 weeks, characterized by intimal thickening, T-cell infiltrates, and vascular cell activation, a response resembling clinical intimal arteritis. The PPARγ agonists 15-deoxy-prostaglandin-J(2), ciglitazone, and pioglitazone reduced intimal expansion, intimal infiltration of CD45RO(+) memory T cells, and plasma levels of inflammatory cytokines. The PPARγ antagonist GW9662 reversed the protective effects of PPARγ agonists, confirming the involvement of PPARγ-mediated pathways. In vitro, pioglitazone inhibited both alloantigen-induced proliferation and superantigen-induced transendothelial migration of memory T cells, indicating the potential mechanisms of PPARγ effects. CONCLUSION: Our results suggest that PPARγ agonists inhibit allogeneic human memory T cell responses and may be useful for the treatment of vascular graft rejection.

Altered immunoglobulin profiles in children with Tourette syndrome.

BACKGROUND: Post-infectious autoimmunity and immune deficiency have been implicated in the pathogenesis of Tourette syndrome (TS). We asked here whether B cell immunity of patients with TS differs from healthy subjects. METHODS: In two independent cross-sectional samples, we compared serum levels of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE in 21 patients with TS from Yale University (17 males, 4 females, 8-16 years) versus 21 healthy controls (13 males, 8 females, 7-17 years); and in 53 patients with TS from Groningen University (45 males, 8 females, 6-18 years) versus 53 healthy controls (22 males, 31 females, 6-18 years), respectively. We also investigated correlations between Ig concentrations and symptom severity. In 13 additional patients (9 males, 4 females, age range 9-14), we established Ig profiles at time points before, during, and after symptom exacerbations. RESULTS: IgG3 levels were significantly lower in Yale patients compared to healthy children (medians 0.28 versus 0.49 mg/ml, p=.04), while levels of IgG2, IgG4, and IgM in patients were lower at trend-level significance (p≤.10). Decreased IgG3 (medians 0.45 versus 0.52 mg/ml; p=.05) and IgM (medians 0.30 versus 0.38 mg/ml; p=.04) levels were replicated in the Groningen patients. Ig levels did not correlate with symptom severity. There was a trend-level elevation of IgG1 during symptom exacerbations (p=.09). CONCLUSION: These pilot data indicate that at least some patients with TS have decreased serum IgG3, and possibly also IgM levels, though only few subjects had fully expressed Ig immunodeficiency. Whether these changes are related to TS pathogenesis needs to be investigated.

Cell-permeable Foxp3 protein alleviates autoimmune disease associated with inflammatory bowel disease and allergic airway inflammation.

Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4(+)CD25(-) T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.

Children with Tourette's syndrome may suffer immunoglobulin A dysgammaglobulinemia: preliminary report.

BACKGROUND: Postinfectious autoimmunity has been implicated in Tourette's syndrome and obsessive-compulsive disorder (TS/OCD), whereas increased frequency of upper respiratory tract infections (URTI) in TS/OCD patients suggests immune deficiency. We hypothesized that antineuronal antibodies may be elevated in patients (reflecting autoimmune processes), and levels of total immunoglobulins (Igs) may be decreased (reflecting immune deficiency). METHODS: We analyzed plasma of TS/OCD patients (n = 24) and healthy age- and sex-matched control subjects (n = 22) by enzyme-linked immunosorbent assay (ELISA) for the levels of total and specific IgG, IgM, and IgA against antigens previously identified in multiple sclerosis (myelin basic protein and myelin-associated glycoprotein) and Sydenham's chorea (ganglioside-GM1, lysoganglioside, and tubulin). RESULTS: Total IgA was decreased in TS/OCD patients (median 115 mg/100 mL) compared with control subjects (141 mg/100 mL; p = .02). Specific IgA against all antigens, except tubulin were also decreased in the patients (MPB 0 vs. 13 [ELISA units [EU]; myelin-associated glycoprotein 29 vs. 44 EU, p = .04; ganglioside GM1 21 vs. 35 EU, p = .01; lysoganglioside 44 vs. 56 EU, p = .03; tubulin 44 vs. 44 EU, p = .8). The levels of total IgA and anti-myelin basic protein (MBP) IgA were significantly lower in the subgroup of pediatric autoimmune neuropsychiatric disorder associated with Streptococcus (PANDAS) cases (n = 10) than in non-PANDAS cases (n = 9; total IgA 98 mg/100 mL vs. 133 mg/mL, p = .03; anti-MBP IgA 1 vs. 6 EU, p = .03) or healthy control subjects (total IgA 141 mg/100 mL, p = .02; anti-MBP IgA 13 EU, p = .005). CONCLUSIONS: At least some TS/OCD patients may suffer IgA dysgammaglobulinemia, possibly rendering the children more prone to URTI.

