CD4⁺ T cell signals delivered during priming and recall phase are required for optimal secondary responses.

<p>(<b>a</b>) A schematic protocol. After 90 days following immunization, total CD8⁺ T cells containing memory CTLs were purified from WT (helped memory CTLs) mice and adoptively transferred equally to the naïve secondary recipients, WT and MHCII^−/− mice (∼15×10⁶/mouse). The MHCII^−/− mice were additionally reconstituted with different types of CD4⁺ T cells (∼15–20×10⁶/mouse) along with CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse). Both groups were boosted with AdVova and assessed for memory CTLs expansion. (<b>b</b>) Naïve B6.1/OTII CD4⁺ T cells were co-cultured with irradiated BM DCova, as detailed in material and methods to generate Th1 cells. Th1 cells were then transferred to naïve congenic WT mice (∼10–15×10⁶/mouse). After 45 days, these cells were triple stained and phenotypically characterized for the expression of activation or memory markers (grey-shaded area) as shown in the figure. Irrelevant isotype-matched Abs were used as control (dotted thin lines). The value in the dot plot indicates % of OVA-specific CD4⁺ T memory cells remaining in total CD4⁺ T cell population. One representative of the two independent experiments is shown. (<b>c</b>) Naïve MHCII^−/− mice were adoptively transferred with helped memory CTLs with naïve OTII T cells (∼1.5×10⁶/mouse), polyclonal CD4⁺ T cells (∼15–20×10⁶/mouse) or polyclonal CD4⁺ T cells containing OVA-specific memory CD4⁺ T cells (∼15–20×10⁶/mouse) and CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047004#pone-0047004-g006" target="_blank">Fig. 6a</a>. Three days later, all the groups were boosted with AdVova and the recall potential of the memory CTLs were assessed 6.5 days later. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD8⁺ T cell population and are representative of two independent experiments with four to five mice per group. *<i>P</i><0.05 or **<i>P</i><0.01, versus MHCII^−/− mice with helped memory CTLs alone.</p

CD4⁺ T cell signals provided during priming and recall phase are required for optimal secondary responses.

<p>(<b>a</b>) A schematic protocol. After 90 days of immunization, total CD8⁺ T cells containing memory CTLs were purified from WT B6 mice with helped CTLs or MHCII^−/− mice with unhelped CTLs, adoptively transferred in equal numbers into the naïve secondary recipients, WT and MHCII^−/− mice (∼15×10⁶/mouse), and assessed for recall potential after boosting. (<b>b</b>) Three days after adoptive transfer of helped or unhelped memory CTLs into naïve WT and MHCII^−/− mice, all the mice groups were boosted with AdVova and monitored for the expansion of memory CTLs 6.5 days later. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD8⁺ T cell population and are representative of two independent experiments with five to six mice per group. **<i>P</i><0.01, versus MHCII^−/− mice with helped or unhelped memory CTLs.</p

Molecular mechanisms of CD4⁺ T-helper signals required for functional AdV-specific memory CTL responses^a.

a<p>One day prior to immunization, MHCII^−/− mice were adoptively transferred with CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse) and naïve polyclonal (∼15–20×10⁶/mouse) or OTII CD4⁺ T cells (∼1.5×10⁶/mouse) with or without designated gene deficiency, as indicated. 120 days later, all the immunized mice were challenged with BL6-10ova tumor cells. Twenty-four days after the challenge, lung tumor colonies were counted and graded. The data are cumulative of two independent experiments, each comprising five to six mice per group.</p

CD4⁺ T cells impact the kinetics of AdV transgene-specific CTL populations.

<p>Following immunization, AdVova-specific CTLs were analyzed in the peripheral blood at different time points by tetramer (<b>a</b>) and intracellular IFN-γ (<b>b</b>) stainings. The values are presented as mean%±SD of OVA-specific tetramer⁺ CTLs (<b>a</b>) or IFN-γ⁺ CTLs (<b>b</b>) in total CD8⁺ T cell population and are representative of two to three independent experiments with three to four mice per group. **<i>P</i><0.01, versus MHCII^−/− mice. (<b>c</b>) Ten days following immunization, the proportions of CFSE^high-OVAI-pulsed target cells lysed by effector CTLs were determined in the spleens by <i>in vivo</i> cytotoxicity assay. The values represent mean %±SD of targets remaining in spleens relative to controls and are representative of two independent experiments with three to four mice per group. (<b>d</b>) On day 75, following immunization, OVA-specific memory CTLs were characterized in spleen for PD-1 expression by flow cytometry. A representative figure from immunized groups along with matching isotype control is shown on the left. The values in the bar diagram represent the mean %±SD of PD-1⁺ tetramer⁺ CTLs in total tetramer⁺ CTL population and are representative of two independent experiments with 3 to 4 mice per group. **<i>P</i><0.01, versus WT mice.</p

Polyclonal CD4⁺ T cells support maintenance of AdVova transgene product-specific CTLs.

