Allogeneic T-cell Proliferation Research Articles

JTA–009, a fully human antibody against human AILIM/ICOS, ameliorates graft–vs–host reaction in SCID mice grafted with human PBMCs

Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator [ICOS]) is the third molecule identified in the CD28 family participating in T-cell activation. AILIM/ICOS has been implicated in both effector and pathogenic T-cell functions, as evidenced by the beneficial effects of AILIM/ICOS blockade in several murine disease models. In the present study, the role of human AILIM/ICOS in T-cell function was investigated using a fully human monoclonal antibody specific to human AILIM/ICOS (JTA-009). The effect of JTA-009 on allogenic T-cell proliferation was examined using human mixed lymphocyte reaction (MLR). To investigate the efficacy of AILIM/ICOS blockade in vivo, a graft-vs-host disease (GVHD) model, in which severe combined immunodeficient (SCID) mice were grafted with human peripheral blood mononuclear cells (PBMCs), was used. In MLR, suppressive effect of JTA-009 on allogenic T-cell proliferation was detected with comparable potency to CD28 blockade by cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig at an intermediate culture phase. JTA-009 acts as a blocking antibody in vivo and inhibited binding of human AILIM/ICOS to mouse AILIM/ICOS ligand (B7h). Treatment with JTA-009 significantly prolonged survival of mice, with reductions of human interferon-gamma levels in blood and number of human cells in spleens. These results demonstrate that human AILIM/ICOS plays a role in the GVHD pathogenesis mediated by human T cells, and its blockade is attractive for abrogating undesired T-cell responses as is well-documented in mice.

Dexamethasone transforms lipopolysaccharide‐stimulated human blood myeloid dendritic cells into myeloid dendritic cells that prime interleukin‐10 production in T cells

Myeloid dendritic cells (MDC) play an important role in antigen-specific immunity and tolerance. In transplantation setting donor-derived MDC are a promising tool to realize donor-specific tolerance. Current protocols enable generation of tolerogenic donor MDC from human monocytes during 1-week cultures. However, for clinical application in transplantation medicine, a rapidly available source of tolerogenic MDC is desired. In this study we investigated whether primary human blood MDC could be transformed into tolerogenic MDC using dexamethasone (dex) and lipopolysaccharide (LPS). Human blood MDC were cultured with dex and subsequently matured with LPS in the presence or absence of dex. Activation of MDC with LPS after pretreatment with dex did not prevent maturation into immunostimulatory MDC. In contrast, simultaneous treatment with dex and LPS yielded tolerogenic MDC, that had a reduced expression of CD86 and CD83, that poorly stimulated allogeneic T-cell proliferation and production of T helper 1 (Th1) cytokines, and primed production of the immunoregulatory cytokine interleukin-10 (IL-10) in T cells. In vitro, however, these tolerogenic MDC did not induce permanent donor-specific hyporesponsiveness in T cells. Importantly, tolerogenic MDC obtained by LPS stimulation in the presence of dex did not convert into immunostimulatory MDC after subsequent activation with different maturation stimuli. In conclusion, these findings demonstrate that combined treatment with dex and LPS transforms primary human blood MDC into tolerogenic MDC that are impaired to stimulate Th1 cytokines, but strongly prime the production of the immunoregulatory cytokine IL-10 in T cells, and are resistant to maturation stimuli. This strategy enables rapid generation of tolerogenic donor-derived MDC for immunotherapy in clinical transplantation.

Gene delivery of indoleamine 2,3‐dioxygenase prolongs cardiac allograft survival by shaping the types of T‐cell responses

