Level Of Antigen-presenting Cells Research Articles

Identification of Five Tumor Antigens for Development and Two Immune Subtypes for Personalized Medicine of mRNA Vaccines in Papillary Renal Cell Carcinoma.

Increasing evidence has revealed the promise of mRNA-type cancer vaccines as a new direction for cancer immune treatment in several solid tumors, however, its application in papillary renal cell carcinoma (PRCC) remains unclear. The purpose of this study was to identify potential tumor antigens and robust immune subtypes for the development and appropriate use of anti-PRCC mRNA vaccines, respectively. Raw sequencing data and clinical information of PRCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The cBioPortal was utilized for the visualization and comparison of genetic alterations. The TIMER was used to assess the correlation between preliminary tumor antigens and the abundance of infiltrated antigen presenting cells (APCs). Immune subtypes were determined by the consensus clustering algorithm, and clinical and molecular discrepancies were further explored for a deeper understanding of immune subtypes. Five tumor antigens, including ALOX15B, HS3ST2, PIGR, ZMYND15 and LIMK1, were identified for PRCC, which were correlated with patients' prognoses and infiltration levels of APCs. Two immune subtypes (IS1 and IS2) were disclosed with obviously distinct clinical and molecular characteristics. Compared with IS2, IS1 exhibited a significantly immune-suppressive phenotype, which largely weakened the efficacy of the mRNA vaccine. Overall, our study provides some insights for the design of anti-PRCC mRNA vaccines and, more importantly, the selection of suitable patients to be vaccinated.

Harnessing the Induction of CD8+ T-Cell Responses Through Metabolic Regulation by Pathogen-Recognition-Receptor Triggering in Antigen Presenting Cells.

Cytotoxic CD8+ T-cells are key players of the immune responses against viruses. During the priming of a CD8+ T-cell response, the activation of a naïve T-cell by a professional antigen presenting cell (APC) involves the induction of various intracellular and metabolic pathways. The modulation of these pathways at the level of APCs or T-cells offers great potential to enhance the induction of robust effector cells and the generation of long-lived memory cells. On the one hand, signaling through pathogen recognition receptors (PRRs) expressed by APCs can greatly influence T-cell priming, and the potential of several PRR ligands as adjuvants are being studied. On the other hand, the engagement of several metabolic processes, at play in APCs and T-cells upon stimulation, implies that modulating cellular metabolism can impact on priming efficacy. Here, we review recent efforts to understand the interplay between PRR mediated signaling and metabolic pathway modulation in this context, through three examples: interplay between TLR4 and fatty acid metabolism, between TLR9 and IDO, and between STING and autophagy. These initial works highlight the potential for harnessing the induction of antiviral CD8+ T-cell responses using synergistic modulation of metabolic and PRR pathways.

The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector

BackgroundAgonists of TLR3 and TLR4 are effective immunoadjuvants for different types of vaccines. The mechanisms of their immunostimulatory action differ significantly; these differences are particularly critical for immunization with non-replicating adenovirus vectors (rAds) based vaccines. Unlike traditional vaccines, rAd based vaccines are not designed to capture vaccine antigens from the external environment by antigen presenting cells (APCs), but rather they are targeted to the de novo synthesis of vaccine antigens in APCs transfected with rAd. To date, there is no clear understanding about approaches to improve the efficacy of rAd vaccinations with immunoadjuvants. In this study, we investigated the immunoadjuvant effect of TLR3 and TLR4 agonists on the level of activation of APCs during vaccination with rAds.ResultsWe demonstrated that TLR3 and TLR4 agonists confer different effects on the molecular processes in APCs that determine the efficacy of antigen delivery and activation of antigen-specific CD4+ and CD8+ T cells. APCs activated with agonists of TLR4 were characterized by up-regulated production of target antigen mRNA and protein encoded in rAd, as well as enhanced expression of the co-activation receptors CD80, CD86 and CD40, and pro-inflammatory cytokines TNF-α, IL6 and IL12. These effects of TLR4 agonists have provided a significant increase in the number of antigen-specific CD4+ and CD8+ T cells. TLR3 agonist, on the contrary, inhibited transcription and synthesis of rAd-encoded antigens, but improved expression of CD40 and IFN-β in APCs. The cumulative effect of TLR3 agonist have resulted in only a slight improvement in the activation of antigen-specific T cells. Also, we demonstrated that IFN-β and TNF-α, secreted by APCs in response to TLR3 and TLR4 agonists, respectively, have an opposite effect on the transcription of the targeted gene encoded in rAd. Specifically, IFN-β inhibited, and TNF-α stimulated the expression of target vaccine antigens in APCs.ConclusionsOur data demonstrate that agonists of TLR4 but not TLR3 merit further study as adjuvants for development of vaccines based on recombinant adenoviral vectors.

