Systemic IL-12 Administration Alters Hepatic Dendritic Cell Stimulation Capabilities

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2–3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections

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

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

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

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

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