Paricalcitol (19-nor-1,25-dihydroxyvitamin D2) and calcitriol (1,25-dihydroxyvitamin D3) exert potent immunomodulatory effects on dendritic cells and inhibit induction of antigen-specific T cells.

Paricalcitol (19-nor-1,25/OH(2)/D(2)), a second generation vitamin D receptor (VDR) activator, is a synthetic analogue of vitamin D3. In contrast to calcitriol, paricalcitol has a reduced effect on intestinal calcium resorption thus avoiding undesirable hypercalcemia. Information about immunomodulatory activity of paricalcitol is scarce. In this study we show that, in all investigated aspects, paricalcitol retains significant immunomodulatory activity, comparable to calcitriol. Both VDR agonists impaired differentiation of immature dendritic cells (DCs) from monocytes. The presence of VDR agonists during DC differentiation abolished their capacity to be activated and, despite potent Toll-like receptor mediated stimulation, VDR agonist-treated DCs remained in the immature state. In accordance with these findings, VDR-treated DCs produced no bioactive IL-12 and had a significantly decreased capacity to induce antigen-specific T cells while the capacity to induce functional Tregs remained unchanged when compared to control DCs. As DCs and T cells play an important role in the pathogenesis of atherosclerosis, in end-stage renal disease patients, paricalcitol should be a VDR agonist of choice for the reduction of the risk of atherosclerosis due to its immunomodulatory effect proven in this study and known limited hypercalcemic effect. The immunomodulatory potency of paricalcitol makes it a drug of interest in the therapy of chronic immune-mediated inflammatory diseases.

In vitro assessment of dendritic cells pulsed with apoptotic tumor cells as a vaccine for ovarian cancer patients.

Surgery and chemotherapy are standard treatments in ovarian cancer, but patients have a high rate of relapse. Dendritic cell (DC)-based vaccines are a new treatment option for elimination of residual tumor disease. We aim to explore the feasibility and immunogenicity of DC vaccines pulsed with autologous irradiated tumor cells from ovarian cancer patients. Monocyte-derived DC were generated and pulsed with autologous tumor-derived bodies, matured and subsequently cocultured with autologous lymphocytes. The ability of DC to activate lymphocytes was evaluated by proliferation and IFN-gamma ELISPOT. Induction of tumor cell apoptosis was optimal at 24 h, and DC pulsing optimal at 4 h. Maturation of DC and proliferation of lymphocytes were achieved in 75% of patients tested. Lymphocyte IFN-gamma production increased in response to tumor antigen-pulsed DC. We show the feasibility of preparing individual DC-based vaccines in ovarian cancer patients and the potential for induction of lymphocyte responses.

Methods of dendritic cell preparation for acute lymphoblastic leukemia immunotherapy in children.

Cell immunotherapy through dendritic cells (DC) presents a hopeful strategy for the treatment of various tumors. The aim of our study was to find which progenitor cells are most suitable for the preparation of dendritic cells in acute lymphoblastic leukemia (ALL) in pediatric patients, whether blasts from bone marrow or dendritic cells generated from peripheral blood mononuclear cells taken at the time of remission after induction chemotherapy. DC generated from the BM blasts of patients with B-ALL and T-ALL (n = 15) at the time of diagnosis expressed low levels of costimulatory molecules and CD markers typical for mature DC. In contrast, DC cultivated from peripheral mononuclear cells of patients (n = 9) had comparable morphology and expression of costimulatory molecules to DC obtained from healthy individuals, which was even higher after tumor lysate pulsing. Autologous lymphocyte proliferation increased after DC blasts lysate pulsation and further after lymphocyte restimulation, showing evidence of induction of specific cytotoxic lymphocytes. When comparing both cell sources for the preparation of DC in patients with ALL, it appears that peripheral mononuclear cells obtained after chemotherapy are more suitable than bone marrow leukemic blasts due to similar morphology, phenotypic, and functional capacity to monocytes of healthy donors. Despite this, it is necessary to take into account individual variability when preparing DC-based vaccines. The final verification of the efficiency of immunotherapy against residual hematopoietic malignant cells in patients with ALL can only be obtained through a clinical study.