Abstract 4531: Systems pharmacology to predict cellular biomarkers and optimize mono- and combination-therapy regimens: Focusing on immune checkpoint targets PD-1, PD-L1 and CTLA-4

Abstract Cancer immunotherapy focuses on stimulating and promoting the immune system to recognize and eliminate cancer cells, with several FDA approvals in recent years. However, identifying patients best suited for specific immune therapies, and determining optimal treatment regimens continue to be a clinical challenge. Using a molecular-detailed computational systems pharmacology model to identify cellular biomarkers and optimize regimens, we may be able to predict the efficacy of regimens in specific patient populations, and expedite drug development for cancer treatment. We developed a cell/receptor-based multi-compartment systems pharmacology model focusing on the immune response against a growing tumor, with the intent to test the effects of immune checkpoint inhibitors against PD-1, PD-L1 and CTLA-4 administered as mono- and combination therapies. Additionally, the model also allows for testing of other immuno-therapies, such as adoptive cell therapies, which can be combined with the checkpoint inhibitors. The model was designed and developed using the SimBiology plug-in in MATLAB. Simulations were performed with parameters that define the immune response at particular tumor stages of melanoma and NSCLC. All results were qualitatively and quantitatively compared to experimental pre-clinical and clinical data for model validation, or used for the generation of predictions suitable for further experimental testing. In silico, we have identified that administrations of the prescribed doses of 1-10 mg/kg of anti-CLTA-4 (based on binding kinetics) effectively saturates the receptors on the T cells, and promotes both an extended life span of the antigen presenting cells (APCs), and the maximum attainable activation levels of the effector T cells. The model further predicts that the effectiveness of anti-CTLA-4 therapy is limited by the immunogenicity of the system (i.e., the antigen intensity level and number of APCs presenting the antigens) in a monotonic fashion. Furthermore, injecting activated APCs without therapy would show a temporary tumor response and a subsequent recovery by the tumor to its original growth trajectory, while raising the antigen intensity had a sustained effect on tumor response. Other simulations indicate that, despite the lack of apparent tumor response, a sustained immune attack may be ongoing in the body; however, the immune activity is proportionally limited by the tumor and regulatory cells. Lastly, several dose-responses and clinical trials were simulated for both combination and monotherapies, and correlated with published clinical trial data. Future work will focus on uncovering the cellular biomarkers responsible for such results, experimentally validating them, as well as simulating optimal combination treatment regimens for future evaluation. Citation Format: Oleg Milberg, Chang Gong, Bing Wang, Paolo Vicini, Rajesh Narwal, Lorin Roskos, Aleksander Popel. Systems pharmacology to predict cellular biomarkers and optimize mono- and combination-therapy regimens: Focusing on immune checkpoint targets PD-1, PD-L1 and CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2017-4531

Immunomodulatory effects of Lactobacillus rhamnosus GG on dendritic cells, macrophages and monocytes from healthy donors

Lactobacillus rhamnosus GG (LGG) is clinically documented in relieving and preventing diarrhea, childhood infections, allergies and atopic eczema but knowledge of its immunomodulatory properties on antigen-presenting cells (APCs) is lacking. Here, immunomodulation of LGG on dendritic cells (DCs), macrophages and monocytes was investigated. Toll-like receptor (TLR) mRNA levels and immunophenotype of LGG-treated APCs, and cytokine secretion levels of culture of LGG-treated DCs and peripheral blood mononuclear cells were examined. LGG reduced TLR 8 mRNA level of APCs, implying that the immunomodulation of LGG may be partly due to the reaction of RNA with APCs. LGG reduced CD16 expression on macrophages and CD11b expression on monocytes. The results obtained demonstrated the effect of LGG on type-1 immune response polarization as observed from elevated production of IL-12 and TNFα across different APC stimulation models, suggesting that LGG may have immunotherapeutic potential in ameliorating and preventing the development of Th2-mediated inflammatory diseases and against infections.

