Decreased antigen uptake by splenic and hepatic DCs following systemic IL-12 treatment.

<p>Leukocytes from IL-12 or vehicle control treated mice were incubated with 100 µg/ml DQ-OVA for 30 min at 37°C and analyzed by flow cytometric analysis gating on cDC (NKp46⁻CD11c⁺Class II⁺) and pDCs (NKp46⁻CD11c⁺Class II⁺mPDCA-1⁺) populations in the spleen (A) and liver (B). Left hand shows a representative histogram sample for an individual mouse displaying the antigen uptake and processing of DQ-OVA by cDC or pDCs (solid line). The shaded line is the respective isotype control. The MFI of a representative sample is shown in the upper right corner of each histogram. The right side graphically displays the mean ± SD of the DQ-OVA MFI from an independent experiment with 3–5 mice/group that has been repeated 3 times with similar results. *, <i>p</i><0.05; Mann-Whitney U test.</p

Increased splenic and hepatic DC numbers following systemic IL-12 treatment.

<p>BALB/c mice were treated with 1 µg/mouse IL-12 (solid square) or vehicle control (VC; open triangle) for 4 consecutive days from day 0, as indicated with arrows, then leukocyte populations analyzed at various days post treatment. The total DC number (NKp46⁻CD11c⁺ Class II⁺), is shown for the spleen (A) and for the liver (B). The results shown are the mean ± SD from 3–5 mice/group at each time point and representative of 3 separate experiments. (C). Differences in DC subsets were examined in the spleen and liver at day 4 for CD11b⁺ (NKp46⁻CD11c⁺Class II⁺CD11b⁺), CD8α⁺ (NKp46⁻CD11c⁺Class II⁺CD8α⁺), and pDCs (NKp46⁻CD11c⁺Class II⁺B220⁺mPDCA-1⁺SiglecH⁺), respectively. Shown is the mean ± SD from 4–5 mice/group and representative of more than 3 independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; Mann Whitney U test.</p

Enhanced T cell proliferation from hepatic DCs following systemic IL-12 treatment.

<p>(A) Differential HA TCR proliferation of naïve splenic and hepatic DC. Purified DC were peptide pulsed and incubated with 2×10⁴ HA-TCR T cells at a 1∶1 ratio for 72 hours and 1 µCi of [³H]thymidine added during the last 18 hours of culture. (B) Allogeneic mixed lymphocyte reaction. Purified splenic and hepatic DCs from BALB/c mice (H-2^d) were cultured at differing DC∶T cell ratios with 1×10⁵ purified T cells from C57BL/6 mice (H-2^b) for 72 hours with 1 µCi of [³H]thymidine added during the last 18 hours of culture. (C) HA TCR Tg model. Purified splenic and hepatic DCs were pulsed with varying doses of HA peptide for 2 hours then cultured with 2×10⁴ HA-TCR T cells at a 1∶1 ratio for another 72 hours. 1 µCi of [³H]thymidine was added to each well during the last 18 hours of culture. Shown is the mean ± SD done in quadruplicates from one of three independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; *** <i>p</i><0.001; Student's T test.</p

Hepatic T cells are differentially modulated compared to splenic T cells following IL-12 therapy in RENCA tumor bearing mice.

<p>(A) Schematic of the treatment cycle for orthotopic RENCA tumor bearing mice, where mice were injected with 1×10⁵ RENCA cells into the kidney capsule then treated with two cycles of IL-12 or VC injections. (B) Mice were euthanized on day 13 following treatment and the weight of the primary tumor was recorded. The activation of CD4 and CD8 T cells (CD3⁺DX5⁻) from the spleen and liver of VC (white) and IL-12 (black) treated RENCA tumor-bearing and VC (grey) and IL-12 (striped) treated non-tumor bearing mice is shown through expression of CD69 (C) and CD44high (D) Shown is the mean ± SEM from two independent experiments with 5 mice/group in each experiment. Purified splenic and hepatic T cells pooled from 2–5 mice/group from RENCA tumor-bearing mice were co-cultured with irradiated RENCA at a 10∶1 ratio for 96 hours followed by IFNγ detection E) and IL-10 (F) in the culture supernatants. The bar graphs represent the means ± SD assayed in triplicate. Shown is a representative experiment that has been repeated twice with similar results. NS not significant, **p<0.01; *** p<0.001; Mann Whitney U test.</p

Altered cytokine expression profile of splenic and hepatic DCs.

