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

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

pMDM100-specific CD8⁺ T cells from Ag^pos mice display reduced functional avidity.

<p>(A) Splenocytes from Ag^neg mice and Ag^pos mice were stimulated with RMA-S cells coated with titrated concentrations of pMDM100 peptide or an irrelevant control peptide, pSV9, in the presence of 10 U/ml IL-2. After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent mean±SD of triplicate values. IFN-γ produced in response to RMA-S loaded with pSV9 was subtracted from all values. Data are representative of two independent experiments. (B) To measure antigen specific proliferation, splenocytes from Ag^neg mice (black squares) and Ag^pos mice (grey circles) were stimulated with RMA-S cells coated with titrated concentrations of pMDM100 peptide or an irrelevant control peptide, pSV9, in the presence of 10 U/ml IL-2 for 2 days, and then pulsed with 0.5 µCi ³[H]-thymidine for 24 hours. The maximal proliferative response by splenocytes from the Ag^neg and Ag^pos mice was 130480.2±9552.9 CPM and 56225.17±2794.9 CPM (triplicate values), respectively.</p

IL-2 and IL-15 efficiently induce Bcl-2 expression and restore the expansion of tolerant MDM2-specific CD8⁺ T cells.

<p>(A) CFSE-labelled splenocytes from Ag^neg and Ag^pos mice stimulated with RMA-S coated with pMDM100 peptide (10 µM) for 72 hrs 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, stained with CD8 and Vβ7 mAbs and subjected to CFSE profiling of gated CD8⁺Vβ7⁺ T cells. (B) 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 and Bcl-2 expression levels were analysed by intracellular staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8⁺Vβ7⁺ population. Following staining of the splenocytes with an appropriate isotype control mAb the MFI of the gated CD8⁺Vβ7⁺ population from the Ag^neg mice and Ag^pos mice was 98 and 97, respectively. Data in A and B are representative of three independent experiments. (C) CD8⁺ T cells isolated from the spleens of Ag^neg and Ag^pos mice were stimulated with RMA-S coated with pMDM100 peptide (10 µM) in the presence or absence of 10 U/ml IL-2 and cell death was determined on the indicated days by staining with annexin-V and propidium iodide and flow cytometric analysis.</p

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