Stromal Interferon-γ Signaling and Cross-Presentation Are Required to Eliminate Antigen-Loss Variants of B Cell Lymphomas in Mice

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60–70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80–100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches

The metastasis-associated gene MTA3 is downregulated in advanced endometrioid adenocarcinomas

The metastasis-associated gene MTA3 has an important function in invasion and metastasis of human cancer cells. Therefore, the aim of this study was to investigate the expression of this protein in endometrial adenocarcinomas and to analyse potential correlations between this nuclear transcription factor and estrogen receptors in endometrial adenocarcinomas. Additionally, we evaluated whether MTA3 might be a prognostic parameter in endometrioid adenocarcinomas. Endometrioid adenocarcinomas were obtained from 200 patients and immunohistochemically analysed for MTA3 and estrogen receptor alpha and beta (ER-alpha and ERbeta) expression. Overall, endometrioid adenocarcinomas of histological differentiation grade 3 demonstrated a significantly lower expression of MTA3 compared to carcinomas of histological grade 1 and 2 (p<0.05). MTA3 expression is reduced in endometrioid adenocarcinomas of poor differentiation, though without any correlation to ER-alpha and ER-beta expression. Furthermore, the expression of MTA3 did not affect progression-free, cause-specific and overall survival. Overall, MTA3 did not constitute an independent prognostic factor in this study, suggesting that MTA3 is not a useful marker to assess and identify high-risk patients with endometrial adenocarcinomas. Still, the downregulation of MTA3 predispose this cell type to be of high metastatic potential after malignant transformation, playing an essential, but as yet unknown role in human endometrial carcinogenesis

IFN-γ responsiveness of lymphoma cells is not required for antigen-dependent lymphoma rejection.

<p>IFN-γ-receptor-deficient λ-huMYC lymphoma cells, either retrovirally transduced (50OVA) (solid line) or untransduced (50PC) (dashed line), were injected s.c. into wild-type mice. STAT1-deficient mice inoculated with retrovirally transduced 50OVA cells served as positive control (dotted line). Mice were monitored for survival (left panel) and cumulative tumor growth (right panel). The data are combined from 2 independent experiments.</p

Host IFN-γ and host IFN-γ signaling are required for rejection of 291OVA cells.

<p>1×10⁵ 291 parental cells (291PC) and retrovirally transduced 291OVA cells were injected s.c. into IFN-γ-deficient (A) and into IFN-γ-receptor- and STAT1-deficient recipient mice (B). Survival (left panels) and corresponding cumulative tumor growth (right panels) were monitored over 100 days. The data are compiled from two independent experiments. (C) Lymphomas developing in IFN-γ-receptor- and STAT1-deficient mice after inoculation of 1×10⁵ 291 parental or 291OVA cells were analyzed by immunohistochemistry for infiltration of CD3-positive cells (peroxidase brown staining) and perforin expressing cells (alkaline phosphatase staining) (left panel) in the same fashion as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#pone-0034552-g002" target="_blank">Figure 2C</a>. 10 animals per group were analyzed and Mann Whitney test was used for comparison.</p

Inoculation of OVA-expressing lymphoma cells into wild-type mice elicits an OVA-specific CD8+ T cell response.

<p>(A) 1×10⁵ CD90-selected splenic T cells of mice challenged with 291OVA cells were restimulated with peritoneal macrophages (APC, antigen presenting cells) in the presence or absence of SIINFEKL peptide (p = 0.020) or with 291 parental (291PC) or OVA-expressing lymphoma cells (291OVA, p = 0.021, Mann-Whitney test, n = 5 animals/group). (B) T cell responses against OVA were monitored at different time points by flow cytometry using SIINFEKL-specific pentamers in the peripheral blood of wild-type mice inoculated with 291OVA cells. The percentage of SIINFEKL pentamer-positive CD8+ T cells increased over time as shown for one representative mouse (B, left panel). Mean values (+/− standard deviation) of percentage of SIINFEKL pentamer-positive CD8+ T cells increased over time as compiled from 5 mice (B, right panel). (C) After inoculation of 1×10⁵ 291 parental or 291OVA cells, the developing lymphomas were analyzed by immunohistochemistry for infiltration of CD3-positive (peroxidase brown staining) and perforin expressing (alkaline phosphatase red staining) cells (C, left panel). CD3− and perforin-positive cells were quantified in blinded fashion by counting 10 high power fields (HPF, 400×) per section. Values are given as a mean ± standard error of the mean (C, right panel). Only CD3-positive and CD3/perforin-double positive cells (CD3/HPF) were regarded as T cells, whereas single perforin-positive cells were considered to be NK cells and excluded from the analysis. For each group 8–15 sections were analyzed. Mann Whitney-U test was used for comparison.</p

Transfer of OVA-expressing tumor cells into wild-type mice results in loss of antigen and induction of MHC on lymphoma cells.