Evidence implicating the commensal microbiota in shaping anti-tumor immunity in melanoma

T cell infiltration of solid tumors is found in a subset of cancer patients and is associated with favorable patient outcomes. This demonstrates the capacity of the immune system to mount protective anti-tumor responses in some cancer patients, yet the underlying mechanisms mediating the presence or absence of a T cell infiltrate are not well understood. Individuals exhibit remarkable diversity in their commensal microbiota, which in turn influences systemic immune responses, yet the role of microbiota in shaping spontaneous immunity against tumors is not known. Here we sought to examine the effect of microbial composition on the immune response to melanoma by comparing subcutaneous B16 melanoma growth in genetically identical C57BL/6 mice derived from two different facilities, Jackson laboratories and Taconic farms, which have been shown to differ in their commensal microbes. We found that B16.SIY tumors exhibited an increased growth rate in mice derived from Taconic (p < 0.02), which was associated with reduced induction of tumor-specific IFN-γ-producing CD8+ T cells (p < 0.009). A markedly increased number of SIY antigen-specific CD8+ T cells was found to accumulate within the tumor microenvironment in Jackson versus Taconic mice (p < 0.03). Strikingly, the differences in tumor growth, endogenous T cell priming, and SIY antigen-specific T cell infiltration were ablated in animals cohoused for at least two weeks prior to injection of tumor cells, consistent with a microbiota-derived effect. We thus asked whether transfer of Jackson fecal material into Taconic mice by oral gavage prior to tumor inoculation was sufficient to confer protective anti-tumor responses. Indeed, Taconic mice that received Jackson feces exhibit dramatically slower tumor growth (p < 0.015) and increased numbers of SIY antigen-specific CD8+ T cells at the tumor site, compared to Taconic mice that received Taconic feces. To pursue mechanisms that might explain these differences, we transferred CFSE-labeled SIY-specific 2C TCR Tg T cells into tumor-bearing Jackson and Taconic mice and tested their proliferation and acquisition of IFN-γ production ex vivo. 2C T cells responding to tumors in Taconic mice produced significantly less IFN-γ than 2C T cells responding to tumors in Jackson mice, likely pointing to a difference at the level of antigen-presenting cells. No difference in Th17 induction was observed. Our data provide evidence implicating the microbiota in shaping anti-tumor immunity in a pre-clinical model of melanoma. Gaining further understanding of the mechanisms underlying this effect may provide the foundation for rational manipulation of commensal microbes as a cancer therapeutic.

Plasmacytoid Dendritic Cells Engagement by Influenza Vaccine as a Surrogate Strategy for Driving T-Helper Type 1 Responses in Human Neonatal Settings

The elicitation of T-helper type 1 (Th1) cellular immunity to eradicate intracellular pathogens is a challenging task because of the interleukin 12 (IL-12) deficit observed in early infancy. Screening cord blood responses to various pediatric vaccines and Toll-like receptor (TLR) agonists for innate responses and CD4(+) T-cell differentiation. We identified that nonadjuvanted inactivated trivalent influenza vaccine (TIV) was able to cosignal T cells for the production of interferon γ (IFN-γ) in a neonatal setting. This process includes the mobilization of neonatal plasmacytoid dendritic cells (pDCs) as antigen-presenting cells (APCs) that efficiently engage Th1 cells in an IL-12-independent but type I IFN-dependent manner. In addition, cord blood pDCs efficiently cross-presented antigen to CD8(+) T cells. Importantly, activation by TIV mainly requires TLR7; however, R848/TLR7- and CpGB/TLR9-activated pDCs, which poorly produced IFN-α, induce neonatal Th2 responses. TLR pathway engagement in pDCs is necessary but not sufficient for a successful neonatal Th1 outcome. We provide evidence of a mature and functional neonatal immune system at the level of APCs and T cells and propose to implement the IFN-α/IFN-γ axis in pediatric vaccination as a surrogate for the defective IL-12/IFN-γ axis.