We investigated the hypothesis that overexpression of indoleamine 2,3-dioxygenase (IDO) by a cardiac allograft may result in a survival advantage of the allograft by creating a tolerogenic microenvironment. An adenoviral vector encoding for murine IDO cDNA (AdIDO) was transfected into murine allogeneic cardiac allografts, and transplantation was performed for evaluation of the effects of local AdIDO transfection on allograft survival. Intragraft IDO expression and lymphocytes infiltration were measured by immunohistochemical and histological analysis. Quantitative polymerase chain reaction assays, mixed lymphocyte reaction and flow cytometric analysis were employed to determine the expression of mRNA for Foxp3, IDO, pro-inflammatory cytokines, allogeneic T-cell proliferation and the proportion of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) from graft-infiltrating lymphocytes and splenocytes of recipients, respectively. Cardiac allografts transfected with AdIDO showed a significant prolonged survival compared to the control groups. Hearts treated with AdIDO exhibited considerable up-regulation of IDO expression, whereas contained significantly reduced transcript levels for interleukin (IL)-2, interferon-gamma and IL-17. These T cells isolated from allografts pre-treated with AdIDO displayed a dramatic reduction of proliferation capacity to alloantigen stimuli and had a significant higher proportion of Tregs compared to the control, as demonstrated by an increase of Foxp3 expression in allografts pre-treated with AdIDO compared to control groups. Overexpression of IDO significantly delays cardiac allograft acute rejection by shaping the types of T-cell responses elicited by alloantigen stimuli.

Interferon-α and viral triggers promote functional maturation of human monocyte-derived dendritic cells

Type I interferons (IFNs) play an important role in the pathogenesis of many autoimmune disorders including psoriasis. In the presence of IFN-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF), monocytes differentiate into dendritic cells (DCs) referred to as IFN-DCs. IFN-DCs potentially mimic DC populations involved in psoriasis and express a wide range of Toll-like receptor (TLR) subtypes. Recently, it was shown that single-stranded RNA (ssRNA) triggers TLR7 and TLR8; therefore we studied ssRNA, as a surrogate for ssRNA viruses and their impact on IFN-DCs. We established culture conditions for IFN-DCs, generated from plastic adherent monocytes using GM-CSF plus IFN-alpha. For DC stimulation ssRNA40, a 20-mer ssRNA oligonucleotide was used. The phenotypic analysis of DC preparations was performed using flow cytometry. The production of various cytokines was analysed by enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction was used to quantify TLR and cytokine gene expression. The ability of IFN-DCs to stimulate allogeneic T-cell proliferation was evaluated in a mixed leucocyte reaction. We found that IFN-DCs express mRNA for TLR7 and TLR8 and that ssRNA stimulation significantly improves their costimulatory molecule expression, stabilizes their phenotype and enhances their capacity to stimulate naive T-cell proliferation. Unstimulated IFN-DCs did not produce bioactive interleukin (IL)-12 and produced low levels of other proinflammatory cytokines. In contrast, ssRNA stimulation led to a significant production of IL-12p70, IL-1beta, IL-6 and tumour necrosis factor alpha. IFN-DCs contained mRNA for IL-12p35, IL-12p40, IL-23p19, IL-27p28 and IL-27EBI, which was further increased by incubation with ssRNA. Our study sheds light on a potential role for IFN-alpha and viral infections in triggering DC populations in psoriasis. These results provide additional data for the better understanding of human autoimmune and antiviral responses and may also have implications for strategies developing cancer immunotherapy.

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Apoptotic cells induce dendritic cell‐mediated suppression via interferon‐γ‐induced IDO

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-gamma expression by DC in association with apoptotic environments. The specific generation of IFN-gamma by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-gamma and IDO blockade demonstrated a role for IFN-gamma and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-gamma-dependent. Blocking transforming growth factor-beta (TGF-beta) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-gamma-induced IDO and TGF-beta.

IVIG effects on immature dendritic cells of Common Variable Immunodeficiency Patients

