Untreated Peripheral Blood Mononuclear Cells Research Articles

Human antigen presenting cells stimulated with Salmonella delivered influenza antigens induce cytokine production and proliferation of human CD4+ T cells in vitro

This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4+ T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3+CD4+ T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3+CD4+ T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1β, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-β) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity.

Effect of ginger extract on expression of GATA3, T-bet and ROR-γt in peripheral blood mononuclear cells of patients with Allergic Asthma

Introduction and objectivesAllergic asthma is a chronic inflammatory disorder of the airways. Th1, Th2 and Th17 cells are the main cells involved in the pathophysiology of asthma. The function of these cells is affected by T-bet, GATA3 and RORγt transcription factors (respectively). Therefore, the aim of this study was to evaluate the effect of ginger (officinal Roscoe) extract on the expression of T-bet, GATA-3 and ROR-γ in peripheral blood mononuclear cells (PBMC) of asthmatic patients, in comparison with healthy volunteers as controls. Materials and methodsIn this case-control study, a total of 50 individuals including 25 patients with severe, moderate and mild allergic asthma and 25 unrelated healthy controls were involved. The PBMCs were isolated and divided into four groups: negative control, two positive controls (Budesonide and PHA) and ginger-extract treated group. After cell treatment and incubation for 48h, PBMCs were isolated and cDNA was synthesized. Gene expressions of T-bet, GATA3 and ROR-γt were evaluated by Real-time PCR. ResultsAccording to the results of this study, hydroalcoholic extract of ginger could reduce the expression of GATA-3, ROR-γt, and T-bet in PBMCs of asthmatic patients in comparison with untreated PBMCs (P values=0.001, 0.001, and 0.002, respectively). It was also shown that the ginger extract could affect T-bet/GATA-3, T-bet/ROR-γt, and ROR-γt/GATA-3 expression ratios. ConclusionsThis study showed that the use of ginger extract could control asthma and decrease the severity of this disease by affecting the main cells involving the symptoms of asthma in the airways.

Effect of cryopreservation on the formation of DNA double strand breaks in human peripheral blood mononuclear cells

Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histon e protein H2AX (γH2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis. As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γH2AX was performed on freshly isolated and cryopreserved PBMCs. A significant reduction on relative γH2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors.

H2S mediates increased interleukin (IL)-1β and IL-18 production in leukocytes from patients with periodontitis

ABSTRACTBackground: The mechanisms involved in the interplay between the bacteria and the host cells in periodontitis are not fully understood.Aim: To investigate the effect of the bacterial metabolite H2S on the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 from periodontitis patients and healthy controls, and to evaluate the composition of the subgingival microbiota with its capacity to produce H2S.Methods: Subgingival bacterial samples from patients with periodontitis (N=32) and healthy controls (N=32) were investigated for H2S production and bacterial composition. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence/absence of 1mM H2S for 24h and cytokine concentrations were measured.Results: Subgingival plaque from periodontitis patients had more H2S producing bacteria and produced more H2S, than healthy controls. PBMCs exposed to H2S secreted significantly more IL-1ß and IL-18 (p<0.0001) than untreated control PBMCs from both groups. PBMCs from the periodontitis patients secreted higher levels of the cytokines, both spontaneously (IL-1ß p=0.0001; IL-18 p=0.09) and after exposure to H2S (IL-1ß p=0.03; IL-18 p=0.04), which is a new finding not previously reported.Conclusions: H2S, from the subgingival microbiota, can contribute to a host inflammatory response through secretion of the pro-inflammatory cytokines IL-1β and IL-18. Since this response differs between individuals, it may also reflect the susceptibility of the host to develop periodontitis.

Chromatin Histone Modifying and DNA Repair Inhibition Enhances the Anti-Myeloma Activity of Melphalan