Are the anatomical sites for vaccine administration selected judiciously?

Route of vaccine administration plays an important role in the development of immune response. Antigen administered via different anatomical sites interacts with diverse subsets of antigen presenting cells. Diverse population of antigen presenting cells directs a drastically different immune response. Initially, the recommended routes for vaccine administration were also selected on the basis of clinical trials conducted for the drug molecules. However, physicochemical and pharmaceutical behaviors of proteins (antigens) and chemical compounds are entirely different. Most of the commercial vaccines are injected in the arm or in the scapular region (deltoid muscle). Vaccine administered to these conventional anatomical sites has failed to induce desired immune response due to lack of optimum level of antigen presenting cells. In this review, we have discussed the importance of the selection of anatomical sites for vaccine administration. Mere selection of an optimum site for vaccine administration may drastically change the immune response of the current marketed formulations without any alteration in their existing production plans.

Effects of Experimental Desynchronosis on the Organs of Immune System in WAG and ISIAH Rats

We studied the influence of abnormal illumination regimen on cell composition of the central and peripheral organs of the immune system in ISIAH rats and control WAG rats. In ISIAH rats, 24-h illumination for 14 days led to more pronounced inhibition of cell proliferation and differentiation in the thymus and more pronounced decrease in splenocyte proliferation and T and B cell counts in the spleen in comparison with WAG rats; however, the level of antigen-presenting cells in the spleen of ISIAH increased. We concluded that ISIAH rats are more sensitive to abnormal illumination regimen than WAG rats. Twenty-four-hour illumination was associated with impairments of central differentiation of T cells and activation of systemic inflammation followed by impairment of differentiation regulation, which can aggravate metabolic dysfunctions in these animals.

The cellular pathophysiology of immune thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is a bleeding disorder with circulating platelet counts of &lt;100 9 109/l, in the absence of any other resultant cause or disorder. This disease is primarily mediated by two major immune mechanisms. The predominant response occurs when immunoglobulin G (IgG) autoantibodies are produced against platelet self‐antigens resulting in platelet opsonization and increased Fc‐receptor‐mediated phagocytosis and destruction by macrophages within the spleen. In addition, these IgG autoantibodies can also mediate megakaryocyte inhibition/destruction within the bone marrow. The second immune response concerns T‐cell‐mediated platelet destruction in the periphery and megakaryocyte destruction/inhibition in the bone marrow. One or both of these mechanisms of immune mediated thrombocytopenia may lead to reduced levels of platelets.ResultsIt appears that ITP is associated with many varied defects at the level of antigen presenting cells, T cells and B cells. The primary defect in active disease appears to be an expansion of pro‐inflammatory Th17 cells with a concomitant deficiency in T and B regulatory cells.ConclusionThis article will summarize the role of cellular autoreactive mechanisms associated with ITP to gain a greater understanding of the pathophysiology of this autoimmune bleeding disorder.

Ikaros Deficiency Enhances Notch Signaling in Host Antigen Presenting Cells and Differentially Regulates Gvhd and GVL Responses After Experimental BMT.