Previous studies have suggested that lyophilized IGIV may inhibit dendritic cell (DC) maturation. We investigated the effects of a newer liquid formulation of IGIV on the peripheral blood monocyte (PBMC)-induced differentiation and function of DC in healthy subjects and CVID patients. Immature DCs were derived from PBMC by negative selection using magnetic beads from five adult CVID patients and six adult healthy subjects. PBMC were incubated with IL-4 and GM-CSF to induce DC maturation. IGIV (0.15 mM Gamunex), DMSO, or BSA was added to cultures. Flow cytometry was performed to analyze the differentiation of PBMC into immature DC using the following monoclonal antibodies: CD14, CD1a, CD40, CD83, CD80, CD86, HLA-DR. DC function was evaluated by lucifer yellow and dextran sulfate for antigen uptake, and allogeneic T-cell responses in mixed lymphocyte culture (MLC) assay for antigen processing. There were no significant differences in the expression of immature DC markers between controls vs. CVID patients. IGIV upregulated CD86 expression (p < 0.001) and CD83 (p < 0.001) in both groups. IGIV suppressed lucifer yellow uptake of imDC but enhanced allogeneic T-cell proliferation in MLC. We were unable to identify any defects in the DC differentiation pathway of PBMC in our CVID patients. Our results suggest that liquid 10% IGIV promotes monocyte-derived DC maturation in vitro to a more mature functional phenotype, in contrast to lyophilized IGIV, as shown by the upregulation of CD83 and CD86, and the enhanced antigen processing ability in MLC.

Peptide-Dependent Inhibition of Alloreactive T-Cell Response by Soluble Divalent HLA-A2/IgG Molecule In Vitro

Induction of peripheral tolerance in an antigen-specific manner is a critical goal of transplant biology. The specificity and avidity of multimerized peptide/major histocompatibilty complexes suggest their potential ability to modulate antigen-specific T-cell sensitization and effector functions. A soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr(368-376)) to form a divalent Tyr/HLA-A2 molecule (Tyr/HLA-A2 dimer), which allowed for specific targeting to the epitope-specific cytotoxic T lymphocytes in bulk alloreactive T cells. Alloreactive T-cell response was induced by coculture of Tyr(368-376) -pulsed T2 cells (T2/Tyr) with peripheral blood lymphocytes of HLA-A2-negative (HLA-A2-ve) sample; five samples of HLA-A2-ve individuals were included in this study. After the coculture in the presence of Tyr/HLA-A2 dimer, the suppression of the dimer on alloresponse was characterized by analyzing allogeneic T-cell proliferation, specific cytolytic activity against the T2/Tyr, and specific Tyr/HLA-A2 tetramer staining. The Tyr/HLA-A2 dimer suppresses alloreactive T-cell response by inhibiting its proliferation and cytotoxicity against specific target T2/Tyr in vitro, and it is interesting that the suppression is peptide-specific. The Tyr/HLA-A2 tetramer staining suggests the reduced function of CD8+ T cell is caused by inhibiting the generation of the epitope-specific alloreactive T cells by the Tyr/HLA-A2 dimer in three samples. Moreover, the existence of epitope-specific but function-negative T cells in the other two samples suggests that another mechanism might exist that is involved in silencing alloreactive responses by the dimer. Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression and may be useful in promoting graft survival.

Modulation of Tryptophan Catabolism by Acute Myeloid Leukemia Cells Acts as a General Mechanism of Immune Tolerance Via the Induction of T Regulatory Cells.

Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed acute myeloid leukemia (AML) patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. Moreover, we demonstrated that IDO-expressing AML cells are capable to promote the differentiation of new CD4+CD25+Foxp3+ T regulatory cells (Treg cells). AML cells may be differentiated into leukemic dendritic cells (AML-DCs) which have increased immunogenicity and may be used as vaccine against leukemia. We in vitro generated DCs from 7 AML samples according to standard procedures and tested IDO expression and activity. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. Similarly to undifferentiated IDO+ AML blasts, IDO-expressing AML-DCs induced a population of CD4+CD25+Foxp3+ Treg cells, which were capable to suppress in vitro allogeneic naïve T-cell proliferation. These data identify IDO-mediated catabolism as a general tolerogenic mechanism in AML cells, including AML-DCs and raise several concerns for the use of AML-DCs as cellular vaccine against leukemia.

Leukemic Blasts Secondary to Myelodysplastic Syndromes Are Molecularly Programmed To Differentiate into Functionally Mature Dendritic Cells.

