Synthesis of mizoribine prodrugs and their in vivo evaluation as immunosuppressive agents.

Mizoribine is a well-known immunosuppressive drug, based on a nucleoside scaffold, that targets inosine-monophosphate dehydrogenase (IMPDH). In an effort to increase its in vivo efficacy, three different types of prodrugs (a phosphoramidate prodrug, a lipophilic ester derivative and an amino acid conjugate) were prepared. Screening of these prodrugs in a rapid whole blood assay revealed that the two ester-based mizoribine prodrugs potently inhibited interleukin 2 secretion. Moreover, these prodrugs were able to prolong graft survival, when evaluated in a mouse model of cardiac allograft transplantation. Strikingly, a combination therapy of these mizoribine prodrugs with tacrolimus had a synergistic in vivo effect.

Intratumoral immunotherapy with anti-PD-1 and TLR9 agonist induces systemic antitumor immunity without accelerating rejection of cardiac allografts.

Immune checkpoint inhibitors, such as programmed cell death 1 (PD-1) blockades, have revolutionized the field of cancer immunotherapy. However, there is a growing concern whether PD-1 inhibitors can be administered safely to transplant recipients with advanced cancer, as the T cells activated by checkpoint inhibitors may become reactive not only toward tumor antigens but also toward donor alloantigen, thereby resulting in allograft rejection. Here, immunotherapy with anti-PD-1/toll like receptor 9 agonist was administered to C57BL/6 mice bearing a cardiac allograft that were receiving maintenance immunosuppression or a PI4KIIIß inhibitor-based tolerogenic regimen. Intratumoral (i.t.), but not systemic, immunotherapy promoted potent anti-tumor responses, but did not accelerate allograft rejection. This effect was associated with a pro-immunogenic effect induced by i.t. immunotherapy resulting in systemic cellular and humoral immune anti-tumor responses. Furthermore, when the tumor and cardiac allograft shared major histocompatibility complex (MHC) antigens, i.t. immunotherapy promoted immune responses directed against tumor and the cardiac allograft resulting in allograft rejection. The anti-tumor effect was compromised by maintenance immunosuppression with cyclosporin A, indicating that an optimal balance between enhanced anti-tumor immunity and decreased transplant immunoreactivity is critical. A clinically relevant approach could be to temporarily withdraw maintenance immunosuppression and/or replace it with a PI4KIIIß inhibitor-based tolerance-inducing regimen to allow for effective immunotherapy to take place.

Reduction of myeloid-derived suppressor cells reinforces the anti-solid tumor effect of recipient leukocyte infusion in murine neuroblastoma-bearing allogeneic bone marrow chimeras.

Allogeneic hematopoietic stem cell transplantation is an emerging treatment option for solid tumors because of its capacity to elicit immune graft-versus-tumor effects. However, these are often limited and associated with GvHD. Adoptive recipient leukocyte infusion (RLI) was shown to enhance anti-tumor responses of allogeneic bone marrow transplantation in murine neuroblastoma (Neuro2A)-bearing chimeras. In contrast to the clinically used donor leukocyte infusion, the RLI anti-tumor effect-elicited by host-versus-graft lymphohematopoietic reactivity-does not cause GvHD; however, the tumor growth-inhibitory effect is incomplete, because overall survival is not prolonged. Here, we studied the anti-solid tumor mechanisms of RLI with the objective to improve its efficacy. Host-versus-graft reactivity following RLI was associated with a systemic cytokine storm, lymph node DC activation, and systemic expansion of host-derived IFN-γ-expressing CD4+ T cells and IFN-γ-and granzyme B-expressing CD8+ T cells, which acquired killing activity against Neuro2A and third-party tumor cells. The tumor showed up-regulation of MHC class I and a transient accumulation of IFN-γ-and granzyme B-expressing CD8+ T cells: the intra-tumor decline in cytotoxic CD8+ T cells coincided with a systemic-and to a lesser extent intra-tumoral-expansion of MDSC. In vivo MDSC depletion with 5-FU significantly improved the local tumor growth-inhibitory effect of RLI as well as overall survival. In conclusion, the RLI-induced alloreactivity gives rise to a host-derived cytotoxic T-cell anti-neuroblastoma response, but also drives an expansion of host-type MDSC that counteracts the anti-tumor effect. This finding identifies MDSC as a novel target to increase the effectiveness of RLI, and possibly other cancer immunotherapies.

Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the ß-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8(+) T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV-infected IFN-γ-deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ-deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.

The graft-versus-neuroblastoma effect of allogeneic hematopoietic stem cell transplantation, a review of clinical and experimental evidence and a perspective on mechanisms.

