Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells

Background

The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia.

Design and Methods

Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels.

Results

We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4⁺CD25⁺ Foxp3⁺ T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4⁺CD25⁺ T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms’ tumor protein.

Conclusions

These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.     

Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia

Background

Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood.

Design and Methods

The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor.

Results

Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro.

Conclusions

Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.     

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases

Background

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies.

Design and Methods

Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions.

Results

Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-α) and also induced allogeneic naive CD4⁺ T cells to proliferate and to produce type 1 cytokines such as interferon-γ and tumor necrosis factor-α. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation.

Conclusions

Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.     

Therapeutic effect of tolerogenic dendritic cells in established collagen‐induced arthritis is associated with a reduction in Th17 responses

Objective

Tolerogenic dendritic cells (DCs) are antigen‐presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen–induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis.

Methods

Tolerogenic DCs were generated by treating bone marrow–derived DCs with dexamethasone and vitamin D₃ during lipopolysaccharide‐induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence‐labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry.

Results

Tolerogenic DCs displayed a semi‐mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen–pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin‐10–producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin‐17–producing γ/δ T cells.

Conclusion

Treatment with type II collagen–pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

     

Successful generation of primary virus-specific and anti-tumor T-cell responses from the naive donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regulatory T cells

Background

One of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire.

Design and Methods

Antigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers.

Results

Using this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO⁺ responder cells resulted in improved priming and expansion of antigen-specific precursor T cells.

Conclusions

In conclusion, we present a robust method for the in-vitro induction and isolation of antigen-specific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.     

Polarization dictates iron handling by inflammatory and alternatively activated macrophages

Background

Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes.

Design and Methods

Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells.

Results

M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize – albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective.

Conclusions

Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).     

Activated macrophages induce hepcidin expression in HuH7 hepatoma cells

Background

Hepcidin is an iron regulatory peptide produced by the liver in response to inflammation and elevated systemic iron. Recent studies suggest that circulating monocytes and resident liver macrophages – Küpffer cells – may influence both basal and inflammatory expression of hepcidin.

Design and Methods

We used an in vitro co-culture model to investigate hepatocyte hepcidin regulation in the presence of activated THP1 macrophages. HuH7 hepatoma cells were co-cultured with differentiated THP1 macrophages for 24 h prior to the measurement of HuH7 hepcidin (HAMP) mRNA expression using quantitative polymerase chain reaction, and HAMP promoter activity using a luciferase reporter assay. Luciferase assays were performed using the wild type HAMP promoter, and constructs containing mutations in BMP/SMAD4, STAT3, C/EBP and E-BOX response elements. Neutralizing antibodies against interleukin-6, interleukin-1β , and the bone morphogenetic protein inhibitor noggin were used to identify the macrophage-derived cytokines involved in the regulation of HAMP expression.

Results

Co-culturing HuH7 cells with differentiated THP1 cells induced HAMP promoter activity and endogenous HAMP mRNA expression maximally after 24 h. This induction was fully neutralized in the presence of an interleukin-1β antibody, and fully attenuated by mutations of the proximal C/EBP or BMP/SMAD4 response elements.

Conclusions

Our data suggest that the interleukin-1β and bone morphogenetic protein signaling pathways are central to the regulation of HAMP expression by macrophages in this co-culture model.     

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Background

A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care.

Design and Methods

Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease.

Results

Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO⁺ CD27⁻) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25^high Foxp3⁺) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease.

Conclusions

Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.     

The IL-10 polarized cytokine pattern in innate and adaptive immunity cells contribute to the development of FVIII inhibitors

Background

Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors.

Methods

Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized.

Results

Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4⁺ and CD8⁺ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10.

Conclusions

The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.     

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

Background

Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions.

Design and Methods

We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells.

Results

The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16^ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E₂-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G.

Conclusions

Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.     