Adoptive immunotherapy of leukemia aims at eradicating the disease through the transfer of leukemia-reactive lymphocytes. Donor lymphocytes infusions (DLI) after allogeneic hemopoietic cell transplantation (allo-HCT) have shown to cure chronic myelogenous leukemia (CML). Unfortunately, when applied to acute leukemias, the DLI approach is far less successful. Strategies to increase the efficacy of adoptive immunotherapy for the cure of acute leukemias are therefore urgently needed. To this aim, blasts from acute myeloid leukemias (AML) can be differentiated in vitro into dendritic cells (leukemic DC or LDC) that directly present leukemic antigens to T lymphocytes. This is commonly achieved by culturing AML blasts with cytokines such as GM-CSF, IL-4 and TNF-α for 7–14 days. LDC can be further induced to functional maturation by exposure to CD40L. The efficacy of these protocols is, however, suboptimal, since a large proportion of AML sub-types do not differentiate into LDC. The addition of calcium ionophore (A23187) has been shown to rescue some of the cases. The aim of this study is to identify the characteristics associated with the ability of blast from AML to differentiate into functionally mature LDC. To this, we studied a cohort of 17 consecutive AML patients. AML blasts were induced to differentiate into LDC either by GM-CSF, IL-4 and TNF- α or by cytokines and A23187. LDC were further matured with CD40L. LDC differentiation was defined by expression of MHC class II, costimulatory (CD80, CD86, CD40, CD70) and adhesion molecules (CD54, CD58). The overall efficacy of LDC differentiation by cytokines was 20%. Maturation of LDC induced by cytokines failed, as ascertained by the lack of up-regulation of CD83 and the chemokine receptor CCR7. Upon exposure of AML blasts to cytokines and A23187, we observed LDC differentiation in more than 90% of the cases. LDC differentiation occurred in 24–48 hrs and was associated with the maintenance of disease markers expression (c-kit, CD34). Interestingly, blasts derived from AML secondary to myelodysplastic syndromes directly differentiated into mature LDC. On the other hand, LDC derived from primary AML were insensitive to the maturative stimulus CD40L. Accordingly, LDC from secondary AML were the most potent inducers of allogeneic T-cell proliferation. The efficacy of the combination of calcium ionophore and cytokines in differentiating myelodysplastic blasts into LDC provides the rational for novel adoptive immunotherapeutic approaches to treat high risk AML. Furthermore, the differential ability of primary leukemic blasts and blasts secondary to myelodysplastic syndromes to rapidly differentiate into functionally mature LDC suggest the existence of molecular factors committing AML cells to the DC lineage. The identification of such factors and their exploitation, for example by gene transfer, will enable the generation of LDC from all cases of AML for the stimulation of the immune system against leukemia.

Uremia impairs monocyte and monocyte-derived dendritic cell function in hemodialysis patients

Patients with chronic renal failure maintained on intermittent hemodialysis have frequent infections and a suboptimal response to vaccinations. Dendritic cells are potent antigen-presenting cells essential for the initiation and maintenance of innate and adaptive immunity. In this study we used uremic sera from hemodialysis patients to measure its impact on monocyte and monocyte-derived dendritic cell function in vitro. Monocytes from healthy and uremic subjects were isolated using immunomagnetic beads and differentiated into dendritic cells in the presence of either complete sera or sera from hemodialysis patients. Dendritic cells from normal patients cultured in uremic sera had decreased endocytosis and impaired maturation. These cells, however, had enhanced IL-12p70 production and increased allogeneic T-cell proliferation compared to cells of normal subjects cultured in normal sera. Monocyte derived dendritic cells of hemodialysis patients cultured in either normal or uremic sera were functionally impaired for endocytosis and maturation but had enhanced IL-12p70 production and allogeneic T-cell proliferation only when cultured with uremic sera. High concentrations of urea in normal sera inhibited all aspects of normal dendritic cell function in vitro. Our study suggests that hemodialysis regimes tailored to remove uremic toxins more efficiently may improve immune functions of these patients.