<p>Bulk lymphocytes from the spleen and liver were incubated with media alone (no treatment; no Tx), 25 µg/ml poly(I:C), 1 µg/ml LPS or 2.5 µg/ml CpG for 18 hours. Splenic DC (A) and hepatic DC (B) populations were gated and intracellular IL-12p40 expression examined by flow cytometric analysis. Shown is a representative sample from an individual mouse in an independent experiment repeated 3 times (3–5 mice/group). Purified splenic and hepatic DC pooled from 5–15 mice were cultured under the same conditions and culture supernatants harvested 48 hours later. Cytometric bead array was performed to detect in the supernatants the levels of IL-12p40 (D), IL-12p70 (D) and TNF (E) from splenic DC and hepatic DC. The bar graph displays the mean ± SEM expressed in pg/ml per 10⁶ cells derived from the pooled data obtained from 3 independent experiments. *, <i>p</i><0.05 (Mann Whitney U Test).</p

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

<div><p>We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.</p></div

CD40/IL-2 Immunotherapy induces massive expansion of bystander memory CD8+ cells and anti-tumor effects are CD8 dependent.

<p>Three C57BL/6 mice per group were treated with IT or PBS/rIgG (control) and effects on CD8+ T cell expansion were quantified by flow cytometric analysis 11 days after the initiation of therapy. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. Six to eight 3LL bearing mice were treated with IT and/or CD8+ T cell depletion to examine CD8+ dependence of anti-tumor effects. (<b>a</b>) Gating strategy for bystander memory CD8+ CD44^high NKG2D+ CD25− cells. (<b>b–e</b>) Expansion of bystander memory CD8+ T cells in the spleen and lymph nodes of IT or vehicle treated mice expressed as total numbers (<b>b,c</b>) or as a percentage of total CD8+ T cells (<b>d,e</b>). Effects of IT and/or CD8 depletion on tumor growth (<b>f</b>) and survival (<b>g</b>).</p

Anti-tumor effects of IT in CD4 knockout mice.

<p>3LL tumor bearing WT or CD4 knockout (B6.129S2-CD4^tm1Mak/J) mice were treated with IT or PBS/rIgG (control) and survival and tumor growth were measured. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. (<b>a</b>) Survival. (<b>b</b>) Mean tumor volume with SEM. (<b>c–f</b>) Growth plots of individual tumors in each group. N = 12 mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

PD-1 expression on central and effector memory CD8+ T cells after IT in CD4+ T cell depleted mice.

<p>Control or CD4+ depleted C57BL/6 mice were treated with IT or PBS/rIgG (control). CD62L and PD-1 expression on memory T-cells was quantified by flow cytometric analysis 11 days after the initiation of IT. (<b>a,b</b>) Examples of the gating strategy for PD-1 expression on the CM and EM components of the memory CD8+ T cell compartment. The majority of cells in the memory compartment are CM in untreated mice (<b>a</b>) and EM in IT treated mice (<b>b</b>). (<b>c</b>) PD-1 expression on CM and EM cells. (<b>d</b>) The composition of the memory CD8+ T cell compartment in control or CD4 depleted mice treated with IT or PBS/rIgG. PD-1+ CM cells (<b>e</b>) and PD-1+ EM cells (<b>f</b>) as a percentage of the total memory CD8+ T cell compartment. Results are representative of two to three independent experiments with 3 mice per group (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001, ****<i>P</i><.0001).</p

Memory CD8+ T cell function after IT in CD4+ T cell deficient models.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and assessed for function of memory CD8+ T cells 11 days after the initiation of IT. NKG2D and granzyme B expression were quantified by flow cytometric analysis. Interferon gamma production was quantified by flow cytometric analysis after <i>in vitro</i> restimulation of splenocytes with PMA/Ionomycin (0.16/1.6 ug/ml) for one hour followed by incubation with golgi stop (0.7 ug/ml) for three hours. CD8+ T cell killing function was assayed by scintillation counting using an <i>in vitro</i> redirected lysis assay with ⁵¹Cr labeled P815 target cells incubated for 30 minutes with 10 ug/mL anti-CD3e. (<b>a,f</b>) NKG2D expression on memory CD8+ T cells in CD4 depletion (<b>a</b>) and knockout (<b>f</b>) models. Representative dot plots for NKG2D+ CD25− gating are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102709#pone-0102709-g001" target="_blank">Figure 1</a>. (<b>b</b>) Representative dot plots for IFNγ+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>c,g</b>) Interferon gamma production by memory CD8+ T cells in CD4 depletion (<b>c</b>) and knockout (<b>g</b>) models. (<b>d</b>) Representative dot plots for Granzyme B+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>e,h</b>) Granzyme B expression by memory CD8+ T cells in CD4 depletion (<b>e</b>) and knockout (<b>h</b>) models. Killing function of splenocytes from CD4 depleted (<b>i</b>) or CD knockout (<b>j</b>) mice expressed as percentage of maximal lysis. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p