Helicobacter pylori directs tolerogenic programming of dendritic cells

Our laboratory has shown that Helicobacter pylori infection in mice triggers an increase in the number of subepithelial lamina propria CD11c⁺ dendritic cells with luminal projections. The physical characteristic of these cells is consistent with their ability to traverse epithelial tight junctions as reported by Maria Recigno.³ Gastric CD103⁺ dendritic cells, which are known to induce mucosal regulatory T cells, were also increased in number, raising the question whether H. pylori infection induces a regulatory T cell-skewed response by way of a bacteria-dendritic cell interaction. In fact, bone marrow-derived dendritic cells underwent tolerogenic programming, skewing the balance between effector and regulatory T cell responses towards regulatory T cell differentiation in a transforming growth factor-β- and interleukin-10-dependent manner. Depletion of regulatory T cell numbers augmented H. pylori-specific effector helper T cell responses, which correlated with a lower degree of H. pylori colonization. These results suggest H. pylori is capable of inducing a regulatory T cell-skewed response that limits the host''s ability to eradicate the bacteria, allowing the H. pylori infection to persist. To better understand the mechanism of H. pylori tolerogenic programming we compared the differential expressions of 34 genes critical for dendritic cell function in bone marrow-derived dendritic cells pulsed with live H. pylori or other Gram-negative bacteria (e.g., Escherichia coli, Acinetobacter lwoffii). Our data imply that H. pylori targets the Toll-like receptor 2 pathway to induce a regulatory T cell-skewed response. In addition, we show that H. pylori-pulsed dendritic cells are capable of inducing the conversion of naïve T cells to regulatory T cells. These observations are evidence of a unique tolerogenic program in dendritic cells that involves active editing of the immune response by H. pylori, favoring its persistence in the gastric mucosa.Key words: regulatory T cells, Th17, immune escape, tolerance, two-photon microscopy     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

Safety Constraints in an Artificial Pancreatic β Cell: An Implementation of Model Predictive Control with Insulin on Board

Background

Type 1 diabetes mellitus (T1DM) is characterized by the destruction of pancreatic β cells, resulting in the inability to produce sufficient insulin to maintain normoglycemia. As a result, people with T1DM depend on exogenous insulin that is given either by multiple daily injections or by an insulin pump to control their blood glucose. A challenging task is to design the next step in T1DM therapy: a fully automated insulin delivery system consisting of an artificial pancreatic β cell that shall provide both safe and effective therapy. The core of such a system is a control algorithm that calculates the insulin dose based on automated glucose measurements.

Methods

A model predictive control (MPC) algorithm was designed to control glycemia by controlling exogenous insulin delivery. The MPC algorithm contained a dynamic safety constraint, insulin on board (IOB), which incorporated the clinical values of correction factor and insulin-to-carbohydrate ratio along with estimated insulin action decay curves as part of the optimal control solution.

Results

The results emphasized the ability of the IOB constraint to significantly improve the glucose/insulin control trajectories in the presence of aggressive control actions. The simulation results indicated that 50% of the simulations conducted without the IOB constraint resulted in hypoglycemic events, compared to 10% of the simulations that included the IOB constraint.

Conclusions

Achieving both efficacy and safety in an artificial pancreatic β cell calls for an IOB safety constraint that is able to override aggressive control moves (large insulin doses), thereby minimizing the risk of hypoglycemia.     

Cardioprotective effects of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a novel free radical scavenger, on acute autoimmune myocarditis in rats

BACKGROUND:

Recent evidence suggests that oxidative stress may play a role in myocarditis.

PURPOSE:

To test the hypothesis that 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone or MCI-186), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats.

METHODS:

MCI-186 was administered intraperitoneally (1 mg/kg/day, 3 mg/kg/day or 10 mg/kg/day) in rats with EAM for three weeks. The results were compared with those from untreated rats with EAM.

RESULTS:

MCI-186 treatment did not affect the hemodynamics of the rats, but did reduce the severity of myocarditis when the heart weight:body weight ratio and the pathological scores were compared. There were significantly fewer myocardial interleukin-1beta-positive cells in rats with EAM treated with MCI-186 (at 3 mg/kg/day and 10 mg/kg/day) than in the untreated rats with EAM.