Repair of DNA and, thus, preservation of the genetic code are critical for normal cellular function. However, tumor cells use DNA repair pathways to develop resistance to chemotherapy. Therefore, inhibiting DNA repair may override this drug resistance. Herein, we further investigate the mechanistic basis of the link between DNA repair efficiency and response to anti-myeloma therapy and provide evidence that histone modifiers might prove efficacious when used in combination with DNA-damaging agents.We studied clonal bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed multiple myeloma (MM) patients (12F/14M; median age 60 years, range 42-66) who responded (n=17) or did not respond (n=9) to subsequent melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. Cells were ex vivotreated with melphalan alone or in the presence of histone deacetylase inhibitor HDACi) vorinostat or the Non-Homologous End-Joining (NHEJ) inhibitor SCR7 and the extent of the DNA damage formation/repair (monoadducts by using Southern blot analysis; interstrand crosslinks by using quantitative-PCR; double-strand breaks repair using immunofluorescence quantification of γH2AX foci by confocal microscopy), the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) and the local chromatin condensation were evaluated. Finally, the expression of a focused panel of 84 genes involved in DNA damage response pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also examined.Both BMPCs and PBMCs from responders to melphalan therapy showed more condensed chromatin structure and slower rates of DNA repair activity (P<0.0022) compared to non-responders. Moreover, apoptosis rates of both BMPCs and PBMCs were inversely correlated with individual DNA repair efficiency, being higher in responders’ cells compared to those of non-responders (P=0.0011). PBMCs from MM patients showed higher looseness of chromatin structure, increased DNA repair activity and higher apoptosis rates compared to healthy controls. Microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients’ responders to subsequent therapy compared to non-responders. Particularly, responders’ PBMCs showed upregulation of ATR, BLM, DDB1, EXO1, FANCA, MRE11A, MSH2, PCNA, RAD50, RAD51, RPA1, XPA, XPC, XRCC1 and TP53 genes and downregulation of ATM, CDC25C, CDKN1A, CHEK2, ERCC1, MPG, PNKP and UNG genes compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that perturbation in the molecular components of DNA damage response pathways plays an important role in the therapeutic action of the genotoxic drugs. Interestingly, co-treatment of cells with melphalan and the NHEJ inhibitor SCR7 significantly reduced the rates of DNA damage repair and increased melphalan sensitivity of both BMPCs and PBMCs. Moreover, co-treatment of BMPCs with melphalan and the HDACi vorinostat resulted in hyperacetylation of histone H4, increased DNA damage burden and higher apoptosis rates of these cells. On the other hand, co-treatment of PBMCs with melphalan in the presence of vorinostat did not significantly alter the melphalan-induced DNA damage burden and the apoptosis rates of these cells. Interestingly, using Western blot analysis, we found that vorinostat decreased the levels of critical DNA repair proteins (Rad50, Mre11, Ku70, Ku86, DNA-PKcs) in malignant BMPCs, while it did not suppressed these DNA proteins in PBMCs, a finding that can explain, in part, the selectivity of vorinostat in causing cell death in malignant cells at concentrations that cause little or no normal cell death.Collectively, responders to melphalan therapy are characterized by slower rates of DNA repair mechanisms, resulting in higher accumulation of the cytotoxic DNA lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. The enhancement of melphalan cytotoxicity by DNA repair modifiers offers a promising strategy toward treatment of MM and improvement of existing regimens. DisclosuresTerpos:Novartis: Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria.

Effects of Resveratrol on Crosstalk between Canonical Β-Catenin/Wnt and FOXO Pathways in Coronary Artery Disease Patients with Metabolic Syndrome: A Case Control Study.

Coronary artery disease (CAD) is the major cause of mortality and morbidity worldwide. The aim of this study was to explore the effect of resveratrol (RES) on Canonical β-catenin/Wnt and forkhead box O (FOXO) pathways in CAD patients. We performed this study on 10 metabolic syndrome patients with three-vessel CAD and 10 sex-aged matched healthy subjects. The effects of RES on β-Catenin, manganese superoxide dismutase (MnSOD), and peroxisome proliferator-activated receptor delta (PPAR-δ) expression were evaluated in peripheral blood mononuclear cells (PBMCs) of participants. RES could increase the MnSOD expression in CAD patients (38%, p < 0.0001). After RES treatment, the MnSOD expression of patients is still non-significantly lower than controls. In both blank and RES treatments, a significant positive correlation between β-catenin and MnSOD mRNA expressions was found in controls, whereas no correlation between these gene expressions was found in untreated PBMCs of CAD patients. However, RES could modestly improve this pathway in CAD. RES could increase the MnSOD activity in healthy and CAD subjects (p = 0.051 and p = 0.009, respectively). Furthermore, in both blank and RES treatments, the significant correlation was found between total β-catenin protein and the MnSOD activity in PBMCs of the controls but not in patients. The cross-talk between β-catenin/Wnt and FOXO pathways was impaired in PBMCs of CAD patients. RES treatment could lead to a modest increase in the MnSOD activity independent of β-catenin/FOXO pathway. Despite a modest improvement in the β-catenin/FOXO pathway after RES treatment, this pathway was not completely repaired in CAD patients.

Changes in DNA Methylation and Chromatin Structure of Pro-inflammatory Cytokines Stimulated by LPS in Broiler Peripheral Blood Mononuclear Cells

The pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α mediate inflammation, which is a protective response by body to ensure removal of detrimental stimuli, as well as a healing process for repairing damaged tissue. The overproduction of pro-inflammatory cytokines can induce autoimmune diseases and can be fatal. The aim of this study was to investigate epigenetic mechanisms in the regulation of pro-inflammatory cytokines expression after lipopolysaccharide (LPS) stimulation of broiler peripheral blood mononuclear cells (PBMC). Gene expression, promoter DNA methylation, and chromatin accessibility of pro-inflammatory cytokines in untreated and LPS-treated PBMC were compared. The expression of epigenetic enzymes DNA methyltransferase (DNMT) 1, histone deacetylase (HDAC), and histone acetylase (HAT) were measured after LPS stimulation. The results showed the activated gene expression of pro-inflammatory cytokines in broiler PBMC stimulated 3 h by LPS. The demethylation of IL-6 gene – 302 and −264 cytosine-guanine (CpG) sites, as well as TNF-α gene -371 CpG site, occurred after LPS treatment (P < 0.05), whereas the methylaiton pattern in the IL-1β gene promoter region was not affected. Otherwise, LPS stimulation relaxed the chromatin structure at IL-1β and IL-6 promoter (P < 0.05). The lower expression of DNMT1 and HDAC2, and higher expression of p300-CBP-associated factor and tat-interaction protein-60, were detected in response to LPS (P < 0.05). Our data indicated that after LPS stimulation for 3 h, IL-1β and IL-6 promoter are remodeled into an accessible structure, and the IL-6 and TNF-α promoter are demethylated at special sites, which possible impact the mRNA expression of pro-inflammatory cytokines.

