Low Levels Of MHC Class Research Articles

Analyzing the role of cancer-associated fibroblast activation on macrophage polarization.

Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFβ. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.

Imprint of Initial Education and Loss of Ly49C/I in Activated Natural Killer Cells of TAP1-KO and C57BL/6 Wildtype Mice.

Natural killer (NK) cells are important effectors of the innate immune system and participate in the first line of defense against infections and tumors. Prior to being functional, these lymphocytes must be educated or licensed through interactions of their major histocompatibility complex class I molecules with self-specific inhibitory receptors that recognize them. In the absence of such contacts, caused by either the lack of expression of the inhibitory receptors or a very low level of major histocompatibility complex class I (MHC class I) proteins, NK cells are hypo-reactive at baseline (ex vivo). After stimulation (assessed through plate-bound antibodies against activating receptors or culture in the presence of cytokines such as interleukin (IL)-2 or IL-15) however, they can become cytotoxic and produce cytokines. This is particularly the case in transporter associated with antigen processing (TAP)-deficient mice, which we investigated in the present study. Transporter associated with antigen processing transports endogenous peptides from the cytosol to the endoplasmic reticulum, where they are loaded on nascent MHC class I molecules, which then become stable and expressed at the cell surface. Consequently, TAP-KO mice have very low levels of MHC class I expression. We present a study about phenotypic and functional aspects of NK cells in two mouse strains, C57BL/6 wildtype and TAP1-KO in spleen and lung. We observed that in both types of mice, on the same genetic background, the initial pattern of education, conferred to the cells via the inhibitory receptors Ly49C/I and NKG2A, was maintained even after a strong stimulation by the cytokines interleukin-2, interleukin-12, interleukin-15 and interleukin-18. Furthermore, the percentages of activated NK cells expressing Ly49C/I and Ly49I were strongly down-modulated under these conditions. We completed our investigations with phenotypic studies of NK cells from these mice.

The Latest Developments in Immunomodulation of Mesenchymal Stem Cells in the Treatment of Intrauterine Adhesions, Both Allogeneic and Autologous.

Intrauterine adhesion (IUA) is an endometrial fibrosis disease caused by repeated operations of the uterus and is a common cause of female infertility. In recent years, treatment using mesenchymal stem cells (MSCs) has been proposed by many researchers and is now widely used in clinics because of the low immunogenicity of MSCs. It is believed that allogeneic MSCs can be used to treat IUA because MSCs express only low levels of MHC class I molecules and no MHC class II or co-stimulatory molecules. However, many scholars still believe that the use of allogeneic MSCs to treat IUA may lead to immune rejection. Compared with allogeneic MSCs, autologous MSCs are safer, more ethical, and can better adapt to the body. Here, we review recently published articles on the immunomodulation of allogeneic and autologous MSCs in IUA therapy, with the aim of proving that the use of autologous MSCs can reduce the possibility of immune rejection in the treatment of IUAs.

Low-dose 2-deoxy glucose stabilises tolerogenic dendritic cells and generates potent in vivo immunosuppressive effects

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and β-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases.

Cutting Edge: Augmenting Muscle MHC Expression Enhances Systemic Pathogen Control at the Expense of T Cell Exhaustion.

Myocytes express low levels of MHC class I (MHC I), perhaps influencing the ability of CD8+ T cells to efficiently detect and destroy pathogens that invade muscle. Trypanosoma cruzi infects many cell types but preferentially persists in muscle, and we asked if this tissue-dependent persistence was linked to MHC expression. Inducible enhancement of skeletal muscle MHC I in mice during the first 20 d of T. cruzi infection resulted in enhanced CD8-dependent reduction of parasite load. However, continued overexpression of MHC I beyond 30 d ultimately led to a collapse of systemic parasite control associated with immune exhaustion, which was reversible in part by blocking PD-1:PD-L1 interactions. These studies demonstrate a surprisingly strong and systemically dominant effect of skeletal muscle MHC expression on maintaining T cell function and pathogen control and argue that the normally low MHC I expression in skeletal muscle is host protective by allowing for pathogen control while preventing immune exhaustion.

