Selective Expansion of Allogeneic Regulatory T Cells by Hepatic Stellate Cells: Role of Endotoxin and Implications for Allograft Tolerance

Anil Dangi, Tina L. Sumpter, Shoko Kimura, Donna B. Stolz, Noriko Murase, Giorgio Raimondi, Yoram Vodovotz, Chao Huang, Angus W. Thomson and Chandrashekhar R. Gandhi
J Immunol April 15, 2012, 188 (8) 3667-3677; DOI: https://doi.org/10.4049/jimmunol.1102460

Abstract

Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines and expressing Ag-presenting and T cell coregulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally occurring immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4+ T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs upregulated Fas (CD95) expression on conventional CD4+ T cells and induced their apoptosis in a Fas/Fas ligand-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell–cell contact and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80, and CD86 and stimulated the production of IL-1α, IL-1β, IL-6, IL-10 and TNF-α by HSCs. Interestingly, production of IL-1α, IL-1β, IL-6, and TNF-α was strongly inhibited, but that of IL-10 enhanced in LPS-pretreated HSC/Treg cocultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxin-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs and reducing inflammation and alloimmunity.

The liver receives 75–80% of its blood supply from the portal vein that conveys food-derived and microbial products, Ags, endotoxins, and xenobiotics and plays a major defense role against potentially harmful factors on a continuous basis. This function is performed by the various hepatic cell populations in a coordinated manner. Despite its repertoire of immunologically active cell types (1, 2), the liver demonstrates a remarkable capacity to impart immune tolerance, as evidenced by viral persistence (e.g., hepatitis B and C), parasite infection (e.g., malaria parasite), and tumor metastasis. Liver tolerance is exemplified by the spontaneous acceptance of hepatic allografts across MHC barriers in animal models (3, 4) and human liver transplants (5, 6). Specific interactions between hepatic cell types and those of the immune system may determine the short- and long-term fate of liver allografts. Thus, donor liver-derived passenger leukocytes (microchimerism) can subvert antidonor T cell responses (7–9). Other liver nonparenchymal cells can also exhibit tolerogenic properties (2). Thus, although hepatic dendritic cells (DCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs) have been studied extensively in this regard (10, 11), many unanswered questions remain concerning mechanisms underlying the tolerogenic properties of liver nonparenchymal cells.

Hepatic stellate cells (HSCs), which are located in the perisinusoidal space (Space of Disse), have been investigated extensively for their role in liver fibrosis (12–14). Under physiological conditions, HSCs are quiescent and store most of the body’s retinoids (∼80%). During liver injury, they undergo activation characterized by the loss of retinoids and transdifferentiation into highly proliferative and fibrogenic myofibroblast-like cells. The role of HSCs in hepatic immunobiology, and in particular transplant immunology, is understudied, despite their ability to produce an array of cytokines/chemokines (14–16) and their expression of Ag-presenting and coregulatory molecules (17–21). Fully activated, IFN-γ–stimulated HSCs can inhibit allogeneic effector T cells (17) and promote IL-2–dependent expansion of regulatory T cells (Tregs) (19). Activated HSCs can also prevent DC-induced activation/proliferation of CD8+ T cells via a CD54-dependent mechanism (20). However, in the healthy donor liver, HSCs are quiescent or may be activated transiently following graft reperfusion (22, 23). Because the endothelial barrier is disrupted during cold ischemic preservation and reperfusion (I/R) of the graft (24, 25), there is direct contact between the cells in the hepatic sinusoids and perisinusoidal HSCs. Thus, the role of quiescent or transitionally activated HSCs in immune regulation may be of importance in the control of alloimmunity following liver transplantation.

We have shown previously (26–28) that LPS stimulates the synthesis of proinflammatory mediators, such as IL-6 and TNF-α, by both quiescent and transitionally activated HSCs. Circulating LPS levels are increased in liver disease and following liver transplantation and remain elevated until graft function is fully restored (29). We hypothesized that LPS-stimulated quiescent or transitionally activated HSCs might regulate the hepatic immune response by influencing allogeneic CD4+ (conventional and regulatory) T cells. We found that HSCs induced Fas ligand (FasL)-mediated death of allogeneic conventional CD4+ T cells, but caused MHC class II-dependent expansion of naturally occurring allogeneic Tregs. The latter effect was augmented when HSCs were pre-exposed to LPS. Coculture with Tregs resulted in augmentation of IL-10 secretion and suppression of production of the proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α by LPS-pretreated HSCs. These data suggest that HSCs may play an important role in immune regulation early after liver transplantation.

Materials and Methods

In vivo exposure to LPS and cold ischemic preservation of livers

Experimental protocols were approved by the University of Pittsburgh Animal Care and Use Committee, in accordance with National Institutes of Health guidelines. Male C57BL/6 (H-2b) (B6) and BALB/c (H-2d) mice (6–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Livers of B6 mice were perfused in situ with cold Belzer University of Wisconsin (UW) solution for 5 min via the portal vein and then harvested and stored in UW solution for 18 h at 4°C. The livers were then reperfused with 30 ml medium containing 16 × 106 purified bulk B6 or BALB/c CD4+ T cells via the portal vein (3 ml/min). The inferior vena cava was ligated below the liver and the perfusate collected from the suprahepatic vena cava. Retention of infused cells in the livers was determined by their enumeration in the outflow perfusate using a hemocytometer (30). In some experiments, B6 mice were treated with LPS (1 mg/kg; i.p.) 6 h prior to perfusion. Control livers, following cold ischemic preservation, were reperfused with medium without cells. The liver lobes were cryopreserved for immunostaining.