The State of Chromatin Condensation, the Expression of Genes Involved in DNA Damage Response and the DNA Repair Capacity Affect the Drug Sensitivity of PBMCs of Myeloma Patients Treated with Melphalan

Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeuticdrugs in the treatment of multiple myeloma (MM). However, the molecular mechanisms contributing to differential response of MM patients to melphalan are poorly understood. Herein, we investigated the underlying mechanisms in processing and repair of melphalan-induced DNA lesions and their potent contribution to the outcome of anti-myeloma therapy.We studied peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed multiple myeloma (MM) patients (8M/7F; median age 61 years) responders (≥PR, n=9) and non-responders (< PR, n=6) to subsequent melphalan therapy. PBMCs from 25 healthy controls were also included in this study (HC; 14M/11F, median age 58 years). PBMCs were ex vivo treated with melphalan and the extent of the DNA damage formation/repair (monoadducts by using Southern blot; ICLs by using a quantitative PCR-based assay; double strand breaks (DSBs) using immunofluorescence quantification of γH2AX foci), the induction of the apoptotic pathway by using a photometric enzyme-immunoassay and the local chromatin condensation by using micrococcal nuclease digestion were examined. Finally, the expression of a focused panel of 84 genes involved in DNA damage response (DDR) pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also evaluated.Following ex vivo treatment of PBMCs with melphalan, in all individuals examined biphasic DNA repair kinetics were observed, including a fast first-phase of repair and a much slower second phase. Interestingly, the accumulation of monoadducts was inversely correlated with the first-phase repair capacity of the PBMCs, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). The second phase repair capacity showed no differences in all individuals analyzed. Also, although ICLs "unhooking" rates were similar in all individuals, accumulation of ICLs was significantly higher in HC compared to responders' PBMCs (P<0.01), due to higher levels of monoadducts that are precursors of ICLs left unrepaired in these cells, resulting in higher formation of ICLs. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICL, which are precursors of DSBs and lower rates of DSB repair in these cells (P<0.01). Again, minimal amounts of DSBs were observed in non-responders.Interestingly, apoptosis rate of PBMCs was inversely correlated with the repair efficiency of both the first-phase monoadducts and the DSBs, with the apoptosis being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). In untreated PBMCs, we found an inverse correlation between the local chromatin condensation and the repair capacity. Therefore, we observed a progressive, significant increase in the looseness of the local chromatin structure, from HC to MM patients, with responders showing more condensed chromatin structure compared to non-responders (all P<0.05). Interestingly, by using a-amanitin, an inducer of chromatin condensation, PBMCs from non-responders showed the DNA repair capacity and the melphalan sensitivity similar to PBMCs from responders, suggesting that the state of the chromatin structure contributes to the response to melphalan therapy.Finally, microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients compared to HC. Particularly, responders' PBMCs showed upregulation of 4 genes (ATR, CHEK2, XPA, XRCC1) and downregulation of 5 genes (ATM, MPG, UNG, CDKN1A, CDC25C) compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that changes in the molecular components of the DDR pathways correlate with the outcome of melphalan therapy.We conclude that the state of chromatin condensation, the expression of genes involved in DDR pathways and the repair capacity of monoadducts and DSBs affect the drug sensitivity of PBMCs and maybe used for the prediction of response of myeloma patients to melphalan therapy. DisclosuresTerpos:Celgene: Honoraria, Other: travel expenses; Novartis: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses. Munshi:celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Janssen-Cilag: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Amgen: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Reduced IRF7 response to rhinovirus unrelated with DNA methylation in peripheral mononuclear cells of adult asthmatics.

BackgroundHuman rhinoviruses are the major cause of asthma exacerbation in both children and adults. Recently, impaired antiviral interferon (IFN) response in asthmatics has been indicated as a primary reason of the susceptibility to respiratory virus, but the mechanism of defective IFN production is little understood to date. The expression of IFN regulatory factor 7 (IRF7), a transcriptional factor for virus-induced type I IFN production is known to be regulated epigenetically by DNA methylation.ObjectiveWe aimed to investigate the expression of IFN-α, IFN-β, and IRF7 in response to rhinovirus infection in the adult asthmatics and evaluate DNA methylation status of IRF7 gene promotor.MethodsTwenty symptomatic adult asthmatics and 10 healthy subjects were enrolled and peripheral blood was collected from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated, cultured, and ex vivo stimulated with rhinovirus-16. The mRNA expressions of IFN-α, IFN-β, and IRF7 were analyzed using real time quantitative polymerase chain reaction. Genomic DNA was isolated from untreated PBMCs and the methylation status of IRF7 gene promotor was investigated using bisulfite pyrosequencing.ResultsThe mean age of asthmatics was 45.4 ± 15.7 years and 40% was male, which were not different with those of control group. Asthmatics showed significantly decreased mRNA expressions (relative expression to beta-actin) of IFN-α and IFN-β compared with normal control. The mRNA expression of IRF7 in the asthmatics was also significantly lower than those in the normal control. No significant difference of DNA methylation was observed between asthmatics and controls in all analyzed positions of IRF7 promotor CpG loci.ConclusionThe mRNA expression of type I IFN in response to rhinovirus was impaired in the PBMCs of adult asthmatics. The mRNA expression of IRF7, transcriptional factor inducing type I IFN was also reduced, but not caused by altered DNA methylation in the IRF7 gene promotor.

