Human intrahepatic regulatory T cells are functional, require IL‐2 from effector cells for survival, and are susceptible to Fas ligand‐mediated apoptosis

Regulatory T cells (Treg) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated Treg [PEM Treg]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg. Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of Treg to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138–150)


Analysis of T reg function, plasticity and response to IL-2 in the hepatic microenvironment
To examine T reg suppressive ability, PEM T reg were co-cultured with CellTrace™ violet (Life Technologies)-labelled CD4 + CD25responder T cells (T resp ) at PEM T reg to T resp ratios ranging from 1:1 to 1:8. The Treg Suppression Inspector (Miltenyi Biotec) was added to the culture according to the manufacturer's instruction to stimulate T cell proliferation. T resp proliferation was determined by flow cytometry on day 5.

ELISA
Concentrations of IL-2 in cell culture supernatants recovered from human biliary epithelial cells, primary stellate cells, hepatic sinusoidal endothelial cells and hepatocytes that were unstimulated or stimulated with TNF-α (10ng/ml) or IFN-g (100ng/ml) and supernatants from isolated liver infiltrating CD3 + CD4 + and CD3 + CD8 + T cells that were unstimulated or stimulated with anti-CD3/anti-CD28 Dynabeads (Life Technologies) for 12 hours as well as cytokines IL6, IL8, IL12, IFN-γ, TNF-α, IL15, IL1β and IL-2 in explanted human liver supernatants were measured by ELISAs according to manufacturer's instructions. Liver supernatants were mostly prepared by culturing liver tissue at 1g/4ml RPMI overnight. For detection of IL-2 it was necessary to prepare higher concentration supernatants (1g or 2g tissue/1ml RPMI). Liver supernatants were filtered and remaining debris removed by centrifugation before loading onto the ELISA.

Immunohistochemistry
Immunohistochemistry staining was performed on 5μm formalin-fixed paraffin embedded sections after antigen retrieval. Peroxidase quenching with 0.3% H 2 O 2 in methanol for 30 minutes was performed prior to microwaving at full power for 15 minutes in high pH antigen unmasking solution (Vector Labs). The sections were then washed in PBS and blocked with casein solution for 20 minutes at room temperature in a humidified chamber. Next slides were incubated at room temperature with anti-human CD8 (rabbit polyclonal antibody; 1:100, Abcam), anti-human CK19; b170; 1:100, Vector Labs), anti-human FAS Ligand (rabbit polyclonal antibody; 1:100, Abcam) or anti-FOXP3 (236A/E7; 1:20 or 1:80 (for dual staining) Abcam) for 1hour. The sections were then incubated with secondary antibody (Vector Impress Universal mouse/rabbit Ig kit, Vector Labs) and developed with DAB substrate (Vector labs). Sections for dual staining were then incubated at room temperature with casein solution for 20 minutes followed by anti-human caspase-3 (rabbit polyclonal antibody; 1:200, Abcam) for 1hour. After washing with TBS-0.1%tween, pH8.2 they were incubated with HRP-conjugated secondary antibody (Vector Impress rabbit Ig kit) for 30 minutes and developed with Vector Blue AP substrate (Vector Labs). Finally sections were counter stained with haematoxylin and mounted. Slides were examined using a Zeiss Axioskop40 microscope under 200x and 400x magnifications.

Confocal microscopy
Frozen liver tissue sections were blocked in casein and primary antibodies including antihuman CD8 (1:100, mouse IgG2b, clone (4B11) Vector Labs) and anti-human CK19 (1:100, mouse IgG1, clone b170 Vector Labs) applied for one hour followed by conjugated secondary antibodies AF555 anti-mouse IgG 2b (Life Technology), and FITC anti-mouse IgG 1 (Life Technology) for 30minutes. They were then incubated with DAPI (1μg/ml, Life Technology) to stain the cell nuclei. Between stains sections were washed for 10 minutes with PBS. All incubation steps were performed in a humidified chamber protected from the light. Slides were visualized by confocal microscopy using LSM software (ZEISS).

TUNEL Assay on tissue sections
Apoptosis was detected by in situ end-labeling of fragmented DNA using TACS DAB In Situ Apoptosis Detection Kit (Trevigen, Inc.). Tissue sections were deparaffinized and pretreated with proteinase K solution for 5 minutes at room temperature. Endogenous peroxidase activity was blocked by 0.3 % H 2 O 2 in methanol for 5 minutes. After pre-incubation in labeling buffer for 5 minutes, the specimens were incubated with brominated nucleotide (BrdU) and terminal deoxynucleotidyl transferase enzyme (TdT) for 1hour at 37°C. Following terminating the reaction by stop buffer, the specimens were incubated with anti-BrdU in Strep dilution for 30 minutes at 37°C. Then the sections were reacted with Strep-horseradish peroxidase for 10 minutes. DAB staining and haemotoxylin staining were applied before light microscopic examination.

Multicolour flow cytometry
Fresh LIL from different human diseased livers, normal liver and post endothelial migrated T reg and CD8 were stained for surface markers of interest were phenotyped using APCH7

Analysis of IL-2 production by liver infiltrating CD4 + and CD8 + T cells
LIL were isolated and separate populations of CD3 + CD4 + and CD3 + CD8 + T cells isolated by fluorescence activated cell sorting with a Moflo Astrios cell sorter (Beckman Coulter) after staining with BV421-anti-human CD3 (UCHT1, BD biosciences), Viogreen anti-human CD4 (VIT4, Miltenyi Biotec) and PerCPVio700 anti-human CD8 (BW135/80, Miltenyi Biotec). Cells were plated at 1x10 6 cells/ml for 12hours in a round-bottomed 96-well plates and stimulated with or without anti-CD3/anti-CD28 coated Dynabeads (Invitrogen) at 4cells/bead. Supernatants were collected at 12hours and stored at -20°C for analysis of IL-2 cytokine production by ELISA using the human IL-2 ELISA Ready-Set-Go kit (eBioscience) according to manufacturer's instructions. After removal of supernatants for measurement of IL-2 production the volume was replaced with fresh culture medium and cells stimulated with or without PMA and ionomycin in the presence of Brefeldin A for 4 hours. Frequencies of IL-2 producing cells were determined by flow cytometry. Cells were fixed with 3% formaldehyde after staining for dead cells with the fixable viability dye eFluor 506 (eBioscience) then stained for intracellular IL-2 using APC anti-human IL-2 (APC, eBiosciene) in 0.1% Saponin solution. Data were acquired using a CyAn flow cytometer and analysed using Flowjo (Tree Star) software.