<p>(A) Systemically growing lymphomas isolated from the spleen after s.c. inoculation of 291GFP or 291OVA cells were analyzed for GFP expression as a marker for antigen expression in comparison to the inoculated cell lines. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s1" target="_blank">Introduction</a> of either GFP alone (291GFP) or OVA-IRES-GFP (291OVA) into lymphoma cells resulted in loss of or strong decrease in GFP expression in outgrowing lymphomas after transfer into wild-type mice (left panel, n = 6). Outgrowing lymphomas in GFP-transgenic recipients (tolerant for GFP) likewise lost GFP expression after inoculation of 291OVA cells, but retained the antigen when 291GFP cells had been inoculated (right panel). Flow cytometric histogram inlets show representative examples. Grey lines represent GFP expression at the time of injection, black lines after harvest of lymphomas from spleen. (B) Left: western Blot and RT-PCR analysis (arbitrary units, A.U.) of lymphomas harvested after s.c. inoculation of 291 OVA cells from either OVA tolerant (actOVA) or wild type (WT) recipients. In contrast to WT recipients OVA tolerant actOVA animals did not select for antigen (OVA) loss variants. Middle: Explanted lymphomas were cocultured with unprimed OT-I cells (1∶1 ratio). OVA positive lymphomas from actOVA recipients activated OT-I T-cells to secrete large amounts of IFN-γ (ELISA of supernatant, n = 4, Mann Whitney test) whereas OVA negatively selected lymphomas form wild type recipients did not stimulate OT-I cells. Right: Coculture of OT-I T-cells with lymphoma cells dervived from actOVA or wild type recipients resulted in induction of the T-cell activation marker CD69 expansion of CD8+ (OT-I) cells and reduction of the number of GFP expressing cells CD19+ lymphoma cells. Representative analysis from lymphoma cells harvested from individual mice. (C) Inoculation of 291OVA cells, but not of 291PC cells, into wild-type (n = 5) and GFP-transgenic mice (n = 5) significantly induced MHC class I (left panel) and class II (right panel) in outgrowing lymphoma cells. The strong increase in MHC class I and II expression in outgrowing lymphomas depended on the presence of OVA in the lymphoma inoculum (Mann Whitney test). Fold induction was calculated by comparing MHC expression on freshly explanted lymphoma cells with the corresponding cell line on the same day.</p

OVA and GFP serve as foreign antigens and mediate rejection of the λ-huMYC lymphoma cell line 291 in wild-type mice.

<p>(A) 291PC cells were transduced with retroviruses expressing IRES-GFP or OVA-IRES-GFP. Fluorescence activated cell sorting for GFP resulted in a purity of 96 to 98% GFP-positive cells, respectively (left panel of A). Expression of OVA was confirmed by Western blot analysis (A, right panel). (B and C) 1×10⁵ λ-huMYC 291PC (parental cells) lymphoma cells (dashed line), retrovirally transduced with IRES-GFP (291GFP) (dotted line), or OVA-IRES-GFP (291OVA) (solid line) were injected s.c. into either wild-type (B) or into GFP-transgenic UBI-GFPtg mice (C). Overall survival (left panels) and tumor growth (right panels) were monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s4" target="_blank">Material & Methods</a> (data are compiled from 3 independent experiments).</p

Defective cross-presentation in cells of the recipient impairs T cell response and enhances lymphoma growth.

<p>Recipient animals were T cell depleted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s4" target="_blank">Materials and Methods</a> and T cell depletion was continued for 28 days. Wild-type (WT) or bm1 mutant recipient mice received 1×10⁵ 291OVA lymphoma cells s.c. together with 1×10⁶<i>in vivo</i> primed OT-I cells. (A) Development of an OVA-specific T cell response is impaired in bm1 recipients. Peripheral blood of animals inoculated with 1×10⁵ 291OVA and 1×10⁶ OT-I cells were analyzed by flow cytometry for the presence of CD90.1-positive cells (OT-I) on the days indicated. Numbers of OT-1 T cells are expressed as percentage of lymphocytes in the peripheral blood. In wild-type mice (solid line, n = 10) adoptively transferred OT-I cells expand more readily (Mann Whitney test) than in bm1 recipients (dashed line, n = 9, p = 0.001 for day 8–27). (B) Disease-free survival and cumulative tumor growth after lymphoma transfer: bm1 recipient mice (dashed line) developed tumors significantly faster and cumulative lymphoma growth was enhanced (right panel). (C) Mean fluorescence of GFP in lymphomas arising in wild-type mice or bm1 mice in the presence and absence of OT-I cells. T cell depletion in the absence of OT-I cells (WT no OT-I, n = 9) resulted in preservation of antigen expression, while adoptive transfer of OT-I cells (WT+OT-I, n = 5) led to selection of antigen-negative lymphoma cells.</p

Reference Measurement Procedures For Alzheimers Disease Cerebrospinal Fluid Biomarkers: Definitions And Approaches With Focus On Amyloid 42

Cerebrospinal fluid (CSF) biomarkers for Alzheimers disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid 42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid 42. © 2012 Future Medicine Ltd

Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid beta42.

Item does not contain fulltextCerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid beta42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid beta42.1 augustus 201