HSC isolation

B6 mouse livers were perfused in situ through the inferior vena cava with 30–40 ml HBSS (without Ca2+) and then digested with 30–40 ml HBSS (with Ca2+) containing collagenase type IV (0.25 mg/ml; Worthington, Lakewood, NJ) and protease (0.50 mg/ml; Sigma-Aldrich, St. Louis, MO) (26, 27). The cell suspension was filtered through 100-μm nylon mesh. Following removal of hepatocytes and cell debris by low-speed centrifugation (50 × g; 2 min), HSCs were purified by Histodenz density gradient centrifugation and suspended in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, and 10% horse serum. Cells were seeded in gelatin-caoted (0.1% in PBS) plates at a density of 0.5 × 106/cm2, and 20 min later, loosely adherent HSCs were harvested and reseeded in new 6- or 96-well flat-bottom plates. Cell purity, as assessed by vitamin A autofluorescence and desmin immunostaining, was >95%. Purity was further confirmed by FACS analysis (LSR Fortessa; BD Biosciences, San Jose, CA) using anti-CD68 (KCs), anti-CD11b (KCs and DCs), and anti-CD11c (DCs and NK cells) Abs (AbD Serotec, Oxford, U.K., or BioLegend, San Diego, CA) (Supplemental Fig. 1).

Phenotypic characterization of HSCs

Cultured HSCs incubated in the absence or presence of 100 ng/ml LPS for 24 h were harvested using trypsin/EDTA, suspended in DMEM, washed with PBS (2% BSA), and stained with the following Abs: MHC class I, MHC class II, CD40, CD80, CD86, ICAM-1, VCAM, B7-H1, and FasL (BioLegend). The cells were then fixed in 4% paraformaldehyde on ice for 20 min, permeabilized with 0.1% saponin for 30 min at room temperature, and stained with rabbit polyclonal anti-desmin Ab (Abcam, Cambridge, MA) at 1:200 dilution, followed by goat anti-rabbit IgG (H+L)-Pacific Blue secondary Ab (Invitrogen-Molecular Probes, Eugene, OR) at 1:400 dilution. In some experiments, expression of intracellular cytokines IL-6, IL-10, or TNF-α was also determined. The stained cells were detected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.1 (Tree Star, Ashland, OR).

T cell isolation and purification

A single-cell suspension of splenocytes of BALB/c mice was treated with 0.84% NH4Cl to lyse RBCs, and then T cells were enriched using a sterile nylon wool fiber column according to the manufacturer’s protocol (Wako, Osaka, Japan). T lymphocytes were stained for CD3, CD4, CD8, CD25 (BioLegend), CD127 (eBioscience, San Diego, CA), and 7-aminoactinomycin D (7-AAD; BD Pharmingen). Stained cells were acquired using a BD FACSAria (BD Biosciences). Dead cells were stained with 7-AAD for exclusion, and the viable CD3+CD8− cells with the following compositions were sorted: 1) bulk CD4+ T cells (CD4+CD25+/− including Tregs); 2) conventional CD4+ T cells (CD4+CD25− only); and 3) natural Tregs (CD4+CD25highCD127low/−) (31). Following sorting, individual cell populations were tested for their purity, which was always >99%. Nearly 95% or more of the sorted Tregs were found to be positive for Foxp3 (data not shown).

Immunofluorescence microscopy

HSCs seeded on coverslips in coculture with Tregs or liver sections (placed on glass slides) were stained with rabbit anti-desmin (Abcam), rat anti-Foxp3 (Alexis), rabbit anti-Ki67 (Abcam), rat anti-CD4, or rat anti-mouse CD31 Abs (BioLegend). Following primary staining, goat anti-rabbit Alexa Fluor 488, goat anti-rat Alexa Fluor 594, or donkey anti-rat Cy3 were used as secondary Abs, whereas DAPI was used as a nuclear stain. Coverslips or sections were mounted using gelvatol. The slides were placed at 4°C overnight before microscopic examination (25).

Coculture of HSCs with allogeneic CD4+ T cells

Overnight-cultured HSCs were treated with 270 μM GdCl3 (Sigma-Aldrich) for 24 h to block the activity of contaminating KCs, if any, and then washed and stimulated with LPS (100 ng/ml) for 24 h. The cells were then washed and cocultured in fresh medium with CFSE-labeled bulk CD4+ T cells, conventional CD4+ T cells, or Tregs (at 1:10 ratio in 96-well flat-bottom plates). CFSE labeling was carried out as described previously (32). Polymyxin B (300 ng/ml; Sigma-Aldrich) was added to the cultures to block the direct effect of any residual LPS on CD4+ T cells. In some experiments, HSCs were cultured in inserts (0.45-μm pore size) and separated from Tregs placed in the bottom portion of the wells (BD Falcon) to determine whether their influence on T cells was due to soluble mediators or required cell–cell contact. Apoptosis was assessed by staining with 7-AAD and Annexin-V, whereas cell proliferation was measured by CFSE dilution assay (32). In some experiments, blocking Abs (anti-FasL, anti–I-A/I-E) or the costimulation blocking agent CTLA4-Ig were added at the start of cultures.

Quantification of cytokines

Cell-free supernatants were collected to measure concentrations of cytokines using a mouse 20-plex Luminex beadset (Biosource International, San Diego, CA) and Luminex 100 IS apparatus (Luminex, Austin, TX).

