Box and whisker plots of the distribution of basal cytokine production by CD4⁺ T cell cultures.

<p>The bottom and top of the box are the 25^th (Q1) and 75^th percentile (Q3), the lower and upper quartiles, respectively, and the band near the middle of the box is the 50^th percentile (the median). The ends of the whiskers represent the smallest datum still within 1.5 the Interquartile Range (IQR = Q3−Q1) of the lower quartile, and the largest datum still within 1.5 IQR of the upper quartile. Outliers are represented as black dots.</p

The amount of IFN-γ released by CD4⁺ T cells correlates with the number of positive wells.

<p>Healthy donors (HD) #8 and #9 were chosen as representative for donors with different amplitude of IFN-γ release by viral Ag-stimulated CD4⁺ T cells. (A) 1×10⁵/well CD4⁺ T cells were cultured with autologous APCs in the absence (6 wells) and in the presence (6 wells) of the EBNA2 peptide. IFN-γ release was measured at days 14 (white columns) and 21 (grey columns) of culture on supernatant pools of the 6 wells. Values are the means of duplicate determinations ± SEM. (B) After a further week of culture (d21), EBNA2-stimulated CD4⁺ T cells were collected, washed and tested for EBNA2-specific recognition by challenge with un-pulsed or peptide-pulsed autologous APCs and IFN-γ specific release was measured after 48 hours. Values are the means of triplicate determinations ± SEM. Responses significantly higher than the blanks (<i>i.e.</i>, no Ag  =  CD4+APC) are indicated as ***p<0,001 (determined by unpaired, one-tailed Student’s <i>t</i> test). (C) CD4⁺ T cells from the same two donors were cultured plating two seeding densities, <i>i.e.</i> 3×10⁴ cells/well (5 un-stimulated wells and 20-stimulated wells) (upper panels) and 1×10⁴ cells/well (10 un-stimulated wells and 60 stimulated wells)(lower panels). At day 14, IFN-γ secretion was measured as above but on single well supernatants. Dots are means of duplicate determinations.</p

Mean, standard deviation (after discarding of the outliers) and threshold for IFN-γ and IL-5 concentration at day 14 in the un-stimulated wells.

a<p>n.p., not performed.</p

Schematic representation of the culture conditions set for estimate of Ag-specific CD4⁺ T cells frequency.

<p>CD4⁺ T cells were purified from total PBMCs and seeded in 96-well plates at 3×10⁴/well in the presence of irradiated autologous APCs (1∶3 ratio) for a total of 0.9×10⁶ CD4⁺ T cells for each condition, <i>i.e</i>., in the absence (−Ag) or in the presence (+Ag) of the relevant peptide. At day 7, low dose IL-2 was added without further Ag stimulation. At day 14, single-well cytokine release (IFN-γ and IL-5) was measured by ELISA.</p

Point and interval frequencies of Ag-activated cytokine-secreting CD4⁺ T cells in the EBNA-stimulated wells.

a<p>Lower, punctual and upper frequencies are indicated as the number of IFN-γ and IL-5 secreting cells/10⁶ total CD4 T cells.</p>b<p>n.p., not performed.</p

Point and interval frequencies of Ag-activated cytokine-secreting CD4⁺ T cells in the HA-stimulated wells.

a<p>Lower, punctual and upper frequencies are indicated as the number of IFN-γ and IL-5 secreting cells/10⁶ total CD4⁺ T cells.</p>b<p>n.p., not performed.</p

The <i>in vitro</i> re-stimulation culture used does not induce priming of naïve CD4⁺ T cells.

<p>Total CD4⁺ T cells were purified from the blood of adult healthy donors and put in culture (1×10⁵/well) in several replicates in the absence (no Ag = CD4+APCs) or in the presence of the primary Ag KHL. PHA stimulation was used as positive control. At days 14 (d14) and 21 (d21) supernatants were collected and groups of six wells were pooled and IFN-γ and IL-5 release quantified by ELISA. For ³H-thymidine incorporation assays data from each well are shown.</p

Characterization of CEA_{177–189/355–367} specific CD4⁺ T cell clones from pt#15.

<p><i>Profile of cytokine secreted (A)</i>. CD4⁺ T cells were cultured with irradiated APC in the presence or the absence of the relevant peptide in a 2-day stimulation assay and the concentration of the indicated cytokines in the supernatants was determined either by CBA (upper panel) or ELISA (lower panel). The data are representative of at least six experiments; for ELISA assays, the data are means of duplicate determination±SD. <i>HLA restriction (B)</i>. CD4⁺ T cells were cultured with irradiated autologous PBMC or HLA-DR matched LCL, as indicated, in the presence or the absence of the relevant peptide (10 µg/ml) and in the absence or the presence of anti-HLA-DR, anti-HLA-DP and anti-HLA-DQ mAbs: after 2 days IL-5 was tested. Upper panel: % inhibition was calculated based on IL-5 secretion by CD4⁺ T cells in the presence of the relevant peptide (2 ng/ml over 0 ng/ml of background level). The data are representative of two (upper panel) and four (lower panel) experiments and are means of duplicate determination±SD. Responses significantly higher than the blanks (<i>i.e.</i>, the basal levels of cytokines secretion from CD4⁺ T cells in the presence of LCL only) are indicated as: ***, p<0.001 (determined by unpaired, one-tailed Student's t test). <i>Dose-response curves (C)</i>. CD4⁺ T cells were cultured with titrated doses of the relevant peptide in the presence of irradiated APC in a 2-day stimulation assay and tested for IL-5 and IL-13 release. The data are means of duplicate determination±SD. <i>Recognition of the native protein (D)</i>. CD4⁺ T cells were cultured in the presence of irradiated PBMC, as APC, pulsed with either the relevant peptide (10 µg/ml) as positive control, or the purified CEA protein (30 µg/ml) or human IgG (30 µg/ml) as negative control in a 2-day stimulation assay and tested for IL-13 release. The data are means of duplicate determination±SD. Responses significantly higher than the blanks (<i>i.e.</i>, the basal levels of cytokines secretion from CD4⁺ T cells in the presence of PBMC only) are indicated as: **, 0.001TCRVβ expression (E). The test was performed with the IO Test Beta Mark kit. Quadrants were set based on isotype control staining. <i>Surface expression of Th2 (CRTH2 and CCR4) and Th1 (CCR5) markers (F)</i>. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.</p

Effect of titrated doses of IL-12 or IL-27 as single agent on the repertoire of cytokine secreted by CEA_{177–189/355–367} specific CD4⁺ T cells.

<p><i>(A).</i> CD4⁺ T cells were cultured with the relevant peptide and irradiated LCL as APC in the presence of increasing concentrations of IL-27 (0-0,1-1-10-100 ng/ml); after 2 days the cytokine release was evaluated by CBA (IL-5 IL-4, and IFN-γ) or ELISA (IL-13). The data for IL-13 are means of duplicate determination±SD. <i>(B).</i> CD4⁺ T cells were cultured and tested, as described above, in the presence of increasing concentrations of IL-12 (0-5-20 ng/ml). The basal level of cytokines secretion of CD4⁺ T cells in the presence of LCL only was subtracted from the sample values and was comprised between 0 and 0,018 ng/ml. The data are representative of at least three experiments.</p

Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4⁺ T cells.

<p>CD4⁺ T cells from pt#43 and ND#11 were cultured in five replicates, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007234#s2" target="_blank">Materials and Methods</a>, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.</p