DN Helios expression in activated CD8 T cells confers a competitive advantage for transduced cells cultured in IL-12.

<p>Data indicate the percentage of CD8 T cells that are transduced from day 2 to 8, in either IL-15 (20 ng/mL) (A) or IL-12 (5 ng/mL) (B). Cells were transduced with either control (gray filled circles) or DN Helios-expressing retrovirus (open triangles), with retrovirally transduced cells expressing GFP. Data are from two independent experiments. Note that the difference between the control and DN-Helios retroviruses at day 2 reflects a difference in the titers of the retroviral stocks used for transduction.</p

CD8 T cells expressing dominant negative Ikaros have a modest advantage in vivo.

<p>(A) Relative abundance of retrovirally transduced cells within a pool of adoptively transferred CD8 T cells containing both transduced and non-transduced cells. Adoptively transferred cells were identified as live, CD8+ MHC class II negative cells that expressed CD45.1, with retrovirally transduced cells further expressing Thy1.1, with abundance measured within the peripheral blood of C57BL/6 recipient mice (which do not express CD45.1 or Thy1.1). Cells were transduced with a control retrovirus (white bar) or a DN Ikaros expressing retrovirus (black bar). To standardize for slightly different starting frequencies, values are standardized relative to day 1 input values for either control or DN Ikaros transduction efficiencies with data showing mean ± SEM with 3–10 mice for control transduced and 6–10 mice for DN Ikaros transduced cells. Mice were bled on day 1, 7, and 39 post-transfer. Statistically significant differences (p<0.05) are indicated, calculated by unpaired t test. (B) Fold change in the abundance of retrovirally transduced cells in C57BL/6J recipient mice from panel A (either control or DN Ikaros transduced) in the blood (defined as the percentage of live, CD8+ MHC class II negative cells that were CD45.1+ Thy1.1+) following injection of either vehicle alone or recombinant IL-12 (1 µg) at day 162 and 165 post-adoptive transfer, with abundance post-treatment measured at day 168 post-transfer. For these studies, at day 1 after adoptive transfer, the frequency of adoptively transferred, retrovirally transduced cells (defined as CD45.1+ Thy1.1+) among CD8 T cells in the peripheral blood was 1.21±0.07% for control and 1.70±0.09% for DN Ikaros transduced cells (mean ± SEM). Among adoptively transferred cells (CD45.1+), the percentage (mean ± SEM) of transduced cells was 42.1±0.7% for control and 49.6±0.5% for DN Ikaros.</p

Immunization with PR8’s NP and alum primes protective immunity to NY1682.

<p>B6 mice were injected i.m. in both hind-legs with PBS (closed triangles) or a total of 10 µg of NP and 100 µg of alum with (open squares) or without (closed squares) 10 µg of MPL. At least 70 days later, these animals were infected with NY1682 i.n. The mice were weighed daily and the percent of original weight of each mouse calculated for each day. The data are combined from two experiments with 4–5 mice per group (A). These mice were bled one day prior to infection with NY1682 or 5 days following infection. The relative units of NP specific IgG1 and IgG2c present in the serum were determined using a NP specific ELISA (B). The percentages (C) or numbers (D) of D^b/NP_366–74 tetramer+ cells present in one lung lobe of these and naïve control animals were examined. In A, significant differences between the PBS control mice infected with NY1682 and those first immunized with PR8’s NP and alum are indicated with *(p<0.05) and **(p<0.01). No significant differences between PBS control mice and those first immunized with PR8’s NP and alum and MPL were found. In C, cells were gated on live CD8+ lymphocytes that were dump negative. Representative plots are shown from 1 experiment with 4 mice per group with numbers in the plot indicating the percentages of D^b/NP_366–74 tetramer+ out of gated live CD8+ cells. In D, each point represents a mouse and the line shows the mean of the group. The X-axis is set at the level of background staining.</p

Gene expression profiles of Ikaros family members in mature CD8 T cells derived from Gene Expression Omnibus repository (GEO) datasets.

<p>(A) Gene expression profile of human CD8 T cell subsets, including naïve (TN), effector memory (TEM) or central memory CD8 T cells (TCM) with data from GSE23321. (B) Gene expression profile of mouse CD8 T cell subsets, including naïve, effector (Eff) or memory CD8 T cells (Mem) with data from GSE10239. Differential regulation of murine Ikzf1 and Ikzf2 gene expression in P14 cells after LCMV infection with data from GSE10239. Eff expression values are the combined average of array data from sorted Klrg1int or Klrg1hi-expressing P14 cells 4.5 days after LCMV Armstrong infection as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057435#s2" target="_blank">Methods</a>. To allow comparison of microarray hybridization intensities across different samples, microarray data were normalized using standard normalization techniques, either the RMA16 (A) or RMA (B) algorithms to normalize two different generations of Affymetrix microarrays. For both human and mouse datasets, all Ikaros family members are significantly expressed above background (DABG p-values <0.05). Gene names refer to the following gene products: Ikzf1, Ikaros; Ikzf2, Helios; Ikzf3, Aiolos; Ikzf4, Eos; Ikzf5, Pegasus.</p

The NP amino acid sequence from PR8 and NY1682 influenza viruses differ in known epitopes.

<p>Sequence alignment for NP proteins from PR8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone.0061775-Grimm1" target="_blank">[46]</a> and NY1682 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone.0061775-Zhou1" target="_blank">[20]</a>. The IA^b and D^b binding peptides are highlighted in red and green respectively.</p

NY1682 infection does not prime T cells specific for immunodominant epitopes from PR8’s NP.