Determination of the suppressive function of HSC-expanded Tregs

Untreated or LPS-pretreated B6 HSCs were cocultured with purified BALB/c Tregs for 5 d in the presence of 200 U/ml IL-2, as described above. Tregs were recovered and, to increase their yield, reincubated with fresh B6 HSCs plus IL-2 for an additional 5 d. Tregs recovered from culture were characterized for their expression of GITR, programmed death-1 (PD-1), LAG-3, CD103, CCR7, CD62L, and intracellular CTLA4 and IL-10. To determine the suppressive potential of HSC-expanded Tregs, B6-Tregs (Ly5.2) were expanded with BALB/c HSCs and then added at ratios of 1:10 and 1:20 in coculture of CFSE-labeled conventional B6-CD4+ T cells (Ly5.2) and γ-irradiated BALB/c splenocytes (as stimulators) in 96-well plates for 96 h (32). To some cultures, no Tregs or naive or anti-CD3/CD28 Ab-activated conventional B6 CD4+ T cells (isolated from Ly5.1/Ly5.2 hybrid mice) were added to serve as negative controls. Suppression was determined by flow cytometry.

Adoptive transfer of Tregs

BALB/c HSC-expanded B6 Tregs were labeled with CFSE and adoptively transferred to B6 mice (1 × 106 Tregs/animal). One week later, the animals were euthanized, cells isolated from spleens and lymph nodes (axillary, inguinal, mesenteric, and renal), and the level of expression of Foxp3 in the CFSE-labeled cells assessed by FACS to determine their stability in vivo.

Adoptive transfer of HSCs

B6 HSCs (untreated or LPS-pretreated) were adoptively transferred to BALB/c mice through the lateral tail vein (5 × 106 cells/mouse). After 5 d, cells from blood, spleens, and lymph nodes were isolated and assessed for total number of the natural Tregs (CD4+Foxp3+Helios+). In some experiments, GFP+-B6 HSCs were adoptively transferred to BALB/c mice, and on day 2 and 5 after cell transfer, spleens and lymph nodes were collected, cryopreserved, and the migration of allogeneic HSCs and their interaction with Tregs were examined by immunohistology.

Statistical analysis

Results are expressed as means ± SD. Statistical significance between groups was determined by Student t test using the Prism 5 software package (GraphPad, San Diego, CA). A p value <0.05 was considered statistically significant.

Results

LPS modulates the expression of Ag-presenting and T cell coregulatory molecules on HSCs

HSCs expressed relatively high levels of surface MHC class I, but their expression of MHC class II as well as T cell costimulatory molecules (CD40, CD80, and CD86) was very low (Fig. 1). LPS significantly increased expression of these molecules, except MHC class I. HSCs also expressed ICAM-1, VCAM, programmed death ligand-1 (B7-H1), and FasL (CD95L). LPS significantly increased surface expression of VCAM and FasL, as well as the intracellular expression of IL-10 in HSCs (Fig. 1).

FIGURE 1.
FIGURE 1.

Immunoregulatory molecules expressed by untreated and LPS-treated HSCs. (A) HSCs isolated from B6 mice were cultured overnight, treated with 270 μM GdCl3 for 24 h, washed and stimulated with 100 ng/ml LPS for 24 h. Cells were then stained for surface (MHC class I, MHC class II, CD40, CD80, CD86, ICAM-1, VCAM, B7-H1, and FasL) and intracellular (IL-10) molecules. Shaded histogram represents isotype control, whereas dotted and solid lines represent untreated and LPS-treated HSCs, respectively. (B) Bar graph shows mean fluorescence intensity (MFI) for each marker. The data are representative of at least three independent experiments. *p < 0.05, **p < 00.01.

Retention of allogeneic CD4+ T cells by the liver

Despite their higher frequency in the liver (33), hepatic CD4+ T cells (30–40% of total intrahepatic lymphocytes in mice) have been understudied compared with CD8+ T cells (34). In this study, we first determined whether HSCs might display enhanced interactions with allogeneic (BALB/c) CD4+ T cells following cold I/R of the B6 mouse liver. Consistent with previous observations (24, 25), LSEC-specific CD31 staining was markedly reduced in the cold I/R livers, indicating disruption of the endothelial barrier (Fig. 2A). Numerous infused allogeneic CD4+ T cells were retained in the liver, and I/R did not increase their retention (Fig. 2B). However, LPS pre-exposed livers (both unpreserved or normal and cold-preserved) retained significantly greater numbers of CD4+ T cells than the untreated livers (Fig. 2B, 2E); only a few CD4+ cells were observed in the control livers that were reperfused without allogeneic cells (Fig. 2C). In contrast to the allogeneic cells, significantly less retention of the infused syngeneic CD4+ T cells was observed in the cold I/R livers (Fig. 2D, 2E). These results demonstrate that the CD4+ T cells observed in the livers (Fig. 2B) were predominantly allogeneic. By high-power imaging, allogeneic CD4+ T cells were found to be retained mainly in the sinusoids of control livers (data not shown), and in close proximity to HSCs in cold-preserved livers (Fig. 2F, 2G). Interestingly, Foxp3+ Tregs were also found in close association with HSCs in cold I/R livers, and this interaction was enhanced upon LPS pretreatment (Fig. 2H, 2I, 2L). In fact, ∼50% of the retained Tregs were closely associated with HSCs. Immunostaining of HSC/Treg cocultures also demonstrated the adherence of greater number of Tregs to LPS-pretreated (Fig. 2K) compared with untreated HSCs (Fig. 2J). These data indicate the ability of the liver to retain allogeneic CD4+ T cells regardless of cold preservation with augmentation of the effect upon LPS pretreatment (35). The data further suggest that HSCs may play an important role in this phenomenon, especially when the endothelial barrier is disrupted due to cold I/R.