Abstract 690Host antigen presenting cells (APCs) are necessary for induction of graft-versus-host (GVH) responses. But the APC autonomous molecular mechanisms that are critical for modulation of GVH are not known. Ikaros (Ik) is a zinc-finger DNA binding protein that is essential for the development of lymphoid cells and hematological malignancies. Because Ik is known to negatively regulate certain dendritic cell (DC), the most potent APCs, functions (Wu et al, Immunity, 1997) we hypothesized that its deficiency in host APCs will reduce GVHD. We generated [B6→B6] and [Ik-/-B6→B6] chimeras and utilized them as recipients in well characterized MHC matched, minor mismatched C3H.SW B6 model of acute GVHD. [B6→B6] and [Ik/-B6 → B6] animals received 9 Gy and were transplanted with 3 × 106 T cells and 5 × 106 BM from either syngeneic B6 or allogeneic C3H.SW donors. Contrary to our hypothesis, [Ik-/-B6 < 0.001). The increase in mortality was associated with a significant increase in GVHD specific clinical severity, histopathological damage of the liver and GI tract, donor T cell expansion, serum levels TNF-αa, IFNγ and IL-17 on days +7 and +14 after BMT. Similar results were obtained in a second, BALB/c→B6 model of GVHD and also in recipient chimeras generated from dominant negative Ikaros (IkDN) mice, demonstrating a role for Ik in a strain independent manner. We next performed systematic analyses of DCs to uncover the mechanisms underpinning our surprising results. Phenotypic analysis of spleen and BM derived DCs demonstrated similar numbers of CD8+ and CD8-CD11c+DCs but a reduced number of pDCs were present in Ik-/- animals when compared with WT animals. Ik-/-DCs demonstrated an immature phenotype as determined by the expression of CD40/80. By contrast, functional analyses of Ik-/- DCs, when stimulated with LPS showed significantly greater secretion of proinflammatory cytokines TNF-αa and IL-6 and greater stimulation of allo-T cells in MLRs when compared with WT B6 DCs. CFSE, annexin and CD107a labeling studies demonstrated that Ik-/-DCs caused greater proliferation without altering the rate of apoptosis or the cytotoxic function of the allo-T cells. To characterize the molecular mechanisms we evaluated the role of putative molecular targets of Ikaros, the Notch signaling pathway because Ikaros and Notch antagonistically regulate target genes (Chari et al. J Immunol 2008 and Bugeon, et al. J Immunol 2008). Both Ik-/- and IkDN DCs, at steady state, showed an increase in the expression of several Notch target genes such as Hes-1, Hex-1, Deltex1, Notch3, Jagged-1 and Jagged-2. Blockade of Notch signaling with γ-secretase inhibitor (DAPT) in MLR mitigated the enhanced allo-stimulatory capacity of the Ik-/- DCs, thus demonstrating a functional relevance for enhanced notch signaling in the absence of Ikaros. We next hypothesized that given the enhanced GVHD response that was associated with increased proliferation and preserved cytotoxicity of allo-T cells, the GVL response will also be enhanced in the recipients with Ik-/- APCs. Unexpectedly, the [B6→ B6] and [Ik-/-B6 → B6] chimeras when transplanted with allogeneic T cells from C3H.SW cells along with two different doses of syngeneic 0.5 or 1×104 MBL-2 tumor cells demonstrated equivalent GVL responses despite greater severity of GVHD. Together our data demonstrate (a) for the first time, to our knowledge, suggest differential regulation of GVHD and GVL at the level of host APCs and (b) show a role for a novel molecular pathway, the Ik-Notch axis, in the host APCs as an important modulator of GVH responses. Disclosures:Maillard:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Foundation: Research Funding; ASH Scholar Awards : Research Funding.

Upregulation of interferon-γ and interleukin-4, Th cell-derived cytokines by So-Shi-Ho-Tang (Sho-Saiko-To) occurs at the level of antigen presenting cells, but not CD4 T cells

Ethnopharmacological relevance So-Shi-Ho-Tang (SSHT) or known as Sho-Saiko-To in Japanese and Xiao-Chai-Hu-Tang in Chinese has been used to treat chronic liver disease and other infections, and its hepatoprotective effects have been widely studied. Aim of the study We tried to investigate the immunomodulatory effect of SSHT on interferon (IFN)-γ and interleukin (IL)-4 and their Th1/Th2 transcription factors in vivo and in vitro since these two cytokines are important in determining the type of cell-mediated inflammatory and humoral responses. Materials and methods SSHT was orally given to BALB/c mice for 7 days and then injected with anti-CD3 mAb intravenously. IFN-γ, IL-4, IL-2 and Th1/Th2-specific transcription factors as well as splenocyte subsets were measured. Splenocytes and CD4 T cells were cultured with anti-CD3 or anti-CD3/anti-CD28 in the presence of SSHT, its constituent herbs and baicalin, and the levels of cytokines and transcription factors were measured by ELISA and western blotting. Results Oral administration of SSHT to mice in response to i.v. anti-CD3 injection enhanced the expression of IFN-γ, IL-4 and IL-2 in the serum and spleen at the secreted protein and mRNA level. This was accompanied by the upregulation of CD69 and CD4 T cell populations by flow cytometry. The upregulation of IFN-γ and IL-4 by SSHT did not occur in anti-CD3/anti-CD28 stimulated CD4 T cells in vitro. However, SSHT was capable of producing the cytokines in anti-CD3 stimulated splenocytes even in the absence of CD28, suggesting a role for some soluble factors produced by antigen presenting cells (APC). In support of this, we found that SSHT increased IL-12 and IL-6 in the same cells. STAT4, but not T-bet, was involved in the upregulation of IFN-γ by SSHT while the increased IL-4 expression was accompanied by a parallel increase in c-Maf but independent of STAT6 and GATA-3. Conclusion These data indicate that the upregulation of IFN-γ and IL-4 by SSHT must occur through some interactions between APC and CD4 T cells. Taken together, the present data provide additional information on some of the immunological mechanisms of SSHT for treatment of liver diseases and infections.