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti-retroviral therapy, it became clear that the majority of emergent HIV-1 is macrophage-tropic and infects CD4⁺, CCR5-expressing cells (R5-tropic). There are three distinct cell populations, R5-tropic, HIV-1-susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue-associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5-tropic HIV-1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α-galactosylceramide (α-GalCer) and R5-tropic HIV-1 [NL(AD8)], the production of HIV-1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5-tropic HIV-1 replication in CD4⁺ NKT cells. Here, we show that co-culturing R5-tropic HIV-1-infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV-1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5-tropic HIV-1 replication and persistence in CD4⁺ NKT cells.

TheIn VitroGenotoxic Effect of Tucuma (Astrocaryum aculeatum), an Amazonian Fruit Rich in Carotenoids

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 μg/mL, some genotoxic effects related to intermediary concentrations (100-500 μg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

The Anti-Kappa Monoclonal Antibody MDX-1097 Synergizes with Immunomodulatory Drugs to Enhance Antibody-Dependent Cell Cytotoxicity of Multiple Myeloma Cells

AbstractAbstract 4012Multiple Myeloma (MM) is a cancer caused by the proliferation of malignant clonal plasma cells in the bone marrow and accounts for 10% of all hematologic malignancies. Recent advances have been made in the treatment and management of MM, however, despite these advances the majority of patients will ultimately relapse and die from their disease within 3–5 years from diagnosis. Several novel therapeutic approaches, including the use of antibody-based therapies, are being investigated to further improve the treatment of MM.MDX-1097 is a chimeric monoclonal antibody being assessed as a single agent in a Phase 2 clinical trial for the treatment of kappa light-chain restricted (κ-type) MM. MDX-1097 binds to the kappa myeloma antigen (KMA), a tumor-specific membrane-associated protein expressed on malignant plasma cells from patients with K-type MM. Previously we have demonstrated that MDX-1097 exerts its anti-tumour effects through multiple mechanisms, including antibody-dependent cell cytotoxicity (ADCC) in the presence of either normal human peripheral blood mononuclear cells (PBMCs) or purified natural killer (NK cells).The immunomodulatory drugs (IMiDs) lenalidomide (Revlimid) and pomalidomide (Actimid) are currently in use or being assessed for the treatment of MM. These IMiDs have been shown to exert their anti-tumor effects both directly, via apoptotic mechanisms, and indirectly via a number of different mechanisms including the augmentation of NK-dependent cellular cytotoxicity.In this study we report that IMiDs and MDX-1097 co-operate to promote enhanced ADCC of MM cells. In vitro treatment of normal PBMCs with IMiDs led to a 1.4-fold higher level of ADCC-mediated cell death of MDX-1097 spiked JJN3 cells (a κ-type MM cell line) compared with vehicle-treated PBMCs from the same donor. Similarly, in vivo lenalidomide exposed PBMCs isolated from a MM patient were, on average, 1.8-fold more effective in killing MDX-1097 spiked JJN3 cells in vitro compared to PBMC obtained from the same patient prior to lenalidomide treatment. Treatment of JJN3 cells with IMiDs resulted in significantly increased cell surface expression of KMA (lenalidomide: 1.9-fold, p < 0.001; pomalidomide: 2.3-fold, p < 0.01). These IMiD-treated JJN3 cells, when spiked with MDX-1097 were 1.7-fold more susceptible to ADCC-mediated cell death in the presence of untreated PBMCs, compared to JJN3 cells treated with vehicle alone. This difference in sensitivity to ADCC mediated cell death is presumably due to increased KMA expression resulting in more binding sites for MDX-1097, therefore facilitating recruitment of PB immune effector cells. Furthermore, combining IMiD-treated PBMCs with IMiD-treated, MDX-1097 spiked JJN3 cells resulted in a further increment in ADCC-mediated JJN3 cell death.This study demonstrates that in vivo and in vitro treatment of PBMCs with IMiDs engages the PB immune effector cells, leading to increased ADCC-induced κ-type MM cell death in vitro in the presence of MDX-1097. IMiDs also increase cell surface expression of KMA, leading to increased MDX-1097 binding and in turn also enhancing ADCC-induced MM cell killing. Our data provides a rationale for the clinical evaluation of a combination therapy involving both IMiDs and MDX-1097 for the treatment of k-type MM. Disclosures:Cuddihy:Immune System Therapeutics Ltd: Research Funding. Asvadi:Immune System Therapeutics Ltd: Employment. Dunn:Immune System Therapeutics Ltd: Employment, Equity Ownership. Spencer:Immune System Therapeutics Ltd: Research Funding.