P1.12-05 Microenvironment Characterization of Small Cell Lung Cancer Xenografts Implanted in Hematopoietic Humanized Mice

With the high mutational burden seen in small cell lung cancer (SCLC) there is the theoretical potential to improve SCLC outcome through immunotherapy. Recent immunotherapeutic clinical trials in SCLC have demonstrated promising results. However, to better understand the immune response and the potential role of immunotherapy in SCLC, immunocompetent models are needed. To this end, we have applied a hematopoietic humanized mouse model, Hu-CB-BRGS to investigate the mechanisms underlying SCLC immunotherapy and develop novel strategies to improve therapeutic efficacy. BALB/c-Rag2nulllll2rynullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. SCLC flank tumors were initiated in BRGS mice using two characterized SCLC cell lines (CDX) and 1 patient derived xenograft (PDX). Upon verification of human T-cell chimerism in the Hu-CB-BRGS mice, SCLC tumors grown in BRGS mice were engrafted into the flanks of Hu-CB-BRGS mice by trocar transfer. Tumor growth was quantified by twice weekly measurement and harvested on reaching 1200 mm3. At harvest, tumor tissue as well as host immune organs (lymph node, spleen) were collected for immunological assessment. Humanized immune system and tumor were evaluated by flow cytometry and immunohistochemistry (IHC). Flank tumors from two CDX tumors (H82 and H187) and one PDX tumor (LX-95) were successfully developed in the Hu-CB-BRGS mice with take rates averaging 82%. SCLC tumor growth rate in Hu-CB-BRGS mice was comparable to that seen in BRGS mice. Although human T cells were well represented in the lymph nodes and spleens of the Hu-CB-BRGS mice, we detected very few tumor infiltrating immune cells in the engrafted SCLC tumors by IHC and flow cytometry as defined by CD45 and CD3, which is consistent with the observations in SCLC patient tumor tissue. Tumor cells, identified by EpCAM expression, expressed low levels of MHC class I, II and PD-L1. PD-1 was expressed by human T cells found in the lymph nodes and spleen of Hu-CB-BRGS mice, while the SCLC xenografts expressed varying levels. We demonstrate that both SCLC PDX and CDX tumors can be grown in the context of a humanized immune system within mouse recipients. SCLC Hu-CB-BRGS mice demonstrate persistence of human immune cells, including T cells and B cells in the immune organs. The xenograft tumor microenvironment included variable human immune infiltrates. The SCLC Hu-CB-BRGS mouse model may be a valuable preclinical platform for testing human specific immune-oncology therapeutics for SCLC patients.

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Correction: Notch Ligand Delta-Like 4-Pretreated Dendritic Cells Alleviate Allergic Airway Responses by Enhancing IL-10 Production.

[This corrects the article DOI: 10.1371/journal.pone.0063613.].

Human Dendritic Cell-Derived Induced Pluripotent Stem Cell Lines Are Not Immunogenic.

Donor-specific induced pluripotent stem cells (iPSC) can be used to generate desired cell types, including naive immune effectors, for the treatment of different diseases. However, a greater understanding of the inherent immunogenicity of human iPSC and their cellular derivatives is needed for the development of safe and effective cell-replacement therapies, given that studies in mouse models claimed that the syngenic mouse iPSC lines can be immunogenic. We report the characterization of the innate and adaptive immune mechanisms in human iPSC lines derived from peripheral blood-derived dendritic cells using a nonintegrating RNA virus, Sendai virus. We show that these iPSC lines express mRNA of TLR molecules and the Ag-presentation pathway intermediates; however, these mRNA are not translated into functional proteins, and these iPSC lines do not induce TLR-mediated inflammatory cytokine responses or inflammasome activation. We also show that these iPSC lines do not activate T cells in an allogenic MLR; however, they express low levels of MHC class I molecules that can efficiently acquire antigenic peptides from their microenvironment and present them to Ag-specific T cells. In addition, we show that these iPSC lines can be efficiently differentiated into hematopoietic stem cell precursors, as well as APC, under appropriate culture conditions. Taken together, our data show that the dedifferentiation of human dendritic cells effectively shuts down their immunogenic pathways and implicates transcriptional and posttranscriptional mechanisms in this process.