FIGURE 2.
FIGURE 2.

(A–E) Disruption of LSEC and retention of allogeneic CD4+ T cells by the liver. Livers of untreated or LPS-treated (1 mg/kg; 6 h) B6 mice were flushed with cold UW solution and either preserved at 4°C for 18 h or used immediately. The livers were reperfused with warm medium alone or medium containing FACS-purified BALB/c bulk CD4+ T cells (16 × 106). The livers were then fixed and cryopreserved for immunofluorescence microscopy. (A) The sections show the presence or absence of endothelial cells in normal untreated, LPS-treated, cold-preserved (I/R), and LPS-pretreated and cold-preserved livers. LSEC (CD31; pink stain) and HSCs (desmin; green stain). Original magnification ×20. Insets show images at higher (original magnification ×100) magnification. (B) The sections show the retention of allogeneic CD4+ T cells by the liver. CD4+ T cells (CD4; pink stain) and HSCs (desmin; green stain). (C) The sections show the host CD4+ T cells in indicated groups following reperfusion with medium only. (D) The sections show the comparatively low retention of infused syngeneic CD4+ T cells. (E) Bar graphs show the retention of CD4+ T cells in the various groups. n = 4 animals/group. (F–L) Close association of allogeneic CD4+ T cells and Tregs with HSCs. (F and G) Sections of cold I/R livers (untreated and LPS pretreated), reperfused with allogeneic CD4+ T cells, showing the close interaction between HSCs (desmin; green stain, arrows) and CD4+ T cells (CD4; pink stain, arrowheads). (H and I) Sections of the cold I/R livers (untreated and LPS pretreated) reperfused with allogeneic CD4+ T cells showing the close interaction between HSCs (desmin; green stain, arrows) and Tregs (Foxp3; pink stain, arrowheads). Original magnification ×100. (J and K) Coculture of HSCs (untreated or LPS pretreated) and purified allogeneic Tregs (on coverslips) showing the close interaction between HSCs (arrows) and Tregs (arrowheads). Original magnification ×100. (L) Bar graph shows the total number of Tregs in the liver and Tregs closely associated with HSCs in liver sections of average area of 0.4 cm2 (n = 4 animals/group). *p < 0.05.

HSCs promote apoptosis of conventional CD4+ T cells via Fas–FasL interaction

Fas–FasL and B7-H1 (PD-L1)–PD-1 interactions promote T cell death (36, 37). We next considered whether control or LPS-stimulated HSCs might induce conventional CD4+ T cell death via interactions of FasL and B7-H1 on HSCs with Fas and PD-1, respectively, on T cells. As shown in Fig. 1, HSCs displayed low levels of FasL and B7-H1, and LPS treatment significantly increased FasL but not B7-H1 expression. Interestingly, Fas expression increased strikingly on conventional CD4+ T cells cocultured with HSCs, regardless of LPS pretreatment (Fig. 3A). However, surface expression of PD1 on these T cells was not detected, without or with coculture with HSCs ± LPS (Fig. 3B). Because PD-1 is expressed on peripheral CD4+ and CD8+ T cells, NKT cells, B cells, monocytes, and some DC subsets upon their activation by cytokines such as IL-2, IL-7, IL-15, and IL-21 (38, 39), our data suggest that HSCs do not activate CD4+ T cells or produce cytokines that induce their expression of PD-1. Furthermore, HSCs did not upregulate the expression of the early activation markers CD69 or CD71 on conventional CD4+ T cells (data not shown). CFSE dilution assay also confirmed that HSCs did not induce proliferation of conventional CD4+ T cells (Fig. 3C). In coculture with untreated or LPS-pretreated HSCs, apoptosis of conventional CD4+ T cells increased significantly and similarly, as indicated by 7-AAD and Annexin V staining (Fig. 3D, upper panel and bar graph). Partial reversal of HSCs ± LPS-induced apoptosis of conventional CD4+ T cells occurred in the presence of anti-FasL–blocking Ab (Fig. 3D, lower panel and bar graph). Apoptosis of conventional CD4+ T cells induced by coculture with HSCs ± LPS was not altered by B7-H1–blocking Ab (data not shown). These results suggest that HSCs induce apoptosis of conventional CD4+ T cells primarily via a Fas-FasL–dependent mechanism.

FIGURE 3.
FIGURE 3.

Interaction of HSCs with allogeneic conventional CD4+ T cells. The expression of Fas (A) and PD-1 (B) on allogeneic CD4+ T cells cultured alone, with HSCs, or with LPS-pretreated HSCs. Bar graph shows the percentage of conventional CD4+ T cells expressing Fas or PD-1 (n = 3). (C) Proliferation profile of the allogeneic CD4+ T cells. CFSE-labeled CD4+ T cells were cultured alone or with untreated or LPS-pretreated (100 ng/ml) HSCs. Bar graph shows the percentage of proliferating CD4+ T cells (mean + SD) (n = 3). (D) Apoptosis of allogeneic CD4+ T cells induced by HSCs. Apoptosis of CD4+ T cells was determined by flow analysis following 3 d coculture with untreated or LPS-pretreated (100 ng/ml) HSCs in the absence or presence of anti-FasL blocking Ab. The Ab was added to the HSC culture at a concentration of 10 μg/ml, 1 h prior to the start of coculture with CD4+ T cells. Bar graph shows the percentage of CD4+ T cells undergoing apoptosis (7-AAD+Annexin V−, 7-AAD+Annexin V+, and 7-AAD−Annexin V+ cells) in the absence or presence of anti-FasL Ab (n = 3). *p < 0.05, **p < 0.01.