Different regulation of T helper 1‐ and T helper 2‐promoting cytokine signalling factors in human dendritic cells after exposure to protein versus contact allergens

Cytokine-dependent T helper 1 (Th1) differentiation versus T helper 2 (Th2) differentiation is controlled by distinct transcription factors. Previously, we have demonstrated that immature human dendritic cells (DC) from blood donors with allergies show rapid phosphorylation of the Th2-associated signal transducer and activator of transcription 6 (STAT6) upon contact with protein allergens. In the present study we investigated whether this process is regulated by the downstream molecules suppressor of cytokine signalling (SOCS) and/or by the factors T-bet and GATA3. Therefore, immature DC of grass or birch pollen-allergic donors were treated with the respective Th2-promoting protein allergens, and, for comparison, with the Th1-promoting contact allergen 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI) or with the antigen tetanus toxoid. Changes in the mRNA levels of SOCS1, SOCS3, T-bet and GATA3 were analysed by quantitative real-time polymerase chain reaction. Exposure of DC to protein allergens led to the up-regulation of the Th2-associated genes SOCS3 and GATA3, whereas the contact allergen MCI/MI preferentially enhanced the expression of the Th1-associated gene T-bet. Treatment of immature DC with the antigen tetanus toxoid increased both Th1- and Th2-associated genes. Our data indicate that polarization of type 1 versus type 2 immune responses takes place already at the level of antigen-presenting cells, involving molecules similar to those used in T-cell polarization.

Inflammation enhances consumption and presentation of transfused RBC antigens by dendritic cells

Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4(+) T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

NKG2D and Natural Cytotoxicity Receptors Are Involved in Natural Killer Cell Interaction with Self‐Antigen Presenting Cells and Stromal Cells

It is thought that human natural killer (NK) lymphocytes should not damage self-tissues due to the inhibiting signal initiated by the engagement of one or another inhibitory receptor superfamily (IRS) members with self-human histocompatibility antigen (HLA)-I. During viral infection, the low expression of self-HLA-I on infected-cells leads to a reduction of the inhibiting signal and thus NK cells kill self-cells (missing self-hypothesis). Here, we have analyzed human NK cell interaction with self-cells as antigen-presenting cells (APC) or stromal cells isolated from bone marrow or skin. Despite the expression of high levels of HLA-I, APC and stromal cells are killed by interleukin (IL)-2-activated NK cells upon lymphocyte function antigen (LFA)1-(intracellular adhesion molecule) (ICAM)1 interaction. The natural cytotoxicity receptors NKp30 and NKp46 are responsible for the delivery of lethal hit to APC, whereas NKG2D-activating receptor, the ligand of the major histocompatibility complex (MHC)-related molecule MICA, and the UL16-binding protein are involved in stromal cell killing. These events are dependent on the activation of phosphoinositol 3-kinase and consequent release of perforins and granzymes. Both bone marrow stromal cells and skin fibroblasts inhibit T cell proliferation to alloantigen or triggering through CD3/T cell receptor complex. Importantly, NK cells can revert this veto effect. Altogether, these findings support the notion that NK cells can recognize self-cells possibly affecting both APC function and interaction between lymphocytes and microenvironment leading to autoreactivity.

