Lack of influenza specific T_CD8+ responses in Batf3^o/o mice.

<p>(A) Pre-gating strategy to identify DC (B–C) DC from the inguinal and mediastinal lymph nodes of wildtype or Batf3^o/o mice on a B6 background were analyzed for their expression of either (B) CD8 and CD205 or (C) CD8 and CD103. Representative plots from 3-pooled mice from two independent experiments are shown. (D–E) B6 and Batf3^o/o mice were infected with PR8. On day 10, the absolute number of influenza specific T cells specific for defined peptide sequences were measured in the spleen (D) or BAL (E). The specific T cell response was elucidated following stimulation without peptides (Nil) or the peptides NP_366–374, PA_224–233, PB1-F2_62–70, or PB1_703–711. Shown is the absolute number of IFNγ⁺ CD8⁺ T cells, calculated using the following equation: cell count x%PI⁻ x%CD8⁺ x%IFNγ⁺. Average is taken from between 5–6 mice per group over two independent experiments and the error shows the SEM.</p

Antigen presentation by DC subsets after influenza infection.

<p>B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8⁺ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c⁺CD8⁻ cells (upper left; left gate) were segregated into CD103⁺CD11b⁻ (upper right; top gate) and CD103⁻CD11b⁺ (upper right; lower gate); and finally CD11c⁻ cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2D^b restricted influenza epitope NP_366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP_366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP_366–374 T cells (5×10⁴) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.</p

PA_224–33 T_CD8+ can be generated in Batf3^o/o mice.

<p>B6 or Batf3^o/o mice were inoculated intraperitoneally with 2.5×10⁶ LPS-treated, PA_224–233 peptide-pulsed B6 bone-marrow-derived DC. 7 days later, the number of PA_224–233 responding T_CD8+ present in the spleen was determined by ICS. Average is taken from 6 mice per group over two independent experiments and the error shows the SEM.</p

Identification and characterization of a novel NY-ESO-1 T_CD8+ epitope.

<p><i>A</i>. PBMCs were collected from patient 8 on day 70 following vaccination with NY-ESO-1 ISCOMATRIX™ vaccine. These cells were cultured with a panel of overlapping NY-ESO-1 18 mer peptides and then tested for responsiveness to each peptide in an ICS assay for IFN-γ. Because the background to control peptides was negligible, the results from individual cultures were plotted as a single combined figure. <i>B, C</i>, T_CD8+ line expanded with NY-ESO-1_79–96 18 mer was tested under FCS-free condition for its reactivity to various HPLC-purified peptides (B) and the minimum peptide NY-ESO-1_88–96 at various peptide concentrations (<i>C)</i>. <i>D</i>, a panel of LCL lines sharing HLA alleles with patient 8 were pulsed with the minimum NY-ESO-1_88–96 peptide, extensively washed, co-cultured with NY-ESO-1_88–96-specific T_CD8+ line and followed with ICS.</p

NY-ESO-1_88–96 is not naturally presented by melanoma cells.

<p><i>A</i>, NY-ESO-1_157–165– and NY-ESO-1_88–96–specific T_CD8+ lines were expanded from PBMCs collected from the previously reported patient 7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#pone.0044707-Davis1" target="_blank">[6]</a> and patient 8 with 18 mer peptides NY-ESO-1_157–174 and NY-ESO-1_79–96 respectively. These T cells were then co-incubated with tumor line (SK-MEL-8) with or without a 48 hr IFN-γ induction (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#s4" target="_blank">Materials and Methods</a></b> for details). The untreated SK-MEL-8 cells were also pulsed with both peptides followed by washing out excessive peptides to serve as a maximum antigen presentation control. Antigen-specific T cell activation was then revealed by tetramer and IFN-γ double staining. Percentage represents antigen-specific, IFN-γ-producing cells amongst total tetramer positive cells (note, the double negative cell population was not included in the percentage calculation). <i>B</i>, the same T_CD8+ lines used in A were also assessed for their affinity by peptide titration. Percentage represents Ag-specific T cells among total CD8⁺ T cells. Similar data were obtained from three similar experiments.</p

NY-ESO-1_88–96 is cross-presented efficiently by DCs from soluble antigen.