LPS-stimulated HSCs induce expansion of allogeneic Foxp3+ Tregs

In all Treg experiments, exogenous IL-2 (200 U/ml) was added because it is necessary for Treg survival, and our preliminary experiments showed no expression of IL-2 by unstimulated or LPS-stimulated HSCs, even when cocultured with Tregs. Incubation of bulk allogeneic CD4+ T cells with untreated HSCs increased the numbers of the Foxp3+ Tregs significantly compared with controls; this effect was enhanced in coculture with LPS-pretreated HSCs (Fig. 4A and bar graph). These Treg populations were positive for Helios, a member of the Ikaros transcription factor family. Helios is preferentially expressed by naturally occurring Tregs, but not by induced Tregs (40) (Fig. 4A). CFSE dilution assay showed proliferation of ∼60 and 75% of Helios+ Tregs in the presence of control and LPS-pretreated HSCs, respectively (Fig. 4B). These data indicate that LPS-stimulated HSCs induce the expansion of Tregs from the bulk CD4+ T cell population. The HSC-induced Treg expansion appeared to be alloantigen-specific because HSCs failed to expand syngeneic Tregs (data not shown).

FIGURE 4.
FIGURE 4.

LPS-stimulated HSCs increase the incidence of Foxp3+CD4+ cells in bulk allogeneic CD4+ T cell cultures. Purified bulk BALB/c CD4+ T cells were cultured for 5 d in flat-bottom 96-well plates alone or with B6 HSCs preincubated without or with 100 ng/ml LPS for 24 h at an HSC/Treg ratio of 1:10 for 5 d. T cells were then recovered for examination by flow cytometry. (A) HSCs increase the incidence of CD4+Foxp3+Helios+ Tregs. (B) Proliferation of Foxp3+Helios+ cells as assessed by CFSE dilution assay. Bar graph shows the absolute numbers of Tregs in the cultures (n = 3). *p < 0.05, **p < 0.01.

We next examined the influence of HSCs on purified Tregs. CFSE-labeled allogeneic Tregs were cultured alone or with HSCs (untreated or LPS-pretreated). Significant expansion of purified Tregs occurred in coculture with untreated HSCs, and this effect was augmented when HSCs were pretreated with LPS (Fig. 5A). In a parallel experiment, separation of HSCs from Tregs in transwell cultures prevented Treg expansion (Fig. 5A and bar graph), indicating the need for cell–cell contact.

FIGURE 5.
FIGURE 5.

Proliferation of purified Tregs by HSCs requires cell contact. (A) Tregs were cultured alone or with HSCs (untreated or LPS-pretreated [100 ng/ml]) at an HSC/Treg ratio of 1:10 for 5 d in the presence of 200 U/ml IL-2. CFSE dilution assay was performed to determine the proliferation of Foxp3+ cells. Proliferation of Tregs was blocked when the cells were separated from HSCs using transwell culture inserts. Bar graph shows the absolute numbers of Treg at the end of culture period (n = 3). (B) HSC-induced Treg expansion is mediated by MHC class II on HSCs. HSCs, preincubated with 100 ng/ml LPS for 24 h, were treated with 5 μg/ml anti-MHC class II (purified anti-mouse I-A/I-E) Ab, 5 μg/ml of the costimulation blocking agent CTLA4-Ig, or control IgG for 30 min. The medium was aspirated, and CFSE-labeled Tregs were added to the culture wells in fresh RPMI-1640 medium. At the end of incubation (day 5), proliferation of Tregs was measured by CFSE dilution assay. Bar graph represents percent Treg proliferation (mean + SD) (n = 3). *p < 0.05, **p < 0.01.

HSC-induced proliferation of Tregs is dependent on MHC class II

Even though HSCs produce a number of mediators, including TGF-β and retinoic acid, that might affect the activation/function of Tregs (41), the data shown in Fig. 5A demonstrated that HSC-induced Treg expansion was contact dependent. Furthermore, incubation of Tregs in medium conditioned by HSCs (± LPS) did not stimulate their expansion (data not shown). Key cell surface-expressed molecules, such as MHC class II and CD80/CD86, have been implicated in Treg proliferation (42–44). LPS increased the expression of MHC class II, CD80, and CD86 on HSCs significantly (Fig. 1). Upon examining the role of these upregulated molecules, blockade of MHC class II, but not the costimulatory molecules, significantly prevented HSC-induced Treg expansion (Fig. 5B and bar graph), suggesting a predominant role of TCR signaling in this effect.

Bidirectional interaction between HSCs and Tregs in the production of cytokines

Cytokines produced in the hepatic microenvironment following transplantation influence the course of allograft acceptance or rejection. Therefore, we ascertained the influence of HSC–Treg interaction on the production of anti-inflammatory (IL-10) and proinflammatory (IL-1α, IL-1β, IL-6, and TNF-α) cytokines. HSCs produced these cytokines spontaneously, and LPS pretreatment increased their production by HSCs significantly (Fig. 6A). Basal production of these cytokines by Tregs was very low/negligible, and, in coculture, their basal release by HSCs was abrogated. Interestingly, production of IL-10 by LPS-pretreated HSCs increased and that of IL-1α, IL-1β, IL-6, and TNF-α was reduced significantly in coculture with Tregs (Fig. 6A). Intracellular staining further confirmed production of these cytokines by HSCs (Fig. 6B). Although we did not observe IL-6 and TNF-α expression by Tregs cultured alone or with HSCs (± LPS) (data not shown), their IL-10 expression increased in coculture with HSCs (Fig. 7A). These data indicate that bidirectional interaction between HSCs and Tregs can markedly affect the balance between pro- and anti-inflammatory cytokine production.