Human Leukocyte Antigen-G5 Secretion by Human Mesenchymal Stem Cells Is Required to Suppress T Lymphocyte and Natural Killer Function and to Induce CD4+CD25highFOXP3+ Regulatory T Cells

Adult bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells that are the subject of intense investigation in regenerative medicine. In addition, MSCs possess immunomodulatory properties with therapeutic potential to prevent graft-versus-host disease (GvHD) in allogeneic hematopoietic cell transplantation. Indeed, MSCs can inhibit natural killer (NK) function, modulate dendritic cell maturation, and suppress allogeneic T-cell response. Here, we report that the nonclassic human leukocyte antigen (HLA) class I molecule HLA-G is responsible for the immunomodulatory properties of MSCs. Our data show that MSCs secrete the soluble isoform HLA-G5 and that such secretion is interleukin-10-dependent. Moreover, cell contact between MSCs and allostimulated T cells is required to obtain a full HLA-G5 secretion and, as consequence, a full immunomodulation from MSCs. Blocking experiments using neutralizing anti-HLA-G antibody demonstrate that HLA-G5 contributes first to the suppression of allogeneic T-cell proliferation and then to the expansion of CD4(+)CD25(high)FOXP3(+) regulatory T cells. Furthermore, we demonstrate that in addition to their action on the adaptive immune system, MSCs, through HLA-G5, affect innate immunity by inhibiting both NK cell-mediated cytolysis and interferon-gamma secretion. Our results provide evidence that HLA-G5 secreted by MSCs is critical to the suppressive functions of MSCs and should contribute to improving clinical therapeutic trials that use MSCs to prevent GvHD.

Immunosuppression by Placental Indoleamine 2,3-Dioxygenase: A Role for Mesenchymal Stem Cells

Background/objectives Mesenchymal stem cells (MSC) can be isolated from human placenta and have the potential to contribute to the immunosuppressive properties of placental tissue. The objectives of this study were to investigate the phenotype and differentiation characteristics of MSC derived from human placenta and evaluate the role of the tryptophan degrading enzyme, indoleamine 2,3 dioxygenase (IDO), in mediating their immunosuppressive affect. Methods MSC obtained from placental tissue (pMSC) were characterised using flow cytometry and tested for multipotency by determining differentiation into all mesenchymal lineages. The immunosuppressive properties of pMSC were tested in allogeneic mixed lymphocyte reactions and IDO expression and activity were measured by semi-quantitative real-time PCR and HPLC respectively. Results Multipotent stem cells were isolated from placenta and displayed chondrogenic, osteogenic and limited adipogenic differentiation. Cell surface antigen expression of pMSC was similar to bone marrow MSC (bMSC) with lack of the haematopoietic and common leukocyte markers (CD34, CD45), and expression of adhesion (CD29, CD166, CD44) and stem cell (CD 90, CD105, CD73) markers. Placental MSC were suppressive of allogeneic T-cell proliferation, an effect which was intensified following IDO induction by IFN-γ. Replenishment of tryptophan or treatment with the IDO-blocker, 1-methyl-tryptophan (1-MT), attenuated the immunosuppressive action of pMSC. Conclusions These results suggest that placental tissue contains MSC, which are phenotypically and functionally similar to bMSC, and that IDO is a key mediator of their immunosuppressive effect. Further investigation is needed to determine if pMSC function effects pregnancy outcome.

Inhibition of Dendritic Cell Maturation by Malaria Is Dose Dependent and Does Not RequirePlasmodium falciparumErythrocyte Membrane Protein 1

Red blood cells infected with Plasmodium falciparum (iRBCs) have been shown to modulate maturation of human monocyte-derived dendritic cells (DCs), interfering with their ability to activate T cells. Interaction between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and CD36 expressed by DCs is the proposed mechanism, but we show here that DC modulation does not require CD36 binding, PfEMP1, or contact between DCs and infected RBCs and depends on the iRBC dose. iRBCs expressing a PfEMP1 variant that binds chondroitin sulfate A (CSA) but not CD36 were phagocytosed, inhibited lipopolysaccharide (LPS)-induced phenotypic maturation and cytokine secretion, and abrogated the ability of DCs to stimulate allogeneic T-cell proliferation. CD36- and CSA-binding iRBCs showed comparable inhibition. P. falciparum lines rendered deficient in PfEMP1 expression by targeted gene knockout or knockdown also inhibited LPS-induced phenotypic maturation, and separation of DCs and iRBCs in transwells showed that inhibition was not contact dependent. Inhibition was observed at an iRBC:DC ratio of 100:1 but not at a ratio of 10:1. High doses of iRBCs were associated with apoptosis of DCs, which was not activation induced. Lower doses of iRBCs stimulated DC maturation sufficient to activate autologous T-cell proliferation. In conclusion, modulation of DC maturation by P. falciparum is dose dependent and does not require interaction between PfEMP1 and CD36. Inhibition and apoptosis of DCs by high-dose iRBCs may or may not be physiological. However, our observation that low-dose iRBCs initiate functional DC maturation warrants reevaluation and further investigation of DC interactions with blood-stage P. falciparum.