<p>(<b>a</b>) Ten days following immunization, total CD8⁺ CTLs containing AdVova-specific effector CTLs were purified from B6.1 mice (CD45.1⁺ background) and adoptively transferred to naïve congenic WT and MHCII^−/− mice (CD45.2⁺ background; ∼15×10⁶/mouse). The OVA-specific tetramer⁺ CTLs were tracked after staining peripheral blood samples with tetramer reagent and congenic marker up to 30 days post-adoptive transfer. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD45.1⁺ adoptively transferred T cell population at the indicated intervals (right panel) and are representative of two independent experiments with five to six mice per group. **<i>P</i><0.01, versus MHCII^−/− mice. (<b>b</b>) Forty-five days after adoptive transfer, the above mice groups were challenged with BL6-10_OVA. Twenty-four days after the challenge, both groups were assessed for tumor protection. Images represent distorted pathology of lungs, showing relative surface tumor burden. (<b>c</b>) Ninety days following the immunization, total CD8⁺ T cells containing AdVova-specific memory CTLs were purified from WT mice and adoptively transferred into naïve WT and MHCII^−/− mice (∼15×10⁶/mouse). The OVA-specific tetramer⁺ CTLs were tracked in peripheral blood samples by tetramer staining. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD8⁺ T cell population at the indicated intervals (right panel) and are representative of two independent experiments with four to six mice per group. **<i>P</i><0.01, versus MHCII^−/− mice. (<b>d</b>) Thirty days after the adoptive transfer, the above mice groups (<b>c</b>) were challenged with BL6-10_OVA and the relative surface tumor burden was assessed 24 days after the challenge as detailed above.</p

AdVova stimulates persistent CD4⁺ and CD8⁺ T cell responses.

<p>Following immunization, the AdVova-specific CTLs (<b>a</b>) and CD44⁺CD4⁺ T cells (<b>b</b>) in the peripheral blood and/or spleens were analyzed by flow cytometry at the indicated time points after tetramer staining, and CD44 and CD4 double marker staining, respectively. The values in the figure (left panel) or line diagram (right panel) are presented as mean%±SD of OVA-specific CD8⁺ CTLs in total CD8⁺ T cell population (<b>a</b>) or of CD44⁺ CD4⁺ T cells in total CD4⁺ T cell population (<b>b</b>), and are cumulative of three independent studies with three to five mice per group. (<b>c</b>) Following immunization, the spleen samples were analyzed for OVA-specific CD4⁺ T cells by intracellular IFN-γ staining at the indicated intervals. The values (% frequencies) are cumulative of two independent experiments with three to four mice per group. *<i>P</i><0.05, versus matching controls.</p

Molecular mechanisms of cognate Th-delivered help responsible for CTL survival and recall responses.

