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

MDM2-specific CD8⁺ T cells are present in the periphery of Ag^pos mice and express reduced levels of TCR and CD8 but high levels of CD44.

<p>(A) Lymph nodes cells from 6–8 week old Ag^pos and Ag^neg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8⁺Vβ7⁺ cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.</p

MDM2-specific T cells from Ag^pos mice display defects in antigen-driven expansion.

<p>(A) CFSE-labelled splenocytes from Ag^neg and Ag^pos mice were stimulated with RMA-S coated with pMDM100 peptide (10 µM) or pSV9 peptide (10 µM) for 48 hrs, stained with CD8 and Vβ7 mAbs and subjected to CFSE profiling by flow cytometry of gated CD8⁺Vβ7⁺ T cells. Data are representative of at least three independent experiments. (B) To measure antigen-specific IL-2 production, splenocytes from Ag^neg mice and Ag^pos mice were stimulated with RMA-S cells coated with pMDM100 peptide (100 µM) or a control peptide, pSV9 (100 µM). After 72 hours, 50 µl of culture supernatant was harvested and murine IL-2 was measured in a CTLL bioassay. Data represent the mean±SD of triplicate values and are representative of two independent experiments. (C) CFSE-labelled splenocytes from Ag^neg mice were stimulated with RMA-S cells coated with pMDM100 or SV9 control peptides for 72 hrs. In the bottom panel, CFSE-labelled T cells were stimulated for 72h with pMDM100 in the presence of unlabelled splenocytes from Ag^pos mice. (D) CFSE-labelled splenocytes from Ag^pos mice were stimulated with RMA-S cells coated with pMDM100 or SV9 control peptides for 72 hrs. In the bottom panel, CFSE-labelled T cells were stimulated for 72h with pMDM100 in the presence of unlabelled splenocytes from Ag^neg mice.</p

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

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