Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Monoclonal T-Cell Receptors: New Reagents for Cancer Therapy

Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate “undifferentiated” T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells

A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

BACKGROUND: Most tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-γ, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-γ production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR. CONCLUSIONS/SIGNIFICANCE: These data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2

Exploiting coreference annotations for text-to-hypertext conversion

The paper describes an annotation scheme for coreference developed within the application context of text-to-hypertext conversion. In this context coference is used (1) for generating document-internal and cross-document hyperlinks, and (2) for resolving anaphoric expressions in order to achieve cohesive closedness in hypertext nodes. We will argue that for the purpose of cross-document linking it is necessary to separate the annotation of coreference relations from the annotation of anaphoric relations. To account for this requirement, we developed a knowledge-based annotation scheme that relates referential expressions in the text to entities in a knowledge representation, which is modeled using XML Topic Maps. 1. Project Framework Converting linear text documents into documents that can be published in a hypertext environment is a complex task requiring conversion software on the technical side as well as conversion strategies and methods on the conceptual side. In the project HyTex1, which is the framework of the approach discussed in this paper, we concentrate on principles and strategies for handling conceptual problem

CTL from Ag^pos mice display defects at the level of cytotoxic effector function that can most effectively be rescued by IL-2.

<p>Splenocytes from Ag^neg mice and Ag^pos mice were stimulated for 3 days <i>in vitro</i> with pMDM100-coated targets in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. (A) CD43 expression levels on the stimulated T cells was analysed by surface staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8⁺Vβ7⁺ population. (B) Granzyme B expression levels on the stimulated T cells was analysed by intracellular staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8⁺Vβ7⁺ population. Following staining with an appropriate isotype control mAb the MFI of the gated CD8⁺Vβ7⁺ T cells from the Ag^neg mice and Ag^pos mice was 398 and 495, respectively. Data in A and B are representative of three independent experiments. (C) Cytolytic activity of stimulated T cells against MDM2-expressing MBL-2 tumor cells and RMA-S targets coated with pMDM100 peptide (10 µM) or a class I binding control peptide, pSV9 (10 µM). Data are representative of two independent experiments.</p

Induction of unresponsiveness limits tumor protection by adoptively transferred MDM2-specific cytotoxic T lymphocytes

There is evidence showing that high avidity CTLs can be more effective than low avidity CTLs for adoptive tumor immunotherapy. Because many T cell-recognized tumor antigens are nonmutated self-proteins, tolerance mechanisms are likely to render high avidity T cells unresponsive or cause T cell elimination by clonal deletion. We recently used the allo-restricted strategy to circumvent immunologic tolerance to a ubiquitously expressed tumor-associated protein, MDM2, and raised high avidity CTLs in humans and in mice. In this study, we investigated whether high avidity MDM2-specific CTLs can mediate tumor protection without causing damage to normal tissues in mice. Although the CTLs prolonged survival of tumor-bearing mice without causing damage to normal tissues, tumor protection was incomplete. We show that tumor growth occurred despite the continued presence of MDM2-specific CTLs and the continued susceptibility of tumor cells to CTL killing. However, analysis of the CTLs revealed that they had been rendered unresponsive in vivo because they did not produce interferon γ in response to antigen-specific stimulation. These experiments suggest that induction of unresponsiveness may be an important mechanism limiting the efficacy of adoptive CTL therapy

pMDM100-specific CD8⁺ T cells from Ag^pos mice display defects in IFN-γ effector function that can be restored by IL-2.

<p>(A) To measure antigen-specific IFN-γ production, splenocytes from Ag^pos mice and Ag^neg mice were stimulated with RMA-S cells coated with pMDM100 peptide (10 µM) or a control peptide, pSV9 (10 µM). After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent the mean±SD of triplicate values. (B) Antigen-specific IFN-γ production by splenocytes from Ag^pos mice stimulated with pMDM100 peptide or a control pSV9 peptide in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent the mean±SD of triplicate values. Data in A and B are representative of three independent experiments. (C) Antigen-specific IFN-γ production upon secondary peptide stimulation of IL-2 rescued T cells was measured by intracellular staining for IFN-γ. T cells were re-stimulated with RMA-S cells coated with pMDM100 (10 µM) or an irrelevant control peptide, pSV9 (10 µM), for 5 hours in the presence or absence of 10 U/ml IL-2, followed by intracellular staining for IFN-γ. Comparable results were obtained when IFN-γ production by IL-2 rescued T cells was measured by ELISA in two independent experiments.</p