Oral administration of the nitroxide radical TEMPOL exhibits immunomodulatory and therapeutic properties in multiple sclerosis models

Therapies with both immunomodulatory and neuroprotective properties are thought to have the greatest promise in reducing the severity and progression of multiple sclerosis (MS). Several reactive oxygen (ROS) and reactive nitrogen species (RNS) are implicated in inflammatory-mediated damage to the central nervous system (CNS) in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxide radical with potent antioxidant activity. The goal of our studies was to investigate the immunomodulatory effects and therapeutic potential of orally-delivered TEMPOL in the mouse EAE model. Mice receiving TEMPOL chow ad libitum for 2weeks prior to induction of active EAE showed delayed onset and reduced incidence of disease compared to control-fed animals. Reduced disease severity was associated with limited microglial activation and fewer inflammatory infiltrates. TEMPOL’s effects were immunomodulatory, not immunosuppressive: T cells produced less interferon-γ and tumor necrosis factor-α, and TEMPOL-fed mice exhibited a shift towards TH2-type antibody responses. Both myeloid and myeloid-dendritic cells of TEMPOL-fed EAE animals had significantly lower levels of MHC class II expression than controls; CD40 was also significantly reduced. TEMPOL administration was associated with an enrichment of CD8+ T cell populations and CD4+FoxP3+ regulatory populations. TEMPOL reduced the severity of clinical disease when administered after the induction of disease, and also after the onset of clinical symptoms. To exclude effects on T cell priming in vivo, TEMPOL was tested with the passive transfer of encephalitogenic T cells and was found to reduce the incidence and peak severity of disease. Protection was associated with reduced infiltrates and a relative sparing of neurofilaments and axons. The ability of oral TEMPOL to reduce inflammation and axonal damage and loss demonstrate both anti-inflammatory and protective properties, with significant promise for the treatment of MS and related neurological disorders.

Immune Privileged Features of Multipotent Mesenchymal Stromal Cells Are Lost after Co-Cultivation with Allogeneic Lymphocytes in Vitro

Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed.The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics.Materials and methodsMSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2.After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA).Data are presented as mean ± standard error. Statistical analysis was performed using Student’s t-test (considered reliable p <0.05).ResultsThe number of cells in cultures of MSCs and IFN-g-MSCs did not differ significantly during the 4 days of observations. The presence of non-activated lymphocytes had no effect on the parameters of growth and viability of MSCs. Co-cultivation of activated lymphocytes with MSCs resulted in a reduction of viable MSCs, to 51.6 ± 5.5% compared with IFN-g-MSCs (68,1 ± 6,2%, p = 0.02). The viability of MSCs cultures without lymphocytes was 74.5% ± 4.8 on day 4 from the beginning of the experiment.HLA-DR expression level gradually increased in IFN-g-MSCs (see table). Co-culturing MSCs with lymphocytes also leads to a gradual increase in HLA-DR expression on MSCs apparently due to IFN-g, produced by lymphocytes. However, in IFN-g-MSCs co-cultured with lymphocytes HLA-DR MFI was significantly lower (p = 0.05) than without lymphocytes. HLA-DR MFI increased about 10 times in MSCs co-cultured with activated lymphocytes regardless of their IFN-g pretreatmentThe data obtained indicate that IFN-g-MSCs did not change in their growth characteristics and had increased immunomodulating properties. IFN-g-MSCs have a greater resistance to activated lymphocytes, which makes them more effective for cell therapy. The results suggest that injected to patient MSCs could have increased level of HLA-DR expression regardless of their initial treatment with IFN-g. The increase of HLA-DR expression on MSCs co-cultured with lymphocytes indicates a possibility of loss of immune privilege by these cells when injected to patient. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intra-adrenal murine TH-MYCN neuroblastoma tumors grow more aggressive and exhibit a distinct tumor microenvironment relative to their subcutaneous equivalents.

In around half of the patients with neuroblastoma (NBL), the primary tumor is located in one of the adrenal glands. We have previously reported on a transplantable TH-MYCN model of subcutaneous (SC) growing NBL in C57Bl/6 mice for immunological studies. In this report, we describe an orthotopic TH-MYCN transplantable model where the tumor cells were injected intra-adrenally (IA) by microsurgery. Strikingly, 9464D cells grew out much faster in IA tumors compared to the subcutis. Tumors were infiltrated by equal numbers of lymphocytes and myeloid cells. Within the myeloid cell population, however, tumor-infiltrating macrophages were more abundant in IA tumors compared to SC tumors and expressed lower levels of MHC class II, indicative of a more immunosuppressive phenotype. Using 9464D cells stably expressing firefly luciferase, enhanced IA tumor growth could be confirmed using bioluminescence. Collectively, these data show that the orthotopic IA localization of TH-MYCN cells impacts the NBL tumor microenvironment, resulting in a more stringent NBL model to study novel immunotherapeutic approaches for NBL.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1663-y) contains supplementary material, which is available to authorized users.