Despite aggressive treatment, patients with high-risk neuroblastoma face high relapse rates and bleak prognoses. Increasing evidence that neuroblastoma cells are or can become immunogenic has stimulated research into novel therapies based on triggering or enhancing tumor immunity. Here we review clinical and experimental studies on this subject, the underlying immune mechanisms and perspectives for clinical application. Allogeneic hematopoietic stem cell transplantation has proven to be of substantial benefit in the treatment of certain leukemias through the generation of a graft-versus-leukemia-effect and has become of interest as a possible treatment for patients with solid tumors, including neuroblastoma.

γ-Hydroxyethyl piperidine iminosugar and N-alkylated derivatives: a study of their activity as glycosidase inhibitors and as immunosuppressive agents.

An efficient and practical strategy for the synthesis of (3R,4s,5S)-4-(2-hydroxyethyl) piperidine-3,4,5-triol and its N-alkyl derivatives 8a-f, starting from the D-glucose, is reported. The chiral pool methodology involves preparation of the C-3-allyl-α-D-ribofuranodialdose 10, which was converted to the C-5-amino derivative 11 by reductive amination. The presence of C-3-allyl group gives an easy access to the requisite hydroxyethyl substituted compound 13. Intramolecular reductive aminocyclization of C-5 amino group with C-1 aldehyde provided the γ-hydroxyethyl substituted piperidine iminosugar 8a that was N-alkylated to get N-alkyl derivatives 8b-f. Iminosugars 8a-f were screened against glycosidase enzymes. Amongst synthetic N-alkylated iminosugars, 8b and 8c were found to be α-galactosidase inhibitors while 8d and 8e were selective and moderate α-mannosidase inhibitors. In addition, immunomodulatory activity of compounds 8a-f was examined. These results were substantiated by molecular docking studies using AUTODOCK 4.2 programme.

Recipient leukocyte infusion enhances the local and systemic graft-versus-neuroblastoma effect of allogeneic bone marrow transplantation in mice.

Allogeneic hematopoietic stem cell transplantation and donor leukocyte infusion (DLI) may hold potential as a novel form of immunotherapy for high-risk neuroblastoma. DLI, however, carries the risk of graft-versus-host disease (GvHD). Recipient leukocyte infusion (RLI) induces graft-versus-leukemia responses without GvHD in mice and is currently being explored clinically. Here, we demonstrate that both DLI and RLI, when given to mixed C57BL/6→A/J radiation chimeras carrying subcutaneous Neuro2A neuroblastoma implants, can slow the local growth of such tumors. DLI provoked full donor chimerism and GvHD; RLI produced graft rejection but left mice healthy. Flow cytometric studies showed that the chimerism of intratumoral leukocytes paralleled the systemic chimerism. This was associated with increased CD8/CD4 ratios, CD8+ T-cell IFN-γ expression and NK-cell Granzyme B expression within the tumor, following both DLI and RLI. The clinically safe anti-tumor effect of RLI was further enhanced by adoptively transferred naïve recipient-type NK cells. In models of intravenous Neuro2A tumor challenge, allogeneic chimeras showed superior overall survival over syngeneic chimeras. Bioluminescence imaging in allogeneic chimeras challenged with luciferase-transduced Neuro2A cells showed both DLI and RLI to prolong metastasis-free survival. This is the first experimental evidence that RLI can safely produce a local and systemic anti-tumor effect against a solid tumor. Our data indicate that RLI may provide combined T-cell and NK-cell reactivity effectively targeting Neuro2A neuroblastoma.

Synthesis of a 2,4,6-trisubstituted 5-cyano-pyrimidine library and evaluation of its immunosuppressive activity in a Mixed Lymphocyte Reaction assay.

A series of novel pyrimidine analogues were synthesized and evaluated for immunosuppressive activity in the Mixed Lymphocyte Reaction assay, which is well-known as the in vitro model for in vivo rejection after organ transplantation. Systematic variation of the substituents at positions 2, 4 and 6 of the pyrimidine scaffold led to the discovery of 2-benzylthio-5-cyano-6-(4-methoxyphenyl)-4-morpholinopyrimidine with an IC(50) value of 1.6 µM in the MLR assay.

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment.

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.

The vitamin D analog, TX527, promotes a human CD4+CD25highCD127low regulatory T cell profile and induces a migratory signature specific for homing to sites of inflammation.

The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3(+) T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4(+)CD25(high)CD127(low) regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.

G-CSF stem cell mobilization in human donors induces polymorphonuclear and mononuclear myeloid-derived suppressor cells.