<p>In A, MoDCs expressing both HLA-A2 and HLA-B18 were cultured for 7 days, and then incubated overnight under the indicated conditions before being co-cultured with the indicated T_CD8+ lines for 5 hrs in the presence of BFA. NY-ESO-1 specific T_CD8+ activation was assessed by tetramer and ICS. IFN-γ producing cells out of total antigen-specific (tetramer positive) T_CD8+ were converted to percentages of maximum activation induced by the respective minimum peptide (peptide activation of NY-ESO-1_157–174 T_CD8+ line and NY-ESO-1_79–96 T_CD8+ line were both 30% to 45% for all three experiments conducted, data not shown) and plotted as “% Maximum activation”. After data conversion, mean values and standard deviations were calculated from data obtained from three similar experiments. In B, one of the control experiments was shown for APCs that were either pulsed with the corresponding peptide or soluble NY-ESO-1 for one hour followed with BFA addition to demonstrate the nature of intracellular cross-presentation for both T_CD8+ epitopes without affecting extracellular peptide presentation. Similar results were obtained twice.</p

NY-ESO-1_88–96-specific T cells are vaccine boosted and utilize polyclonal T cell receptors.

<p>PBMCs from patient 8 collected before (day 0) and after (day 70) vaccination with NY-ESO-1 ISCOMATRIX™ vaccine were expanded with 18 mer NY-ESO-1_79–96 and the T cells were assessed by ICS (A). A similar T cell line expanded from day 70 PBMC sample from patient 8 was first stimulated with NY-ESO-1_88–96 peptide, then split into multiple wells and stained with anti-CD8 and a panel of antibodies specific to various TCR Vβ families separately, followed with ICS for IFN-γ (B). Graph indicates the percentage of NY-ESO-1_88–96-specific (IFN-γ-producing) T cells expressing the indicated TCR Vβ families.</p

T_CD8+ response to HLA-B18/NY-ESO-1_88–96.

<p>Melanoma patients from three clinical trials (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#s4" target="_blank">Materials and Methods</a>) with detectable anti-NY-ESO-1 antibody responses and HLA-B18 expression were selected for the screen. T cells from PBMC samples post vaccination (or placebo controls that did not receive the NY-ESO-1 ISCOMATRIX™ vaccine but received diluents) were expanded with 18 mer NY-ESO-1_79–96 peptide for 12∼15 days and assessed with NY-ESO-1_88–96 in an ICS assay (only ICS results <0.1% are shown as negative “–” indicated by ‘*’). For patients who showed positive T_CD8+ response to this epitope (>0.1%, data not shown) in their post vaccination samples, pre- and post-vaccination PBMC samples were then expanded the same way side-by-side in a second screen intended to determine whether the response was a result of the vaccination. The peptide-specific T_CD8+ in the second screen were assessed with the specific HLA-B18/NY-ESO-1_88–96 tetramer. Tetramer results >0.1% of total CD8⁺ T cells with a discrete staining pattern are shown; and those results <0.1% are shown as “-”. Pre – pre-existed response; Boosted – vaccine-boosted response; ND – not determined, Pre-vac, prior to vaccination; Post-vac, after vaccination.</p

NY-ESO-1_88–96 is directly presented by tumor cells expressing high level of immunoproteasome.

<p>Five melanoma lines, including SK-MEL-8, were left untreated or treated with either IFN-γ for 48 hrs or were further infected with rVV.NY-ESO-1 for 5 hrs. The tumor cells were then co-cultured with T cell lines specific for NY-ESO-1_157–174, NY-ESO-1_88–96 and Melan A26–35 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#pone-0044707-g003" target="_blank">Figure 3</a>. The purity of the T_CD8+ lines were 42%, 88% and 68% respectively (data not shown). The antigen presentation results are shown in A and the western blot results for LMP2, LMP7 and the loading control GAPDH for the corresponding tumor lines and the treatment conditions are shown in B. The FACS analysis results of the cell surface HLA molecules as Mean Channel Fluorescence intensity (MCF) are shown in C. The MCF values for HLA-A2 and B18 were about 100 for the FITC-conjugated secondary antibody alone; and those values for the All Class I group for the PE-conjugated secondary antibody alone were about 300. Similar data were obtained from three similar experiments.</p