The Measure of DAMPs and a role for S100A8 in recruiting suppressor cells in breast cancer lung metastasis

To determine whether there was a relationship between damage associated molecular pattern molecule (DAMP) expression and recruitment of suppressor cells to sites of metastasis we measured relative expression of DAMPs, regulatory T cells (Tregs), and myeloid derived suppressor cells (MDSC) in mice at various stages of breast cancer progression using the 4T1 model. Although S100A8 was expressed at relatively low levels in the tumor cells, expression was 100-fold higher in the lung and liver which are common sites of metastasis for this tumor. Despite the relatively high level of S100A8 expression in the lungs of naïve mice, the level of expression increased further and was significantly elevated after only 7 days of tumor growth. The same pattern was observed for MDSC, and both S100A8 and MDSC expression peaked in the lungs of mice following 21 days of tumor growth. Characterization of MDSC from the lungs revealed expression of RAGE, and the cells were capable of migrating in a dose-dependent manner toward S100A8. In addition, the MDSC expressed low levels of MHC Class I, MHC Class II, CD80, and secreted TGF-β. Taken together, these data suggest that expression of S100A8 in the lungs may facilitate recruitment of MDSC, which may in turn aid in establishing a metastatic niche capable of suppressing a localized immune response.

B Cells and CD40/CD154 Interactions Are Required for CD8 T Cell Recovery Following Antibody-Mediated Lymphoablation.

Antibody-meditaed lymphoablation is commonly used as an induction therapy in transplant patients. However, recipients eventually reconstitute their T cell repertoire, develop anti-donor immune responses and reject transplanted organ. The goal of this study was to investigate the requirements for T cell recovery following lymphoablation in a mouse model of heterotopic cardiac transplantation. We previously reported that memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts (0.2% vs 3-5% of spleen CD8 T cells recovered by d. 10 posttransplant) and in non-transplanted B6 mice. Analogous findings were made in recipients treated with anti-CD8 ± anti-CD4 mAbs demonstrating that the requirement for CD4 cell help is a common feature of antibody-mediated lymphoablation. In addition, memory CD8 T cell reconstitution depended on CD4 T cell help in non-transplanted B6 mice and in B6 isograft recipients treated with mATG. Treatment with blocking anti-CD154 mAb plus mATG inhibited memory CD8 T cell recovery in B6 heart allograft recipients similar to the levels observed after CD4 T cell depletion. Conversely, agonistic anti-CD40 mAb restored expansion of memory CD8 T cells following mATG treatment of CD4 T cell depleted heart allograft recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFa suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.

B cells are required for CD8 T cell recovery after antibody-mediated depletion (TRAN3P.895)

Abstract Antibody-mediated lymphoablation is used as an induction therapy in transplant patients. We investigated the requirements for T cell recovery following lymphoablation in a mouse model of heart transplantation. Memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts and in non-transplanted B6 mice. Treatment with blocking anti-CD154 mAb plus mATG inhibited CD8 T cell recovery, while agonistic anti-CD40 mAb restored memory CD8 T cell expansion after mATG treatment of CD4 T cell depleted recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted &amp;gt;90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFα suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.

The Antigenic Determinant That Defines Thymic Nurse Cells Is Expressed by Thymic Epithelial Progenitor Cells.

Stromal thymic epithelial cells with the multicellular structure unique to thymic nurse cells (TNCs) express the pH91 antigen on their cell surfaces. The multicellular TNC-complexes develop through an intimate association between αβTCR+CD4+CD8+ thymocytes and pH91-expressing cortical epithelial cells. TNCs participate in MHC-restriction and exhibit epithelial cell progenitor characteristics. In this report, we show that as early as E11.5 stage of thymus development, the pH91 antigen is expressed in association with K8, K5, Foxn1, and p63. The expression of these epithelial progenitor markers along with the pH91 antigen persists throughout thymic development in the murine thymus. At E13.5, pH91+ cells express relatively low levels of MHC class II. After E17.5, the first multicellular TNC complexes are recognizable along with increased cell surface expression of MHC class II. Our data suggest that epithelial cells bearing the “progenitor phenotype” develop into the multicellular TNCs.