The role of myeloid-derived suppressor cells (MDSC) is emerging in transplantation. An expansion of myeloid progenitor cells with suppressive capacity has been reported to occur as a bystander phenomenon in the course of allogeneic hematopoietic stem cell transplantation (allo-HSCT) protocols, particularly, in mice during bone marrow chimerism induction and in human stem cell donors during G-CSF-mobilization protocols. Hypothesizing that such 'regulatory myeloid cells' play a role in regulating post-transplant T-cell alloreactivity, we performed a phenotypical and functional characterization of these cells in peripheral blood stem cell grafts of G-CSF-treated donors. We demonstrate that expanding myeloid cells in the peripheral blood of G-CSF-mobilized donors comprise the typical phenotype of the mononuclear and polymorphonuclear MDSC-subtypes that were recently described in cancer patients, and that both MDSC-subsets have the capacity to regulate alloreactive T-cell responses in-vitro. This study provides the basis for investigating the clinical relevance of MDSC and MDSC-subtypes in human allo-HSCT.

Establishment of operational tolerance to sustain antitumor immunotherapy.

BACKGROUND: Commonly used immunosuppressive drugs are efficacious in the prevention of transplanted solid organ rejection but, are complicated by an increased rate of malignancies. Treatment of the latter with cancer immunotherapy is frequently associated with increased graft loss from rejection. METHODS: B16 melanoma was inoculated subcutaneously (s.c.) in the ear of host B6 mice carrying a heterotopic allogenic murine heart. Intratumoral (i.t.) immunotherapy with anti-programmed death-1 (PD-1) and a toll-like receptor 9 (TLR9) agonist was conducted to treat cancer. Cyclosporine A (CsA) therapy or replaced by other immunosuppressive agents was conducted to preserve heart allograft tolerance, respectively. RESULTS: Here we show that long-term allograft-bearing mice receiving CsA therapy and challenged with host-type melanoma resist cancer immunotherapy and reject their grafts after CsA withdrawal. However, when CsA therapy is replaced by intermittent administration of the PI4KIIIß inhibitor UCB9608, effective antitumor immunity is induced while preserving graft tolerance. UCB9608 switch combined with i.t. immunotherapy resulted in donor-specific tolerance with preserved third-party responses. This operational tolerance may not be dependent on regulatory T cells (Tregs). CONCLUSIONS: Immune effects induced by UCB9608 switch following CsA therapy allow for antitumor immunity, opening up new approaches to establish donor-specific operational tolerance in solid organ transplantation with cancer.

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells.

BACKGROUND: Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants. DESIGN AND METHODS: We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8(+) T and NK cells, and intracellular cytokine staining. RESULTS: Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8(+) T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras. CONCLUSIONS: In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8(+) T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.

1,25-Dihydroxyvitamin D3 alters murine dendritic cell behaviour in vitro and in vivo.

BACKGROUND: Differentiation and maturation of dendritic cells yield a cell type with the ability to prime immune responses towards defence and destruction. 1,25(OH)2D3, the active form of vitamin D3, fosters the development of tolerogenic dendritic cells. This study aimed to evaluate the effects of 1,25(OH)2D3 on murine dendritic cell behaviour in vitro and in vivo. METHODS: Dendritic cells were differentiated from bone marrow cells of female C57Bl/6 mice in the presence or absence of 10(-8) M 1,25(OH)2D3 for 8 days (IL4 and GM-CSF). Maturation was induced for 48 h (IFNγ, LPS and BALB/C islet homogenate antigen). RESULTS: Bone marrow-derived dendritic cells displayed a different surface marker profile in the presence of 1,25(OH)2D3 with decreased MHC II, CD86 and CD80 and increased CCR5, DEC205, F4/80 and CD40, as well as lower IL6 and IL12 expression upon LPS/IFNγ stimulation. T-cell proliferation was significantly reduced when exposed to islet antigen-loaded 1,25D3-DCs as compared to control dendritic cells and IL4, IL10, TNFα and TGFß levels were increased. In vivo, transfer of islet antigen-loaded control dendritic cells resulted in priming of the immune system and hyperacute islet allograft rejection (4/4), whereas this was prevented in 5/7 mice treated with islet antigen-loaded 1,25D3-DCs. CONCLUSION: We conclude that in vitro 1,25(OH)2D3 exposure alters dendritic cell behaviour, converting them into a cell type that drives T cells away from destruction towards a regulatory phenotype.

Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents.

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.

Immunization with alloantibodies-covered melanoma cells induces regional antitumor effects that become systemic when combined with 5-FU treatment.