Diagnostic and Prognostic Utility of Measuring Tumor Necrosis Factor in the Peripheral Circulation of Patients With Immune-Mediated Sensorineural Hearing Loss

To characterize levels of tumor necrosis factor (TNF; formerly known as tumor necrosis factor α), a well-established proinflammatory cytokine, in patients with immune-mediated sensorineural hearing loss (IM-SNHL) and to determine the role of this cytokine in identifying steroid-responsive hearing loss. Prospective case-control study. Tertiary care academic medical center. A total of 11 control subjects and 85 patients with clinical and audiometric characteristics of IM-SNHL (autoimmune inner ear disease and sudden SNHL combined) treated with corticosteroids were enrolled in the study. Patients were categorized as steroid responders (n = 47) and steroid nonresponders (n = 38). Peripheral venous blood was used to determine the total amount of plasma TNF by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) were isolated and treated with in vitro dexamethasone. Treated and untreated PBMCs were then analyzed for release of soluble TNF protein into conditioned supernatants as well as expression of TNF messenger RNA (mRNA). Mean plasma levels of TNF, unstimulated and dexamethasone-stimulated PBMC-secreted levels of TNF, and TNF mRNA levels in unstimulated and dexamethasone-stimulated PBMCs. Steroid nonresponders had the highest mean baseline plasma levels of TNF compared with steroid responders and control subjects (27.6, 24.1, and 14.4 pg/mL, respectively) (P = .03). For patients with IM-SNHL with a high baseline plasma levels of TNF (>14.4 pg/mL), the mean TNF secreted by PBMCs was 59.1 pg/mL, which decreased to 7.2 pg/mL with in vitro dexamethasone stimulation in the responder group, while the mean TNF secreted by PBMCs was 11.2 pg/mL, which slightly increased to 11.7 pg/mL with in vitro dexamethasone stimulation in the nonresponder group (P = .04). The level of TNF can be used as both a diagnostic and prognostic cytokine for IM-SNHL. For patients presenting with a sudden change in hearing threshold, a high baseline plasma TNF from the peripheral circulation is supportive of the diagnosis if it is greater than 18.8 pg/mL, with a positive predictive value higher than 97%. In addition, this study demonstrates that for patients with IM-SNHL and high plasma levels of TNF, their clinical response to oral glucocorticoids can be predicted by their in vitro PBMC response to dexamethasone. This algorithm may further guide optimal medical treatment and possibly avoid the deleterious adverse effects of administering glucocorticoids to those patients who would not benefit from their effect.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of anti-RBC autoantibodies with or without complement activation. However, the underlying mechanism for the development of AIHA remains largely unclear. In this study, we carefully evaluated the potential role of Th17 cells in the development of AIHA. We found an elevated frequency of Th17 cells in patients with AIHA, which were closely correlated with their disease activity, including the level of anti-RBC IgG antibodies, hemoglobin, serum C3, and lactate dehydrogenase activity. Furthermore, we observed that interleukin (IL)-17 was also closely correlated with the disease activity in AIHA patients. To further elucidate the potential role of Th17 cells in induction of AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA. Notably, we found that Th17 cells affected development of AIHA by enhancing the adaptive humoral responses. Specifically, we found that adoptive transfer of Th17 cells heightened the initial anti-rat RBC antibody responses and concomitantly increased the onset of AIHA. In addition, invivo neutralization of IL-17 abrogated the development of AIHA, while initiation of anti-rat RBC IgG responses and induction of AIHA in IL-17(-/-) mice were impaired. Our findings suggest that Th17 cells contribute to the development of AIHA, which could facilitate our better understanding of AIHA pathogenesis and provide clues to developing novel forms of immunotherapy against AIHA.

The Immunoregulatory Effects of Caffeic Acid Phenethyl Ester on the Cytokine Secretion of Peripheral Blood Mononuclear Cells From Asthmatic Children

Asthma is a chronic inflammatory disease of the airways for which current treatments are mainly based on pharmacological interventions, such as glucocorticoid therapy. Our objective was to study the immunoregulatory effects of caffeic acid phenethyl ester (CAPE, a phytochemical synthesized from propolis) on cytokine secretion of peripheral blood mononuclear cells (PBMCs) from asthmatic children. PBMCs from asthmatic children (5.5 ± 3.3 years old, n=28) and healthy children (5.6 ± 2.8 years old, n=23) were co-cultured with CAPE in vitro with and without phorbol-12-myristate-13-acetate-ionomycin. Our results show that predominant interleukin 4 (IL-4) and interferon-gamma secretion of cultured supernatant were detected in healthy donors compared with asthmatics. In the presence of phorbol-12-myristate-13-acetate-ionomycin, with or without CAPE treatment, the asthmatic children showed significantly decreased levels of IL-10 secretion compared with the healthy controls. However, CAPE significantly decreased IL-10 and interferon-gamma in healthy donors. There was a slight but not statistically significant reduction of IL-4 secretion in CAPE-treated PBMCs compared with untreated control PBMCs from the healthy children. Our data also shows that CAPE significantly enhanced transforming growth factor-beta 1 production from PBMCs from asthmatic children. The immunoregulatory effects of CAPE on human PBMCs may be through the induction of regulatory T cells, as evidenced by the enhanced transforming growth factor-beta 1 production from PBMCs from asthmatic children in our study.

NFkB activation promotes immune activation in HTLV-I-associated myelopathy / tropical spastic paraparesis