<p>(<b>a</b>) DCova(K^b–/–)-stimulated Th(pMHC-I^-/-) cells were stained with anti-pMHC I Ab and DCova-stimulated Th(CD40L^-/-) were stained with anti-CD40L Ab (solid thick lines; bottom panels) using DCova-stimulated Th cells as a positive control (solid thick lines; top panel). Stained cells were analyzed by flow cytometry. Irrelevant isotype-matched Abs were used as control (dotted thin lines). Value in each panel represents the percentages of positive staining cells versus the control. One representative of two independent experiments is shown. (<b>b</b>) Both Th and Th(IL-2^-/-) cells were re-stimulated with OVA_323-329-pusled LB27 cells, and IL-2 secretion was assessed in the culture supernatants. The values represent mean%±SD of IL-2 secreted in supernatant and are cumulative of two independent experiments performed in triplicates. (<b>c</b>) Approximately 5x10⁶ effector CTLs were i.v. transferred with or without Th, Th(CD40L^-/-), Th(IL-2^-/-) or Th(pMHC I^-/-) (2x10⁶) cells to CD4⁺ T cell-deficient mice, and analyzed for survival 6 and 60 days later, and recall responses on the 4^th day after boosting. The values represent frequencies of tetramer⁺ CTLs in total CD8⁺ T-cell population, and are cumulative of three independent studies with three to four mice per group. The horizontal bars indicate means. * or **, <i>p</i> <0.05 or 0.01, respectively, versus Th cell-helped CTLs in Ia^b-/- mice. (<b>d</b>) Approximately 5x10⁶ effector CTLs alone that received help during priming period or 5x10⁶ effectors CTLs with or without 2x10⁶ Th cells (i.e., help after CTL priming) were i.v. transferred to WT and Ia^b-/- mice. In addition, some Ia^b-/- mice groups were transferred with effectors CTLs with or without effector Th, Th(IL-2^-/-), Th(CD40L^-/-) or Th (pMHC-I^-/-) cells (2x10⁶each) as indicated. After 100 days, all the mice were challenged i.v. with highly metastasizing BL6-10_OVA tumor cells. On 24^th day of tumor challenge, the numbers of metastasized tumor colonies in lungs were counted and presented as mean%±(SD). The values represent number and percentage of tumor-bearing mice. The data are cumulative of three independent experiments with three to four mice per group. Note: The data of all PBS-injected control mice, which showed >100 tumor colonies, is not shown. (<b>e</b>) Assessment of OVA-specific IFN-γ⁺ CTLs in the spleen of tumor-challenged mice. Approximately 5x10⁶ effector CTLs with Th cells (2x10⁶) or without Th cells were adoptively co-transferred into WT or CD4⁺ T cell-deficient mice. Hundred days later, all the groups were challenged with BL6-10_OVA tumor cells. On 24^th day of tumor challenge, the spleen samples were analyzed by intracellular IFN-γ⁺ staining to assess CTL-mediated tumor protection. The data represent cumulative frequencies of ova-specific IFN-γ⁺ CTL in total CD8⁺ T cell population. **, <i>p</i> < 0.01 versus unhelped CTLs in Ia^b-/- mice. One representative of the three independent experiments with three to four mice per group is shown.</p

Th cell help provided during the priming or transitional period enhances effector CTL survival and transition to memory development.

<p>(<b>a</b>) Experimental Design. To determine direct impact of Th cells on effector CTL fates after priming, effector CTLs and Th cells with or without specific gene deficiency were adoptively transferred to WT or CD4⁺ T cell-deficient mice. All mice were monitored for CTL survival and memory CTL recall responses during the memory phase. Memory recall responses were assessed by boosting with DCova 60 days following the adoptive transfer or by challenging with highly metastasizing BL6-10_OVA 100 days post-transfer. (<b>b</b>) Approximately 5x10⁶ effector CTLs alone that received help during the priming period or 5x10⁶ effectors CTLs without or with 2x10⁶ Th cells (i.e. help after CTL priming) were i.v. transferred into WT or Ia^b-/- mice as indicated. Six and sixty days later, the transferred CTLs were monitored in the tetramer assay. On the 60^th day, all the groups were boosted with 1x10⁶ DCova and monitored for memory CTL expansion 4 days later. The values represent mean%±(SD) of tetramer⁺ (Ova-specific) CTLs in total CD8⁺ T cell population, and are cumulative of three independent experiments with three to five mice per group. * or **, <i>p</i> < 0.05 or 0.01, respectively, versus unhelped CTLs. (<b>c</b>) Influence of Th cell numbers on effector CTL survival and memory CTL development. Approximately 5x10⁶ effector CTLs alone (none) or 5x10⁶ effector CTLs together with 2x10⁶ (low) or 5x10⁶ (high) Th cells were adoptively transferred to WT or CD4⁺ T cell-deficient mice and assessed 60 days later in the peripheral blood by tetramer assay. The values represent mean%±SD of tetramer⁺ CTLs in total CD8⁺ T cell population, and are cumulative of two independent experiments with four to five mice per group. * or **, <i>p</i> < 0.05 or 0.01, respectively, versus the low Th cell dose. (<b>d</b>) Blood samples from WT mice transferred with effector CTLs and Th cells or from CD4-deficient mice transferred with effector CTLs alone were collected 6 and 60 days post-transfer, respectively. The samples were processed for tetramer staining along with staining with a panel of biotin-conjugated Abs specific for effector or memory markers, and streptavidin-PE-Texas Red and PE-Cy5. PE-tetramer-positive CD8⁺ T cells in the circles (panels b and e and panels j and m) from WT and Ia^b-/- mouse groups were further assessed for expression of memory CTL markers such as CD44, CD62L and IL-7R or inhibitory PD-1 molecule (histogram grey filled overlays), respectively. Irrelevant isotype-matched Abs were used as control (dotted thin lines). Value in each panel represents the percentages of positive staining cells versus the control. One representative of two independent experiments is shown.</p