Alloantibodies, in particular immunoglobulin G (allo-IgG), confer a rejection advantage to tumors sharing the same major histocompatibility complex (MHC) in mice. However, when administrated intratumorally, this effect can only be achieved in combination with dendritic cells (DCs) activation. Here, we developed high titer allo-IgG by multiple rounds of immunization with allogenic B16 melanoma cells, which allows for the strong binding with B16 cells. We demonstrate that B16 cells incubated with these allo-IgG (referred to as allo-IgG-B16) become highly immunogenic, which release tumor antigens that are efficiently presented by classic DCs in lymph nodes (LNs). Injection of allo-IgG-B16 turns the tumor into an immune hot one and even elicits a systemic antitumor response when used together with 5-fluorouracil (5-FU). This systemic response is tumor-specific and relies on the critical site - LNs. Our findings provide a rationale for the use of allo-IgG in cancer treatment.

Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes.

The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using different activation models. CADA inhibited lymphocyte proliferation with low cellular toxicity in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads, phytohemagglutinin or anti-CD3 antibodies. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. Immunosuppression by CADA was characterized by suppressed secretion of various cytokines, and reduced CD25, phosphoSTAT5 and CTPS-1 levels. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4­1BB protein biosynthesis by inhibition of its co-translational translocation into the ER in a signal peptide-dependent way. Taken together, this study demonstrates that CADA, as potent down-modulator of human CD4 and 4­1BB receptor, has promising immunomodulatory characteristics. This would open up new avenues toward chemotherapeutics that act as selective protein down-modulators to treat various human immunological disorders.

Synthesis, immunosuppressive activity and structure-activity relationship study of a new series of 4-N-piperazinyl-thieno[2,3-d]pyrimidine analogues.

The synthesis of a new series of 4-N-piperazinyl-thieno[2,3-d]pyrimidines is described. The synthetic route allows introducing structural variety at positions 2, 4 and 6 of the scaffold. Evaluation of their immunosuppressive activity in a Mixed Lymphocyte Reaction (MLR) assay revealed that the most potent compound has an IC(50)-value of 66 nM and therefore deserves attention for further medicinal chemistry optimization.

Donor Lymphocyte-Derived Natural Killer Cells Control MHC Class I-Negative Melanoma.

Natural killer (NK) cells provide a natural defense against MHC-I-negative tumors, such as melanoma. Donor lymphocyte infusion (DLI) containing NK cells, a form of adoptive immunotherapy used after allogenic bone marrow transplantation (allo-BMT), promotes antitumor immune responses but is often associated with life-threatening complications such as graft-versus-host disease (GvHD). Here, we showed that without prior allo-BMT, DLI provoked melanoma control associated with the infiltration and persistence of the transferred NK cells. This allograft acceptance did not correlate with an increase of GvHD; instead it correlated with the expansion and activation of tumor-infiltrating NK cells that expressed the cytotoxic molecules (e.g., IFNγ and granzyme B) and maturation signatures (e.g., CD11bhiCD27lo and KLRGhi/CD43hi). The development of beneficial tumor-infiltrating NK cells of DLI origin required host CD4+ T-cell help in part by producing IL2, as well as by limiting regulatory CD4+ T cells (Treg). IL2 blockade impaired the NK-dependent melanoma control, which could not be rescued by IL2 administration beyond CD4+ T-cell help. Our findings linked NK allograft acceptance-CD4+ T-cell help crosstalk to melanoma development without the need of allo-BMT. We thereby helped define that tumor-infiltrating NK cells of DLI origin may serve as effective therapeutic targets for controlling melanoma.

Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D.

Structural analogues of vitamin D have been put forward as therapeutic agents able to exploit the immunomodulatory effects of vitamin D, without its undesired calcemic side effects. We have demonstrated that TX527 affects dendritic cell (DC) maturation in vitro, resulting in the generation of a tolerogenic cell. In the present study, we aimed to explore the global protein changes induced by the analogue in immature DC (iDC) and mature human DC and to correlate them with alterations in DC morphology and function. Human CD14(+) monocytes were differentiated toward iDC or mature DCs, in the presence or absence of TX527 (10(-8) M) (n=4). Protein samples were separated into two different pH ranges (pH4-7 and 6-9), analyzed by 2-D DIGE and differentially expressed spots (p<0.01) were identified by MALDI-TOF/TOF (76.3 and 70.7% in iDC and mature DCs, respectively). Differential protein expression revealed three protein groups predominantly affected by TX527 treatment, namely proteins involved in cytoskeleton structure, in protein biosynthesis/proteolysis and in metabolism. Moreover, protein interactome-network analysis demonstrated close interaction between these different groups (p<0.001) and morphological and functional analyses confirmed the integrated effect of TX527 on human DCs, resulting in a cell with altered morphology, cell surface marker expression, endocytic and migratory capacity.