FIGURE 6.
FIGURE 6.

Production of cytokines by LPS-stimulated HSCs and in HSC/Treg cocultures. (A) The supernatants from untreated or LPS-pretreated (100 ng/ml) HSCs, or from their cocultures with Tregs, were aspirated after 5 d incubation and the accumulated cytokines measured via Luminex assay (n = 3). (B) Intracellular IL-6, TNF-α, and IL-10 were stained in unstimulated or LPS-prestimulated (100 ng/ml) HSCs cultured alone or with Tregs for 5 d. The experiment was performed twice with essentially similar results. *p < 0.05, **p < 0.01.

FIGURE 7.
FIGURE 7.

HSCs favor retention of a regulatory phenotype and suppressive activity of proliferating Tregs. (A) Purified Tregs cocultured with HSCs (unstimulated or LPS-stimulated [100 ng/ml]) were recovered, stained, and analyzed for the expression of the indicated molecules by flow cytometry. Shaded histogram, isotype control; solid lines, freshly isolated Tregs; dotted lines, HSC-expanded Tregs; dashed lines, LPS-pretreated HSC-expanded Tregs. (B) HSC-expanded allo-Tregs potently suppress the proliferation of syngeneic conventional CD4+ T cells. Purified naive conventional B6 CD4+ T cells (Ly5.2; prestained with CFSE) were cocultured with γ-irradiated BALB/c splenocytes (at a ratio of 10:1). Freshly isolated B6 Tregs or Tregs recovered from cocultures with untreated or LPS-pretreated BALB/c HSCs were added at the indicated ratios to the conventional CD4+ T cell/splenocyte coculture. As negative controls, no Tregs or naive or anti-CD3/CD28 Ab-activated conventional B6 CD4+ T cells (coexpressing Ly5.1 and 5.2) were added at 1:10 ratio. Following incubation for 96 h, proliferation was measured in CD45.2+ CD4+ T cells, while excluding Foxp3+ as well as CD45.1 and CD45.2 double-positive cells. The numbers in the panels indicate the incidence of proliferating CD4+ T cells as a percentage of total CD4+Foxp3− cells (n = 2). (C) In vivo stability of the HSC-expanded Tregs following their adoptive transfer (measured on the basis of their Foxp3 expression). Freshly isolated, BALB/c HSC-expanded (untreated or LPS-pretreated), and anti-CD3/CD28-expanded B6 Tregs were labeled with CFSE and adoptively transferred to syngeneic B6 mice (1 × 106/animal). At 1 wk, expression of Foxp3 in labeled cells was determined in spleen and lymph nodes using FACS. Numbers in panels denote mean fluorescence intensity. n = 2.

HSC-expanded Tregs retain regulatory properties and exhibit potent suppressive function

The principal function of Tregs is maintenance of homeostasis by the suppression of exaggerated immune responses. Therefore, we examined Tregs for expression of molecules that are responsible for suppressive function. About 80–90% of HSC-expanded Tregs maintained Foxp3 expression (data not shown) and were uniformly positive for GITR (Fig. 7A), a potent T cell costimulatory receptor and regulator of Treg function, deficiency of which abrogates the suppressive function of Tregs and increases T cell proliferation (45). HSC-expanded Tregs expressed high levels of PD-1 (surface) and CTLA-4 (intracellular), which are implicated in induction of allograft tolerance by suppressing the activation, proliferation, and survival of CD4+ and CD8+ T cells (37, 46). The proliferating Tregs were positive for LAG-3, a protein expressed by activated natural Tregs and an augmenter of their regulatory function (47), CD103 (integrin αEβ7), a marker for memory/activated Tregs, and CCR7 and CD62L, the molecules implicated in their migration in nonlymphoid tissues (48).

We then tested the suppressive function of Tregs cocultured with HSCs (unstimulated and LPS stimulated). Tregs that proliferated in coculture with untreated or LPS-pretreated HSCs caused greater suppression of alloreactive CD4+ T cell proliferation than freshly isolated Tregs. Only ∼20% alloreactive CD4+ T cells proliferated when cultured with Tregs harvested from HSC cocultures (untreated and LPS pretreated, respectively) in contrast to ∼35% that proliferated with freshly isolated Tregs at the same suppressor/effector cell ratio (Fig. 7B). In contrast, the addition of naive or activated conventional B6 CD4+ T cells (coexpressing Ly5.1 and Ly5.2) (negative controls) instead of Tregs (at 1:10 ratio) demonstrated very mild inhibition of proliferation (Fig. 7B). These data suggest that in vitro HSC-expanded Tregs retain a suppressive phenotype and function. Upon examining their stability in vivo following adoptive transfer, HSC-expanded Tregs located in the spleen and lymph nodes were found to express a high level of Foxp3 even after a week in comparison with the anti-CD3/CD28 Ab-expanded Tregs (Fig. 7C). These data indicate superior suppressive potential and in vivo stability of HSC-expanded Tregs.