<p>B6 mice were infected (bottom) or not (top) with NY1682 i.n. and 25 days later the percentages of D^b/PA_224–38, D^b/NP_366–74 CD8 T cells, or IA^b/NP_311–25 CD4 in the MLN were examined (A). The numbers are the percentages of tetramer+CD44^hi cells out of gated CD8+ or CD4+ live cells that were also dump negative. The serum from these animals was tested for the presence of IgG1 and IgG2c antibody that bound to recombinant PR8 NP with each line representing one mouse (B).</p

The NP specific memory T cell response is similar regardless of the adjuvant combination used.

<p>The numbers of memory NP specific memory CD8 (A, B) or CD4 (C, D) T cells were determined in the spleens (A, C) and popliteal lymph nodes (B, D) of B6 mice immunized with NP protein and alum and MPL or alum at least 70 days previously. Cells were gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone-0061775-g001" target="_blank">Figure 1</a>. The data are combined from two independent experiments with 4 mice per group indicated by each point, the line shows the mean of the group. The X axis is set at the level of detection. n.s. not significant. The expression of memory markers on either NP specific CD8 (E) or CD4 (F) T cells was examined in the spleen (CD8 T cells) or popliteal lymph nodes (CD4 T cells) at least 70 days after immunization. Cells are gated on naive/CD44 low CD8 or CD4 T cells (filled histogram) or on D^b/NP_366–74 tetramer positive CD8 memory T cells or IA^b/NP_311–25 tetramer positive CD4 memory T cells from mice immunized with NP protein and alum (black dashed line) or alum and MPL (red line). Tetramer positive cells were gated on live CD8 or CD4 positive cells that were negative for B220, F4/80, MHC II and either CD4 or CD8 respectively. The data show representative plots from 1–2 experiments with 4 mice per group.</p

CD8 T cells expressing DN Ikaros have enhanced expression of CD25 (IL-2 receptor alpha chain) in an IL-2 independent manner.

<p>(A) Relative abundance of CD25 in CD8 T cells determined by flow cytometry, analyzing cells that were mock transduced (gray), transduced with control vector (blue, Thy1.1+ cells in MiT transduced cultures) or with DN Ikaros retrovirus (red, Thy1.1+ cells in MiT-DN Ikaros transduced cultures). Cells were cultured in mIL-2 (20 ng/mL) (top), mIL-12 (5 ng/mL) (middle) or no exogenous cytokine (bottom). (B) DN Ikaros expression does not enhance CD69 expression in activated CD8 T cells cultured in mIL-12 (5 ng/mL). Data representative of two (IL-2) to five (IL-12) independent experiments. (C) Anti-IL2 antibody effectively neutralizes IL-2 in vitro, as measured by decreased cell size (forward scatter, FSC) or decreased CD25 following culture of activated CD8 T cells in IL-2 (20 ng/mL), comparing treatment with either an isotype control antibody (black) or an anti-IL2 antibody (gray). (D) DN Ikaros-transduced cells cultured in IL-12 (5 ng/mL) have enhanced CD25 expression in an IL-2 independent manner. Data show CD25 expression in DN Ikaros-transduced cultures at day 4 or 6 of culture, in cells treated with isotype control (black) or anti-IL2 antibody (red), relative to cultures treated with an isotype antibody (gray); results representative of two independent experiments.</p

NP protein delivered with alum provides optimal protection from influenza A infection.

<p>B6 mice were immunized i.m. in both hind-legs with PBS (closed triangles) or a total of 10 µg of NP protein delivered alone (closed diamonds), or with 10 µg of MPL (closed circles), 100 µg alum (closed squares), or both adjuvants (open squares). At least 70 days later, these animals were infected with PR8 influenza A i.n. Mice were weighed daily and the percent of original weight calculated (A) or the amount of virus present in one lung lobe examined 4–5 days after infection (B). Data are combined from two separate experiments with 4–5 mice per group. In A, significant differences between the PBS control mice infected with PR8 and the experimental groups are indicated by the following symbol on the indicated days post-infection. Significant differences in mice immunized with NP and alum are indicated by **(p<0.01) and ***(p<0.001). Significant difference in mice immunized with NP and MPL are indicated by ## (p<0.01). Significant differences in mice immunized with NP and alum and MPL are indicated by $(p<0.05),$$(p<0.01),$$$ (p<0.001). No significant difference between control PBS mice and those immunized with NP protein were found. In B, * = p<0.05.</p

NP protein delivered with alum primes a specific T and B cell immune response.

<p>B6 mice were immunized i.m. in both hind-legs with a total of 10 µg of NP protein delivered with or without 100 µg of alum. The percentages (A, B) or numbers (C, D) of D^b/NP_366–74 CD8 or IA^b/NP_311–25 CD4 T cells were examined in the spleen or the two popliteal lymph nodes (DLN) 9 days later. Cells are gated on CD8+dump negative live cells (A) or CD4+dump negative live cells (B) with the number in the plot indicating the percentage of CD8 or CD4 cells that are CD44^hi tetramer+ as indicated by the gate. In C and D each point represents a mouse and the line shows the mean of the group. The X axis is set at the level of detection, determined by staining cells from naïve animals with the MHC tetramers. Serum from these animals was used to examine the level of NP specific IgG1 antibody (E), with each line representing one animal. These data are combined from 2 independent experiments with 3 mice per group.</p