SHIP-deficient mice provide insights into the regulation of dendritic cell development and function

Dendritic cells (DC) play a critical role in establishment and maintenance of central and peripheral tolerance. Despite intensive research, our knowledge of the molecular mechanisms regulating DC development and function is limited, thus hindering our ability to generate appropriate DC populations for manipulating immune tolerance. We utilized mice deficient in the SH2-containing inositol-5-phosphatase (SHIP) to examine the role of cytokine signaling in DC development and function. We analyzed the phenotype of both primary and bone marrow (BM)-derived DC (BMDC) using flow cytometry. In addition, cytokine production was measured using cytometric bead arrays and the ability of DC to induce allogeneic T-cell proliferation was assessed using thymidine incorporation assays. We demonstrated that spleen DC isolated from SHIP-deficient mice are increased in number and have an altered phenotype. In vitro analyses revealed that SHIP-deficient BM cells give rise to a higher frequency of myeloid, but not plasmacytoid, DC due to both an increased progenitor frequency and enhanced cytokine sensitivity. The BMDC exhibit an altered phenotype that correlates with a reduced capacity to induce allogeneic T-cell proliferation. Addition of interleukin-6 to WT BM cultures during DC differentiation partially induces a KO phenotype. These studies suggest that myeloid and plasmacytoid DC progenitors are differentially sensitive to signaling pathways in which SHIP is involved. Moreover, they suggest that interleukin-6 may have an important role in regulating the phenotype and function of myeloid DC.

Blockade of RAGE Suppresses Alloimmune Reactions In Vitro and Delays Allograft Rejection in Murine Heart Transplantation

The receptor for advanced glycation endproducts (RAGE), a multiligand member of the immunoglobulin superfamily, interacts with proinflammatory AGEs, the products of nonenzymatic glycation and oxidation of proteins; high-mobility group box 1 (HMGB1), also known as amphoterin and S100/calgranulins to amplify inflammation and tissue injury. Previous studies showed that blockade of RAGE suppressed recruitment of proinflammatory mechanisms in murine models. We tested the hypothesis that RAGE contributes to alloimmune responses and report that in vivo, acute rejection of fully allogeneic cardiac allografts in a murine model of heterotopic cardiac transplantation is significantly delayed by pharmacological antagonism of RAGE. In parallel, allogeneic T-cell proliferation in the mixed lymphocyte reaction is, at least in part, RAGE-dependent. These data provide the first insights into key roles for RAGE in allorecognition responses and suggest that antagonism of this receptor may exert beneficial effects in allogeneic organ transplantation.

The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16 INK4A-cyclin D1/cdk4 and p21 waf1, p27 kip1-cyclin E/cdk2 pathways

Mesenchymal stem cells (MSCs) have been shown to down-regulate T-cell responses. However, the mechanisms underlying remain unknown. In this study, we report that BALB/c bone marrow-derived MSCs inhibit the proliferation of allogeneic T-cells in mixed lymphocyte reactions (MLR), This inhibition is dependent on cell–cell contact, and do not induce apoptosis. Furthermore, cell-cycle analyses reveal that T-cells, in the presence of MSCs, are arrested in the G0/G1 phase through. The blockage of phosphorylation of retinoblastoma protein (Rb), mediated by the p16 INK4A-cyclin D1/cdk4 complex and p21 waf1, p27 kip1-cyclin E/cdk2 complex pathway. Our results suggest that MSCs may perform a crucial function in the maintenance of immune homeostasis, via direct regulation of the clonal expansion of activated T-cells. The novel T-cell regulatory mechanism exhibited by MSCs may prove useful in a variety of therapeutic applications.