The provision of Th help causes distinct changes in expression of genes that regulate apoptosis, favoring effector CTL survival.

<p>(<b>a</b>–<b>c</b>) Apoptosis pathway-focused gene expression in helped versus unhelped CTLs. Total RNA from purified population of helped or unhelped CTLs was isolated, reverse transcribed to cDNA, and subjected to PCR array. Heat map showing relative gene expression in helped or unhelped CTLs where intensity of color towards red indicates up-regulation and green indicates down-regulation. One representative figure from each group in two independent experiments is shown. Each sample was run on duplicates using pooled cDNA samples derived from two to four mice per group. (<b>d</b>–<b>e</b>) Statistically significant changes in gene expression (at least three-fold mRNA difference compared to naïve OTI CD8⁺ T cells), up- (elevated-top) or down-regulated (reduced-bottom) and their overlap between helped or unhelped CTLs purified from CD4⁺ T cell-sufficient or CD4⁺ T cell-deficient mice are shown, as indicated. See Tables S2a and S2b for gene-expression data. (<b>f</b>) Validation of PCR array results by qRT-PCR. cDNA samples from helped or unhelped CTLs used for PCR array were subjected to qRT-PCR using SYBR Green detection protocol. mRNAs up- or down-regulated in helped or unhelped CTLs purified from CD4⁺ T cell-sufficient or CD4⁺ T cell-deficient mice are shown as indicated.</p

Immunoblot analysis of the expression and phosphorylation status of pro-survival and pro-apoptotic proteins.

<p>(<b>a</b>) Lysates prepared from helped or unhelped CTLs obtained from WT or CD4⁺ T cell-deficient mice were subjected to SDS-PAGE, and transferred to the nitrocellulose membrane. Western blotting was performed with a panel of Abs specific for β-actin, Bcl-2, Bcl10, Akt1, NF-κB-p65, phosphorylated-Akt1 and -NF-kB-p65, cleaved Caspases-3 and -7, or NFATc1 transcription factor and analyzed by the ODYSSEY densitometer. Densitometric values were normalized on the β-actin control and n-fold changes of normalized targets in the helped CTLs of WT or Ia^b-/- mice relative to the unhelped CTLs in Ia^b-/- mice are shown below the corresponding lanes. Data are derived from samples pooled from four to six mice in each group of the first experiment. The results of the second experiment are consistent with the first experiment (data not shown). One representative of two independent experiments is shown. (<b>b</b>) Representative FACS plots of helped CTLs from WT or CD4-deficient mice, or unhelped CTLs from CD4-deficient mice. Blood samples from helped CTLs in WT and Ia^b-/- mice and unhelped CTL in Ia^b-/- mice were collected 16 days of adoptive transfer and processed for triple staining. Value in panel a, b and c represents percentage of tetramer⁺-and CD8⁺-specific double-positive cells in the total of CD8⁺ T cell population. The tetramer⁺-and CD8⁺-specific double-positive cell populations were gated (in the circle) for analysis of TRAIL expression. Histogram overlays (right panels) of CD8⁺ tetramer⁺-gated helped CTLs from WT (histogram green filled overlays; right top panel) or CD4-deficient mice (histogram red filled overlays; right bottom panel) and CD8⁺ tetramer⁺-gated unhelped CTLs from CD4-deficient mice (histogram grey filled overlays; right top and bottom panel). The mean of fluorescence intensity (MFI) was calculated from triplicate values. The value in each panel represents the mean of MFI ± SD of positive staining cells (grey) versus the controls with MFI of 0.5 for green and MFI of 0.8 for red, respectively. One representative of two independent experiments is shown.</p