HSCs preferentially increase Tregs in lymph nodes in vivo

To ascertain the in vivo relevance of HSC-induced Treg expansion, we adoptively transferred B6 HSCs (untreated or LPS-pretreated) into BALB/c mice. After 5 d, naturally occurring Tregs (CD4+Foxp3+Helios+) were assessed in blood, spleen, and lymph nodes. The number of these Tregs in the blood decreased in mice that received HSCs (Fig. 8A, 8B). Although the proportion of Tregs in total CD4+ T cells increased (Fig. 8A), their absolute numbers decreased significantly in the spleens of mice that received HSCs (Fig. 8B). Interestingly, Tregs increased significantly in lymph nodes of mice that received HSCs, the effect being greater when LPS-pretreated HSCs were transferred (Fig. 8A, 8B). Immunostaining for Ki67 showed proliferation of Foxp3+ Tregs in the lymph nodes (Fig. 8C), but not spleens (data not shown). Tregs were found in close proximity of adoptively transferred GFP+ HSCs in the lymph nodes and spleen on day 2 (Fig. 8D, 8E), and a small number could be observed in lymph nodes even on day 5 (data not shown). These data indicate the potential of HSCs to recruit and expand Tregs in vivo.

FIGURE 8.
FIGURE 8.

In vivo migration of HSCs and proliferation of Tregs (CD4+Foxp3+Helios+). Untreated or LPS-pretreated B6 HSCs were adoptively transferred to BALB/c mice (5 × 106 HSCs/mouse). After 5 d, blood, spleen, and lymph nodes were collected for Treg enumeration. (A) Percent incidence of Tregs in various compartments (gated on total CD4). (B) Bar diagram shows the absolute numbers of Tregs (n = 3 animals in each group). (C) Immunohistology (day 5) of lymph nodes of BALB/c mice that received B6 HSCs by adoptive transfer shows proliferating Tregs (Foxp3 cells [pink], Ki67 [green], and nuclear stain DAPI [blue]) indicated with arrowheads. Controls did not receive adoptively transferred HSCs. (D and E) Immunohistology of lymph nodes and spleens of BALB/c mice that received GFP+ B6 HSCs by adoptive transfer (day 2) and control showing the migration of HSCs (green, arrow) closely associated with Foxp3+ cells (red, arrowhead) (n = 4 animals/group). Original magnification ×100. *p < 0.05, **p < 0.01.

Discussion

CD4+CD25+Foxp3+ Tregs suppress the activation/proliferation of autoreactive CD4+ and CD8+ T cells and control allograft rejection, infection-induced immune responses, and inflammatory diseases (49). Expansion of Tregs in a murine model of spontaneous liver allograft acceptance and their potential role in hepatic immune tolerance (50) have been reported. Thus, the differential effects of perisinusoidal HSCs observed in this study (apoptosis of naive conventional CD4+ T cells and expansion of Tregs) indicate that HSCs may play a critical role in liver allograft acceptance/tolerance, especially because the endothelial barrier is disrupted due to cold-ischemic storage and reperfusion of the graft (24, 25). However, even when the sinusoidal endothelium is intact, HSCs can interact with cells in the sinusoids via their cytoplasmic processes penetrating through the SEC fenestrations (13). In this regard, hepatocytes that are located beneath HSCs have been shown to interact with T cells through fenestrations in LSECs (51). Indeed, we observed close association of CD4+ T cells with HSCs in vivo and in vitro, which may be due to the expression of ICAM-1 and VCAM by HSCs (Fig. 1) (21, 52, 53). HSCs are also a principal source of fibronectin (54) that can recruit lymphocytes in the reperfused liver graft by interacting with α4β1 (55). Moreover, retinoic acid, stored mainly by HSCs, enhances the expression of integrin α4β7 on naive CD4+ T cells, which causes their homing toward nonlymphoid tissues (56). Furthermore, HSCs produce CXCL9 (MIG) and CXCL10 (IP-10) (data not shown) that can recruit CXCR3+CD4+ T cells following cold liver I/R injury (57). Together, these observations suggest that HSCs may constitute a major hepatic cell type in regulating immune responses. Indeed, the role of HSCs in immune regulation is exemplified by their ability to protect cotransplanted islet allografts from rejection (58) and enhance hepatocyte engraftment (59).

An important consideration regarding the role of HSCs in hepatic immune regulation is their strong reactivity to LPS, levels of which are elevated pre- and posttransplantation (29). In this study, both untreated and LPS-pretreated HSCs induced apoptosis of conventional allogeneic CD4+ T cells. This property of HSCs contrasts with that of KCs, SECs, and hepatocytes that induce the proliferation of CD4+CD25− T cells (60). Although it is unclear why upregulation of Fas expression on naive conventional allogeneic CD4+ T cells occurred without their activation/proliferation, our data are consistent with inhibition of activation/proliferation, together with apoptosis, of naive conventional CD4+ T cells due to FasL–Fas cross-linking in the presence of TCR ligation (36, 61). The B7-H1–PD-1 pathway has also been reported to be important for the inhibition of T cell proliferation and their apoptosis (37). Fully activated HSCs have been shown to induce apoptosis of DC-activated CD4+ T cells, in part via a B7-H1/PD-1–dependent mechanism, as a result of enhanced B7-H1 expression on HSCs and that of PD-1 on DC-stimulated allogeneic CD4+CD25− cells (17). However, we did not observe such an effect of HSCs on conventional CD4+ T cells. It appears from our studies that quiescent HSCs do not induce PD-1 expression on naive conventional CD4+ T cells (Fig. 3C). Together with the low expression of B7-H1 on quiescent HSCs, this may be the reason why the PD-1–B7-H1 pathway did not play a role in quiescent HSC-induced apoptosis of naive conventional allogeneic CD4+ T cells. Thus, our results indicate that Fas–FasL interaction may be a key mechanism underlying quiescent HSC-induced elimination of conventional allogeneic CD4+CD25− T cells. Our data also support the concept (62) that auto- or alloreactive T cells in target organs are activated mainly by nonprofessional APCs and deleted by apoptosis through the Fas/FasL pathway in the absence of adequate costimulation.