Acute Myeloid Leukemia-Derived Dendritic Cells Express the Immunoregulatory Enzyme Indoleamine 2,3-dioxygenase.

Acute myeloid leukemia (AML) cells may be differentiated into dendritic cells (DC) which have increased immunogenicity, but retain some immunosuppressive features of leukemic cells. Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed AML patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. We, then, in vitro generated DCs from 7 AML samples (AML-DCs) in the presence of GM-CSF, IL-4 and TNF-α. The cells we obtained were morphologically and phenotypically semi-mature DCs expressing CD40, CD80, CD86, HLA-DR and CD1a molecules and they were more efficient to induce T-cell proliferation and type 1 cytokine production than primary AML blasts. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. These data identify IDO-mediated catabolism as a tolerogenic mechanism in AML-DCs and have clinical implications for the use of AML-DCs as cellular vaccine against leukemia.

Superior Immunomodulatory Effects of Intravenous Immunoglobulins on Human T-cells and Dendritic Cells: Comparison to Calcineurin Inhibitors

Prophylactic administration of anti-HBs intravenous immunoglobulins (IVIg) in hepatitis B infected-liver transplant patients protects against acute rejection. To explore the suitability of intravenous immunoglobulins (IVIg) as prophylaxis of acute rejection and graft-versus-host disease (GVHD) after allograft transplantation, the effects of IVIg and calcineurin inhibitors (CNI) on human blood-derived T-cells and DC were compared. T-cells were stimulated with phytohemagglutinin (PHA) or allogeneic spleen antigen-presenting cells (APC) and T-cell proliferation and cytokine production were determined in presence or absence of IVIg or CNI. Immature blood dendritic cells (DC) were stimulated in presence or absence of IVIg or CNI, and allogeneic T-cell stimulatory capacity, cell death, and phenotypic maturation were established. IVIg and CNI equally inhibited T-cell proliferation and IFN-gamma production after PHA stimulation or allogeneic stimulation. CD8+ T-cells were preferentially affected by both IVIg and CNI after allogeneic stimulation. Like CNI, addition of IVIg at later time points after T-cell activation suppressed mitotic progression of responding T-cells. IVIg-treated DC were suppressed in their capacity to stimulate allogeneic T-cell proliferation by 73+/-12%, whereas DC-function was not affected by CNI. The decreased allogeneic T-cell stimulatory capacity of IVIg-treated DC correlated to induction of cell death in DC and decreased up-regulation of CD40 and CD80. In vitro IVIg functionally inhibit the two principal immune cell-types involved in rejection and GVHD, i.e. T-cells and DC, whereas CNI only suppress T-cells. By targeting both T-cells and DC, IVIg may be a promising candidate for immunosuppressive treatment after allograft transplantation.

CD40 stimulation induces differentiation of acute lymphoblastic leukemia cells into dendritic cells.

Despite the very high percentage of long-term remissions in acute lymphoblastic leukemia (ALL) in children, some of them suffer from recurrence of the disease. New treatment modalities, e.g. effective geno- and immunotherapy are needed. The use of neoplasmatic cells to present tumor antigens is one of the approaches in cancer vaccines. ALL cells lack the expression of costimulatory molecules and are poor antigen presenting cells (APCs) for T-cell activation. CD40/40L interaction stimulates B-cells to proliferate, differentiate, upregulate costimulatory molecules and increase antigen presentation. The aim of the study was to test the hypothesis that ALL cells can be turned into professional APCs by CD40L activation. Children with B-cell precursor ALL were enrolled into the study. Mononuclear cells from bone marrow or peripheral blood were stimulated with CD40L and interleukin 4. 1) after culture we noted upregulation of all assessed costimulatory, adhesion and activatory molecules i.e. CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II; 2) CD40L activated ALL cells induced proliferation of allogeneic T-cells (measured by [(3)H]thymidine incorporation). These results confirm the possibility of enhancing the immunogenicity of ALL cells with the CD40L system and indicate that this approach can be used in immunotherapeutic trials.