Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.

OBJECT: Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. METHODS: Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. RESULTS: The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern. CONCLUSIONS: This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Patterns of de novo allo B cells and antibody formation in chronic cardiac allograft rejection after alemtuzumab treatment.

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.

Interleukin-15 receptor blockade in non-human primate kidney transplantation.

BACKGROUND: Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. METHODS: Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. RESULTS: Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8 T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. CONCLUSIONS: Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8 T cells and natural killer cells in the peripheral blood.

Characterization of B cells in muscle-specific kinase antibody myasthenia gravis.

OBJECTIVE: To characterize B-cell subsets in patients with muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). METHODS: In accordance with Human Immunology Project Consortium guidelines, we performed polychromatic flow cytometry and ELISA assays in peripheral blood samples from 18 patients with MuSK MG and 9 healthy controls. To complement a B-cell phenotype assay that evaluated maturational subsets, we measured B10 cell percentages, plasma B cell-activating factor (BAFF) levels, and MuSK antibody titers. Immunologic variables were compared with healthy controls and clinical outcome measures. RESULTS: As expected, patients treated with rituximab had high percentages of transitional B cells and plasmablasts and thus were excluded from subsequent analysis. The remaining patients with MuSK MG and controls had similar percentages of total B cells and naïve, memory, isotype-switched, plasmablast, and transitional B-cell subsets. However, patients with MuSK MG had higher BAFF levels and lower percentages of B10 cells. In addition, we observed an increase in MuSK antibody levels with more severe disease. CONCLUSIONS: We found prominent B-cell pathology in the distinct form of MG with MuSK autoantibodies. Increased BAFF levels have been described in other autoimmune diseases, including acetylcholine receptor antibody-positive MG. This finding suggests a role for BAFF in the survival of B cells in MuSK MG, which has important therapeutic implications. B10 cells, a recently described rare regulatory B-cell subset that potently blocks Th1 and Th17 responses, were reduced, which suggests a potential mechanism for the breakdown in immune tolerance in patients with MuSK MG.

Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.

The liver kinase B1 is a central regulator of T cell development, activation, and metabolism.

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

Regulatory T cells depress immune responses to protective antigens in active tuberculosis.

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.

HBHA-specific IFN-{gamma} Synthesis at the Site of Infection during Active Tuberculosis in Humans.

RATIONALE: Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVE: The mycobacterial antigen heparin-binding-hemagglutinin (HBHA) induces strong interferon-gamma (IFN-gamma) responses by circulating lymphocytes from Mycobacterium tuberculosis latently infected subjects, and low responses associated with CD4(+) regulatory T (Treg) cells in TB patients. Here, we investigated HBHA-specific IFN-gamma responses at the site of the TB disease. METHODS: Bronchoalveolar lavages, pleural fluids and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary and/or pleural TB. HBHA-specific IFN-gamma production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS: The percentages of HBHA-induced IFN-gamma(+) alveolar and pleural lymphocytes were higher for pulmonary (P<0.0001) and for pleural (P<0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-gamma. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSION: TB patients display differential effector and regulatory T cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared to a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.

Immunological Signatures Identifying Different Stages of Latent Mycobacterium tuberculosis Infection and Discriminating Latent from Active Tuberculosis in Humans

Objectives: One third of the world population is considered latently infected with Mycobacterium tuberculosis(LTBI) and sterilizing this reservoir of bacteria that may reactivate is required for tuberculosis (TB) elimination. Thegroup of individuals with LTBI is heterogeneous with some of them being more at risk to develop TB disease thanothers. Improved diagnosis of subjects with LTBI is needed, allowing to differentiate subjects with LTBI from thosewith active TB, and to select among LTBI subjects those who are more at risk to develop active TB. We havecharacterized at the cellular level both the quantitative and qualitative T cell responses to different mycobacterialantigens in selected populations of infected subjects in order to identify new biomarkers that could help to identify M.tuberculosis-infected subjects and to stratify them in risk groups for reactivation of the infection.Methods: Lymphoblast frequencies and cytokine production (IFN-γ, TNF-α, IL-2) among CD4+ and CD8+ T cellswere analyzed by flow cytometry after in vitro stimulation with the latency antigen heparin-binding haemagglutinin(HBHA) or early-secreted antigen Target-6 (ESAT-6) of peripheral blood mononuclear cells from clinically wellcharacterized M. tuberculosis-infected humans (28 LTBI, 22 TB disease,12 controls). The LTBI group definedaccording to the Center for Disease Control guidelines was subdivided into QuantiFERON-TB Gold in-Tube (QFT)positive and negative subgroups.Results: Similar to TB patients, QFT+ LTBI subjects had higher proportions of HBHA-induced TNF-αsingle+ CD4+lymphocytes than QFT- LTBI subjects (p<0.05). Compared to LTBI subjects, TB patients had higher frequencies ofESAT-6-induced CD8+ lymphoblasts (p<0.001), higher proportions of ESAT-6-induced IFN-γ+TNF-α+ CD4+ Tlymphocytes (p<0.05), and lower proportions of HBHA-induced IFN-γ+TNF-α+IL-2+ (p<0.05) CD4+ T lymphocytes.Conclusions: These data provide new biomarkers to discriminate active TB from LTBI, and more interestingly,help to identify LTBI subjects with increased likelihood to develop TB disease.