Evidence suggests that HTLV-I-induced immune activation plays a key role in the pathogenesis of HAM/TSP. The HTLV-I-encoded transactivating protein Tax is known to activate nuclear factor kappa B (NFkB), a key host signaling pathway regulating immune response, but the contribution of the NFkB pathway to the immune activation associated with HAM/TSP has yet to be fully defined. We examined NFkB activation in peripheral-blood mononuclear cells (PBMC) from subjects with HAM/TSP, and tested the effect of NFkB inhibition on key ex vivo correlates of immune activation in HAM/TSP. We examined the role of NFkB activation during immune activation associated with HAM/TSP by using small molecule NFkB inhibitors, including a newly developed selective inhibitor of NFkB, PBS-1086. NFkB activation was assessed in peripheral-blood mononuclear cells (PBMC) from subjects with HAM/TSP and in healthy donors (HD). Nuclear translocation of the NFkB RelA was significantly higher in PBMC from subjects with HAM/TSP compared to HD (p=0.032) following short-term (20 h) culture, indicating increased activation of the NFkB pathway in HAM/TSP. Treatment with the small molecule inhibitor PBS-1086 reduced NFkB activation (p<0.01). PBS-1086 reduced expression of CD25 and CD69 in HAM/TSP PBMC as well as phosphorylation of STAT5 in a dose-dependent manner (p<0.01 for all). PBS-1086 also inhibited spontaneous lymphoproliferation of HAM/TSP PBMC in a dose-dependent manner (p=0.0286). PBS-1086 treatment resulted in a mean proviral load reduction of 20% compared to untreated PBMC in a 72 h culture These results indicate that NFkB activation plays a critical upstream role in the immune activation associated with HAM/TSP, and identify the NFkB pathway as a potential therapeutic target for immune modulation in HAM/TSP.

Reduced production of noggin by immune cells of patients with relapsing–remitting multiple sclerosis

Multiple sclerosis (MS) plaques are characterized by neurodegeneration, astrogliolis, the presence of immature oligodendrocytes and infiltrating immune cells. Recent studies revealed a putative role for noggin in both neurogenesis and oligodenrocytes development. In order to study the potential of peripheral immune cells to contribute to neurogenesis in MS, we studied the mRNA expression, protein secretion and regulation profile of noggin in peripheral blood mononuclear cells (PBMCs) of untreated patients with relapsing–remitting MS (RR-MS), interferon-β (IFN-β) treated RR-MS patients compared to matched healthy controls (HC).Basal levels of noggin mRNA expression, determined by quantitative real-time PCR were lower in untreated patients than in HC. No differences were found between untreated patients and IFN-β treated patients. Similarly, the secreted levels of noggin, detected in 24h PBMCs supernatants by ELISA, were decreased in untreated RR-MS patients than in HC. Again no significant differences were found between untreated patients and IFN-β treated patients. Stimulation with anti-CD3/CD28 mAbs increased noggin mRNA expression in untreated patients but not in HC. However, noggin mRNA levels in untreated patients PBMCs stimulated with anti-CD3/CD28 did not reach noggin levels in unstimulated PBMCs of HC. Purification of monocytes (CD14+) and T cells (CD3+ cells) by magnet-activated cell separation has demonstrated that noggin mRNA is predominantly expressed in CD3+ cells in both HC and in RR-MS patients. This pattern also appeared in protein level of noggin, tested by Western blot. The incubation of the PBMCs with TNF-α increased the expression of noggin only in HC group. In conclusion, T cells possess the potential to participate in the induction of neurogeneration by the production of noggin. This potential seems to be defective in immune cells of RR-MS patients as there is reduced mRNA expression and protein secretion levels of noggin, insufficient stimulatory effect of CD3/CD28 stimulation and unresponsiveness to TNF-α in these patients PBMCs.

Antitumor Activity of An RNA-Based Agonist of TLR7 and 8 In Preclinical Models of Hematological Malignancies

AbstractAbstract 421Treatment of hematological malignancies, non-Hodgkins lymphoma in particular, has evolved greatly in the past decade with the advent of rituximab, proteasome inhibitors and other new agents. Nonetheless, the need remains for effective agents with novel mechanisms of action, which lack cross-resistance to current standards of care. Agents that regulate host immune function may contribute to the control of hematological tumors by affecting innate and acquired immune responses targeting cancer cells. In this context, Toll-like receptors (TLR) are ideal targets for combination therapy as they play a key role in modulating immune responses and can influence the tumor microenvironment through induction of cytokines/chemokines and enhance antibody-dependent cell cytotoxicity (ADCC), a key mechanism involved in rituximab antitumor effects. We have shown that a novel class of synthetic oligoribonucleotide compounds, acting as TLR7 and 8 agonists, induce potent T helper 1-type immune responses in preclinical models (Proc Natl. Acad. Sci. USA, 104: 13750, 2007) and exert significant antitumor effects (Mol Cancer Ther. 9: 1788, 2010). In this study, we have evaluated a combination strategy involving a TLR7/8 dual agonist with rituximab, bortezomib or cyclophosphamide, in human xenograft and murine syngeneic lymphoma models. We performed ADCC assays using human peripheral blood mononuclear cells (PBMCs) stimulated with the agonist as effectors and rituximab-coated Raji, a B cell lymphoma, or Granta-519, a mantle cell lymphoma tumor cell line, as targets. Combining agonist-treated PBMCs with uncoated Raji cells or untreated PBMCs with rituximab-coated Raji cells resulted in about 35% cytotoxicity. When agonist-treated PBMCs were combined with rituximab-coated Raji cells, cytotoxicity was enhanced to 65%. Agonist treatment of PBMCs alone did not induce killing of uncoated Granta-519 cells whereas treatment with rituximab caused 25% cytotoxicity. Combining agonist treatment with rituximab coating of Granta-519 cells increased cytotoxicity to 51%. Thus, the agonist significantly enhances the ADCC activity of rituximab. NOD-SCID mice implanted i.v. with Raji cells were treated with 50 mg/kg agonist (s.c., twice weekly for 5 weeks), 10 mg/kg rituximab (i.p., twice weekly for 5 weeks), the combination of the two agents or PBS starting at day 3. The control group of mice that received PBS developed a disseminated systemic disease with a median survival 18.5 days and died within 24 days. Treatment with agonist or rituximab alone lead to moderate tumor growth inhibition with a median survival of 24 days (p = 0.043) or 36 days (p < 0.01), respectively. However, all mice in both groups developed lymphoma and died within 74 days. Treatment of mice with combination of agonist and rituximab resulted in long term tumor-free survival of 63% of the treated mice (p = 0.0005 compared to antibody alone group) with a median survival of 82 days. Similar antitumor effects were observed in HBL-2 mantle cell lymphoma xenografts in Rag2 mice. Flow cytometry analysis of human CD20+, CD19+ and CD20−CD19+ cells in the peripheral blood of mice bearing HBL-2 tumors showed that treatment with the agonist or rituximab lead to a 19% and 70% reduction of lymphoma cells, respectively. In contrast, treatment with agonist and rituximab increased clearance of total human cells to 97%. Interestingly, although rituximab was very effective in clearing CD20+ cells, it did not impact the number of CD20−CD19+ cells which accumulated over time and account for 8% of the total lymphoma cell population in the blood stream. Treatment with agonist and rituximab, significantly reduced the number of circulating CD20−CD19+ cells. The agonist also showed synergistic antitumor activity when combined with either cyclophosphamide or bortezomib in murine B-cell lymphoma xenografts from Em-myc transgenic mice and A20 lymphoma in BALB/c mice, respectively. Combination treatment in both models resulted in significant tumor growth inhibition compared to cyclophosphamide (p = 0.035) or bortezomib (p = 0.0001) alone. Our data suggest that this novel dual agonist of TLR7 and TLR8 could enhance the antitumor activity of rituximab as well as other standard agents in the treatment of non-Hodgkins lymphoma and other hematological malignancies. Based on these data we have selected the dual agonist IMO-4200 as lead candidate for clinical development. Disclosures:No relevant conflicts of interest to declare.