Factors governing the activation of adoptively transferred donor T cells infused after allogeneic bone marrow transplantation in the mouse

AbstractMurine models of bone marrow transplantation were used to study the mechanisms governing the activation of donor lymphocyte infusions (DLIs) manifesting as lymphohematopoietic graft-versus-host (LH-GVH) and graft-versus-leukemia (GVL) reactivities. We demonstrate here that established mixed chimerism influences the potency of DLI-mediated alloreactivity only in the MHC-mismatched but not MHC-matched setting. In the MHC-matched setting, high levels (≥ 40%) of residual host chimerism correlated negatively with DLI-mediated alloreactivity irrespective of the timing of their administration, the donor's previous sensitization to host antigens, or the level of residual host APCs. In vivo administration of Toll-like receptor (TLR) ligands was required to maximize DLI-mediated LH-GVH and GVL reactivities in chimeras with low levels (≤ 15%) of residual host chimerism. In contrast, coadministration of DLI with antigen-presenting cell (APC) activators was insufficient to augment their LH-GVH response in the presence of high levels of host chimerism unless the host's T cells were transiently depleted. Together, these results show the cardinal influence of donor-host incompatibility on DLI-mediated GVH responses and suggest that in MHC-matched chimeras, the induction of optimal alloreactivity requires not only donor T cells and host APCs but also TLR ligands and in the presence of high levels of host chimerism depletion of host T cells.

Notch ligand mRNA levels of human APCs predict Th1/Th2-promoting activities

Although the contribution of Notch ligands expressed by antigen-presenting cells (APCs) to the Th1/Th2 differentiation has been suggested in mouse studies, their nature in humans remains obscure. To assess the issue, we examined correlation of Notch ligand mRNA levels of human APCs with Th1/Th2-promoting stimulations, i.e. Th1/Th2 adjuvant activities. The expression patterns of human dendritic cells (DCs) and leukemic cell line models of APCs differed partly from those previously observed in mouse DCs and by cellular types and maturation stages. Most strikingly, Th2 adjuvants enhanced the Delta1 expression on human APCs but did not induce Delta4 expression, which was induced by a Th1 adjuvant. The differential expression patterns suggest a distinct role of these Delta members in Th1/Th2 differentiation and their predictive potential for Th1/Th2-promoting activities.

Higher Numbers of Blood CD14 + Cells before Starting Conditioning Regimen Correlate with Greater Risk of Acute Graft-versus-Host Disease in Allogeneic Stem Cell Transplantation from Related Donors

Host antigen-presenting cells (APCs) have been shown to induce acute graft-versus-host disease (aGVHD) in experimental models. In this study, we investigated whether pretransplantation blood levels of host APCs, such as plasmacytoid and myeloid dendritic cells and monocytes, correlate with the development of aGVHD. A total of 89 consecutive patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-matched related (n = 48) or unrelated (n = 41) donors were enrolled in the study. Blood samples were analyzed by flow cytometry before initiating the conditioning regimen. In related donor transplants, patient–donor sex mismatch and monocyte levels significantly correlated with aGVHD grade II–IV in both univariate and multivariate analyses. Similar results were not observed in recipients of matched unrelated transplants, possibly due to use of antithymocyte globulin (ATG) or differences in graft source in these patients. In conclusion, pretransplantation recipient monocyte levels are relevant to the development of GVHD in HSCT from related donors.

Proteasome inhibitor‐induced apoptosis in human monocyte‐derived dendritic cells

Proteasome inhibitors possess potent antitumor activity against a broad spectrum of human malignancies. However, the effects of these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effects of proteasome inhibitors on dendritic cells (DC), antigen-presenting cells playing a key role in the initiation of immune responses. Exposure to the proteasome inhibitors bortezomib, MG132 or epoxomicin was found to promote apoptosis of human monocyte-derived DC and to reduce the yield of viable DC when given to monocytes early during differentiation to DC. DC apoptosis via proteasome inhibition was accompanied by mitochondria disruption and subsequent activation of the caspase cascade. Up-regulation and intracellular redistribution of Bcl-2-associated X protein (Bax), a pro-apoptotic Bcl-2 family protein, were observed in DC treated with these compounds and represent a suitable mechanism leading to activation of the intrinsic apoptotic pathway. Finally, active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by proteasome inhibitors, since the translation inhibitor cycloheximide blocked all of the steps of the observed apoptotic response. In conclusion, induction of apoptosis in DC may represent a novel mechanism by which proteasome inhibitors affect the immune response at the antigen-presenting cell level.

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators.

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.