CONCLUSION:

MCI-186 ameliorated acute EAM in rats, suggesting that free radicals may be involved in the development of myocarditis.     

Identifying type 1 and type 2 diabetic cases using administrative data: a tree-structured model

Background:

To date, few administrative diabetes mellitus (DM) registries have distinguished type 1 diabetes mellitus (T1DM) from type 2 diabetes mellitus (T2DM).

Objective:

Using a classification tree model, a prediction rule was developed to distinguish T1DM from T2DM in a large administrative database.

Methods:

The Medical Archival Retrieval System at the University of Pittsburgh Medical Center included administrative and clinical data from January 1, 2000, through September 30, 2009, for 209,647 DM patients aged ≥18 years. Probable cases (8,173 T1DM and 125,111 T2DM) were identified by applying clinical criteria to administrative data. Nonparametric classification tree models were fit using TIBCO Spotfire S+ 8.1 (TIBCO Software), with model size based on 10-fold cross validation. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of T1DM were estimated.

Results:

The main predictors that distinguished T1DM from T2DM are age <40 years; International Classification of Disease, 9th revision, codes of T1DM or T2DM diagnosis; inpatient oral hypoglycemic agent use; inpatient insulin use; and episode(s) of diabetic ketoacidosis diagnosis. Compared with a complex clinical algorithm, the tree-structured model to predict T1DM had 92.8% sensitivity, 99.3% specificity, 89.5% PPV, and 99.5% NPV.

Conclusion:

The preliminary predictive rule appears to be promising. Being able to distinguish between DM subtypes in administrative databases will allow large-scale subtype-specific analyses of medical care costs, morbidity, and mortality.     

γδ T-cell killing of primary follicular lymphoma cells is dramatically potentiated by GA101, a type II glycoengineered anti-CD20 monoclonal antibody

Background

Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies.

Design and Methods

We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays.

Results

Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ.

Conclusions

In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.     

A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation

Background

There is evidence suggesting that sirolimus, in combination with tacrolimus, is active in the prevention of graft-versus-host disease. Sirolimus-based immune suppression may suppress alloreactive T cells, while sparing the survival and function of regulatory T cells.

Design and Methods

We conducted a randomized trial to compare the impact of sirolimus/tacrolimus against that of methotrexate/tacrolimus on the prevention of graft-versus-host disease and regulatory T-cell reconstitution.

Results

Seventy-four patients were randomized 1:1 to sirolimus/tacrolimus or methotrexate/tacrolimus, stratified for type of donor (sibling or unrelated) and the patients'' age. The rate of grade II-IV acute graft-versus-host disease at 100 days was 43% (95% CI: 27-59%) in the sirolimus/tacrolimus group and 89% (95% CI: 72-96%) in the methotrexate/tacrolimus group (P<0.001). The rate of moderate/severe chronic graft-versus-host disease was 24% (95% CI: 7-47%) in the sirolimus/tacrolimus group and 64% (95% CI: 41-79%) in the methotrexate/tacrolimus group (P=0.008). Overall survival and patient-reported quality of life did not differ between the two groups. On days 30 and 90 post-transplant, sirolimus-treated patients had a significantly greater proportion of regulatory T cells among the CD4⁺ cells in the peripheral blood, and isolated regulatory T cells were functional.

Conclusions

These data demonstrate that sirolimus/tacrolimus prevents grade II-IV acute graft-versus-host disease and moderate-severe chronic graft-versus-host disease more effectively than does methotrexate/tacrolimus, and supports regulatory T-cell reconstitution following allogeneic hematopoietic cell transplantation. Trial registration: ({"type":"clinical-trial","attrs":{"text":"NCT00803010","term_id":"NCT00803010"}}NCT00803010)Key words: tacrolimus, sirolimus, methotrexate, combination therapy, GVHD prophylaxis