In Vivo Antagonistic Effects of Dexamethasone On Lenalidomide-Induced NK Cell Activation.

Abstract 1639Poster Board I-665Dexamethasone (dex) and lenalidomide (len) is a potent treatment for multiple myeloma (MM). In vitro, len directly inhibits MM tumor cell proliferation via cell cyle arrest, and can also costimulate T cells and augment natural killer (NK) cell activity, leading to enhanced anti-tumour immunity. Conversely, dex also directly inhibits MM cell proliferation but is profoundly immuno-suppressive and may therefore subvert the full capacity of len to act via immune mechanisms against MM. We previously reported that MM patients responding to len-dex combination show an increase in Treg numbers, and little evidence in recovery of their B and T cell numbers (Quach et al. Blood 2008; 112: abstract 1696). We have since undertaken a prospective and systematic analysis of NK cell number and function in MM patients treated with len-dex, and evaluated the mechanisms by which dex downregulates len-induced NK activation in in vitro assays using patients' and normal donors' blood samples. 25 relapsed MM patients (aged 58-77 years) were treated with low dose len (15mg Days 1-21 of each 28-day cycle) and dex (20mg/day, Days 1-4,9-12,17-20). After a median of 9 (2-19) cycles, 19 patients responded (24% CR/VGPR, 52% PR). At baseline, NK cell numbers and function [assessed by % lysis of 51Cr labelled K562 target cells at 50 (effector):1 (target) ratio] in MM patients were similar to age matched controls (0.2 vs. 0.3× 105/ml in controls, p=0.09 and 49% K562 cell lysis vs. 58% in controls, p=0.44 respectively) (fig.1A). Whilst NK cell numbers slightly increased in vivo after len-dex treatment [2.0 (baseline) vs. 3.9×105/l (cycle 6), p=0.04, paired t test] (fig.1A), mean NK cell function progressively decreased compared to baseline after 6 and 9 len-dex cycles [mean 49% K562 cell lysis at baseline vs. 28% after 6 cycles (p=0.007) and 31% after 9 cycles (p=0.02)] (fig.1B). Following 72 hours of in vitro treatment with len (10mM), there was increased NK function in healthy donor peripheral blood mononuclear cells (PBMC) [mean 54% K562 cell lysis from len-treated PBMC vs. 38% lysis in untreated PBMC, p=0.04] (fig. 2). In PBMCs from MM patients at baseline, ex vivo treatment with len (10mM) did not significantly increase NK cell function [mean 47% K562 cell lysis (untreated) vs. 52% (len treated), p=0.17], nor did it increase NK cell function after 6 len-dex treatment cycles [mean 32% K562 cell lysis (untreated) vs. 30% (treated), p=0.4].Conversely, dex (0.1mM) decreased NK cell function in healthy donors' PBMC [mean 7.6% K562 cell lysis (dex treated) vs. 38% (untreated) p=0.01], even in the presence of len [mean 7% K562 cell lysis (len+dex) vs. 38% (untreated), p=0.002] (fig. 2). Dex-induced in vitro NK inhibition was dose dependent and could be rescued by the addition of IL-2 to normal donors [mean 7.6 % K562 cell lysis (dex) vs. 28% lysis (Dex +IL2),p=0.03] as well as PBMC from MM patients at baseline [mean lysis 16% (dex) vs. 59% (Dex+IL2) p=0.0002]. However, IL-2 was less able to rescue dex-induced NK dysfunction in PBMC from patients post 6 treatment cycles compared to patients at baseline [mean 59% K562 cell lysis (baseline) vs. 28% (C6), p=0.03]. Dex induced NK dysfunction was reversible as NK cell function recovered after a 3 days dex washout. In summary, NK function in MM patients, whilst similar to healthy controls at baseline, progressively decreases after prolonged len-dex treatment despite a clinical response. The observed decrease in NK function in vivo and in vitro is directly due to the effects of dex, which could not be reversed by the NK activating effects of len. Our results suggest that the efficacy of len and dex co-therapy is not due to augmentation of NK cytolytic activity, due to the immunosuppressive effects of dex against NK cells. This suggests that alternative dosing schedules of dex, after initial induction with len and dex co-therapy, may optimise len-induced immunostimulation of NK cells and subsequent sustained disease control via anti-MM immunity. [Display omitted] [Display omitted] DisclosuresLynch:Celgene Corporation: Employment. Prince:Celgene Corporation: Research Funding.