APLP2 regulates the expression of MHC class I molecules on irradiated Ewing’s sarcoma cells

Ewing’s sarcoma (EWS) is a pediatric cancer that is conventionally treated by surgery, chemotherapy, and radiation therapy. Innovative immunotherapies to treat EWS are currently under development. Unfortunately for EWS patients, when the disease is found to be resistant to current therapeutic approaches, the prognosis is predictably grim. Radiation therapy and immunotherapy could potentially synergize in the eradication of EWS, as some studies have previously shown that irradiation increases the presence of immune receptors, including MHC class I molecules, on the surface of tumor cells. However, EWS cells have been reported to express low levels of MHC class I molecules, a phenotype that would inhibit T-cell mediated lysis. We have previously demonstrated that the transgene-driven overexpression of amyloid β (A4) precursor-like protein 2 (APLP2) reduces the expression of MHC class I molecules on the surface of human cervical carcinoma HeLa cells. We thus examined whether endogenously expressed APLP2 downregulates MHC class I expression on EWS cells, particularly upon irradiation. We found that irradiation induces the relocalization of APLP2 and MHC class I molecules on the surface of EWS cells, redistributing cells from subpopulations with relatively low APLP2 and high MHC class I into subpopulations with relatively high APLP2 and low MHC class I surface expression. Consistent with these findings, the transfection of an APLP2-targeting siRNA into EWS cells increased MHC class I expression on the cell surface. Furthermore, APLP2 was found by co-immunoprecipitation to bind to MHC class I molecules. Taken together, these findings suggest that APLP2 inhibits MHC class I expression on the surface of irradiated EWS cells by a mechanism that involves APLP2/MHC class I interactions. Thus, therapeutic strategies that limit APLP2 expression may boost the ability of T cells to recognize and eradicate EWS in patients.

A transplantable TH‐MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma

Current multimodal treatments for patients with neuroblastoma (NBL), including anti-disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well-explored autologous mouse model for NBL is the TH-MYCN model. However, the immunobiology of the TH-MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH-MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH-MYCN-derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild-type and Rag1(-/-) mice, showing an important role for NK cells in the natural anti-NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti-GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH-MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.

Developmental maturation of innate immune cell function correlates with susceptibility to central nervous system autoimmunity

MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two-week-old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid-derived suppressor cells and plasma-cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti-inflammatory IL-10. When used as APCs, splenocytes from 2-week-old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T-cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2-week-old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T-cell-mediated CNS autoimmune disease.

Notch ligand delta-like 4-pretreated dendritic cells alleviate allergic airway responses by enhancing IL-10 production.

The Notch pathway plays a role in the processes of cell proliferation, differentiation, and apoptosis, which affect the development and function of various organs. Dendritic cells (DCs), as professional antigen-presenting cells (APCs), induce T cell activation and promote T cell differentiation by antigen stimulation. Research has shown that Notch ligand delta-like 4 (Dll4) in APCs is associated with stimulation of a Th1-type response. However, the regulatory roles of Dll4 in the activation and function of DCs have yet to be clearly elucidated. In this study, we demonstrated that activation of Dll4-pretreated bone marrow-derived DCs by performing ovalbumin (OVA) stimulation expressed a high level of interleukin (IL)-10 without diminishing IL-12 production. By contrast, the proinflammatory cytokines, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, decreased in Dll4-pretreated DCs by performing either lipopolysaccharide (LPS) or OVA stimulation. Compared to fully mature DCs, lower levels of MHC class II CD40 and higher levels of CD80 and CD86 molecules were expressed in these semi-mature like DCs. Dll4 Notch signaling also enhanced Notch ligand mRNA expression of Dll1, Dll4, and Jagged1 in DCs. Dll4-modified DCs exhibited a reduced capacity to stimulate the proliferation of OVA-specific CD4+ T cells, but actively promoted large amounts of IL-10 production in these activated T cells. Furthermore, immunomodulatory effects of Dll4-modified DCs were examined in an established asthmatic animal model. After adoptive transfer of OVA-pulsed plus Dll4-pretreated DCs in OVA-immunized mice, OVA challenge induced lower OVA-specific immunoglobulin E (IgE) and higher IgG2a antibody production, lower eotaxin, keratinocyte-derived chemokine (KC), IL-5, and IL-13 release in bronchial alveolar lavage fluid, attenuated airway hyper-responsiveness, and promoted higher IL-10 and interferon (IFN)-γ production in the spleen. In summary, our findings elucidate the new role of Dll4 in the phenotype and function of DCs and provide a novel approach for manipulating T cell-driven deleterious immune diseases.