Basophil activation tests for the diagnosis of food allergy in children.

BACKGROUND: Positive skin prick tests (SPT) for food allergens and specific IgE (sIgE) in serum indicate sensitization but do not enable distinction between sensitized but tolerant and clinically allergic patients. OBJECTIVE: Herein, we evaluate the clinical relevance of basophil activation tests (BATs) for peanut or egg allergy diagnosis. METHODS: Thirty-two peanut-allergic, 14 peanut-sensitized (sIgE(+) and/or SPT(+) to peanuts) but tolerant children and 29 controls with no history of an adverse reaction to peanuts were included. Similarly, 31 egg-allergic, 14 egg-sensitized children (sIgE(+) and/or SPT(+) to egg white) and 22 controls were studied. Flow cytometric analysis of CD63 expression or CD203c upregulation on basophils and the production of leukotrienes (LT) were performed in response to an in vitro crude peanut extract or ovalbumin (OVA) challenge. RESULTS: After in vitro peanut challenge, the basophils from peanut-allergic children showed significantly higher levels of activation than those from controls (P<0.001). After OVA challenge, a similar distinction (P<0.001) was observed between egg-allergics and controls. Interestingly, the majority of egg- or peanut-sensitized children failed to activate basophils, respectively, in response to OVA and peanut challenge. The sensitivity of the CD63, CD203c and LT assay was 86.7%, 89.5% and 76.0% with a specificity of 94.1%, 97.1% and 94.6% for peanut allergy diagnosis. The corresponding performances of BATs applied to egg allergy diagnosis were 88.9%, 62.5% and 77.8% for the sensitivity and 100%, 96.4% and 96.4% for the specificity. CONCLUSION: Neither conventional tests nor BATs are sensitive and specific enough to predict food allergy accurately. However, BATs may helpfully complete conventional tests, especially SPT, allowing improved discrimination between allergic and non-allergic individuals.

Are the reference values of B cell subpopulations used in adults for classification of common variable immunodeficiencies appropriate for children?

Detailed phenotypic characterization of B cell subpopulations is of utmost importance for the diagnosis and management of humoral immunodeficiencies, as they are used for classification of common variable immunodeficiencies. Since age-specific reference values remain scarce in the literature, we analysed by flow cytometry the proportions and absolute values of total, memory, switched memory and CD21(-/low) B cells in blood samples from 168 healthy children (1 day to 18 years) with special attention to the different subpopulations of CD21(low) B cells. The percentages of total memory B cells and their subsets significantly increased up to 5-10 years. In contrast, the percentages of immature CD21(-) B cells and of immature transitional CD21(low)CD38(hi) B cells decreased progressively with age, whereas the percentage of CD21(low) CD38(low) B cells remained stable during childhood. Our data stress the importance of age-specific reference values for the correct interpretation of B cell subsets in children as a diagnostic tool in immunodeficiencies.

Both CD4+ and CD8+ lymphocytes participate in the IFN-y response to filamentous hemagglutinin from Bordetella pertussis in infants, children and adults

Infant CD4+ T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8 + T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8 + T-cell responses upon Bordetella pertussis infection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γ secretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4 + and CD8 + T lymphocytes are involved in IFN-γ production. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4 + lymphocytes were the major source of this cytokine. IFN-γ synthesis by CD8 + cells was CD4 + T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γ synthesis by CD4 + cells was sometimes inhibited by CD8 + lymphocytes, suggesting the presence of CD8 + regulatory T cells. The role of this dual IFN-γ secretion by CD4 + and CD8 + T lymphocytes in pertussis remains to be investigated. © 2012 Violette Dirix et al.

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e. dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.