Alloanergization of HLA-Mismatched Human Donor T Cells Results in the Generation of Allosuppressive CD4+ T Regulatory Cells Both in Vitro and in Vivo

The alloreactivity of T cells contained in either donor bone marrow (BM) or peripheral blood mononuclear cells (PBMC) can be selectively reduced by alloanergy induction via ex vivo coculture with HLA-mismatched stimulator PBMC in the presence of costimulatory blockade (CSB). Alloanergization of human PBMC via allostimulation and CSB with humanized anti-B7.1 and anti-B7.2 monoclonal antibodies (MoAbs) can reduce alloresponses by 2 logs in vitro without abrogation of virus- or tumor-associated antigenspecific T cell responses. We are currently using this strategy to generate non-alloreactive donor PBMC for use in a Phase I clinical study of delayed infusion of alloanergized donor PBMC to improve immune reconstitution after haploidentical hematopoietic stem cell transplantation (HSCT). In addition to direct induction of anergy (hyporesponsiveness) in alloantigen-specific donor T cells, studies of alloanergization by CSB in both murine and human donor T cells suggest that an additional mechanism of active suppression may contribute to reduced alloresponsiveness. However, the mediators of suppression of human alloresponses after alloanergization have not been fully defined, nor has expansion of allosuppressive cells in vivo after HSCT containing alloanergized donor T cells been demonstrated. We report here that alloanergization of human donor PBMC by HLA-mismatched allostimulation with CSB with anti-B7.1 and -B7.2 MoAbs results in acquisition of potent dose-dependent and specific allosuppressive function. Alloanergized PBMC suppressed alloresponses of allonaïve and alloprimed PBMCs, requiring cell: cell contact for efficacy, whereas proliferative responses to 3rd-party allostimulators and to CMV were not significantly suppressed. Alloanergization of PBMC resulted in an increased frequency of CD4+ cells with a FOXP3+ CD25hi CD127lo T regulatory cell (alloTreg) phenotype, expressing CD45RO, CD62L and CCR4, intracellular CTLA4 but not IL10. Depletion and labeling studies demonstrated that alloTreg originated from the natural T regulatory cell (NTR) pool. Alloanergization increased alloTreg frequency, which was further increased upon repeat allostimulation to up to 20% of CD4+ donor T cells. AlloTreg were the primary mediators of the observed allosuppression. Depletion of alloTreg largely abrogated the allosuppressive capacity of alloanergized PBMC. High purity 2-step immunomagnetic-based cell selection was used to directly demonstrate alloTreg allosuppressive function. Although purified CD4+CD25hi CD127lo NTR from resting donor PBMCs were able to suppress alloresponses, purified alloTreg from allorestimulated alloanergized donor PBMCs demonstrated increased allosuppressive function and specificity on a per cell basis. We therefore investigated the frequency and function of CD4+ alloTregs in vivo in 3 patients (pts) who had received large doses of haplo donor T cells contained in BM alloanergized via allostimulation and CSB using humanized anti-B7.1 and -B7.2 MoAbs on a prior clinical study. A marked in vivo expansion of CD4+ alloTreg occurred in all pts early post-HSCT (D+30-100) reaching frequencies of up to 30% of CD4+ cells, and was associated with rapid reconstitution of absolute CD4+ T cells in 2 of the 3 pts assessed. AlloTreg were donor-derived with a phenotype identical to that found in vitro. Finally, alloTreg from 2 pts were purified by flow cytometric sorting, and specifically suppressed recipient-directed alloresponses of untreated donor PBMC. Furthermore, depletion of alloTreg increased recipient-specific alloresponses in donorderived CD4+ cells indicating that alloreactive cells had not been deleted at this stage. Despite receiving a median 7 × 107 CD3+ haploidentical donor T cells/kg, 0/3 pts developed severe acute GvHD indicating that alloreactivity was controlled in vivo. This is the first data to demonstrate that ex vivo alloanergization of HLA-mismatched donor T cells prior to haploidentical HSCT results in a marked expansion of functional donor-derived alloTreg in vivo. This may represent an active mechanism by which alloresponses can be abrogated after alloanergized HLA-mismatched T cell infusion and suggests alloanergization could be further exploited to generate non-alloreactive donor T cells containing high frequencies of alloTreg for adoptive immunotherapy to improve HLA-mismatched HSCT.