It has been reported previously (19) that culture-activated (7–10 d) mouse HSCs (i.e., cells that are retinoid deficient and fibrogenic), once they have been stimulated with IFN-γ for an additional 72 h, cause expansion of Tregs in an IL-2–dependent manner. By contrast, the activation state of HSCs examined in the current study was very low and likely achieved soon after liver transplantation due to the release of free radicals and inflammatory cytokines (14–16). Furthermore, no IFN-γ could be detected in the supernatants of HSC cultures (control or LPS-stimulated) or HSC/Treg cocultures (data not shown). Thus, our data indicate that HSCs can cause Treg expansion both with or without LPS stimulation and in an IFN-γ–independent manner. Considering the distinct effects of LPS and IFN-γ on cytokine production by HSCs, it is reasonable to speculate that the effects of LPS and IFN-γ in causing Treg expansion and promotion of tolerance may be additive or synergistic.

Wiegard et al. (60) reported that KCs, but not SECs or hepatocytes, caused Treg expansion. Interestingly, however, KC-expanded Tregs were less potent than freshly isolated Tregs in suppressing CD4+ T cell proliferation. In contrast, we found that Tregs expanded by HSCs were much more potent in suppressing CD4+ T cell proliferation than freshly isolated Tregs. This can be explained by the retention of a suppressive phenotype and enhanced Foxp3 expression by HSC-expanded Tregs (Fig. 7). Additionally, the suppressive activity of Tregs was reversed when KCs were stimulated with LPS (60), whereas the magnitude of suppressor activity of Tregs expanded by untreated and LPS-stimulated HSCs in this study was similar. This observation is interesting because Foxp3 expression is directly linked to Treg function (63), and Tregs from genetically engineered mice that express low levels of Foxp3 lose suppressive activity and gain effector cell functions (IL-2, IL-4, and IFN-γ production) simultaneously (64). The mechanisms underlying this interesting paradox are the topics of future investigation.

IL-10 inhibits the synthesis of many proinflammatory cytokines and chemokines, including TNF-α and IL-6 (65), and reduces Ag-specific CD4+ and CD8+ T cell activation and responses (66, 67). We observed constitutive and LPS-induced production of IL-10 by HSCs that was enhanced in coculture with Tregs. Previous work (67, 68) has shown that liver DCs and KCs, as well as LSECs, produce IL-10 and that LPS stimulates IL-10 synthesis by human KCs and SECs. It has been postulated that a mechanism underlying the inhibition of Treg suppressor activity by DCs or KCs is due to IL-6 released by the latter cell types (60, 69). In this study, we found that LPS-pretreated HSCs produced comparatively large amounts of IL-6. However, this effect was abrogated when Tregs were present in the cocultures. Interestingly, the release of other proinflammatory cytokines (IL-1α, IL-1β, and TNF-α) was also reduced significantly in cocultures of LPS-pretreated HSCs and Tregs, and IFN-γ and IL-4 were not detected. In contrast, secretion of IL-10 increased significantly in the same cocultures. These data, and the increased hepatic expression of IL-10, concurrent with decreased IFN-γ, IL-6, and TNF-α expression in ConA-tolerant mice (70), suggest that a reciprocal relationship between proinflammatory cytokines and anti-inflammatory IL-10 may be important in hepatic immune tolerance. The mechanism(s) underlying increased IL-10 and decreased proinflammatory cytokine expression in Treg/LPS-pretreated HSC cocultures is unclear, but increased IL-10 has been shown to suppress IL-6 production by SECs and KCs (71). Thus, a similar regulatory effect of IL-10 on proinflammatory cytokine production observed in the current study cannot be ruled out. Together, the bidirectional interaction between LPS-stimulated HSCs and Tregs may be important in promoting tolerance and reducing inflammatory responses.

Our study demonstrates an absolute requirement for cell–cell contact and MHC class II- dependence for Treg expansion by HSCs. Even though constitutive expression of MHC class II is low on HSCs, it increases significantly upon LPS stimulation, which is consistent with enhanced Treg expansion by LPS-pretreated HSCs. Irla et al. (72) reported recently that MHC class II-dependent, myelin Ag-specific interaction of plasmacytoid DCs with naturally occurring Tregs in lymph nodes caused their expansion during autoimmune encephalomyelitis. The role of Tregs in this latter model was indicated by exacerbated pathology in MHC class II-deficient mice. Mast cells have also been shown to stimulate proliferation of naturally occurring Tregs in an MHC class II-dependent manner (73). Experimental evidence also suggests the role of coregulatory molecules (for instance CD80/CD86 and B7-H1) in Treg expansion (74, 75). However, almost complete inhibition of HSC-induced Treg expansion by anti-MHC class II Ab suggests predominance of TCR ligation in this phenomenon. Moreover, mast cell-induced Treg expansion is also unaffected by anti–B7-H1 Ab (73). Although our data demonstrate that MHC class II-dependent expansion of Tregs by HSCs may be an important mechanism of hepatic immune regulation, we note that KCs also express MHC class II constitutively (11). Thus, a similar MHC class II-dependent effect of KCs on Treg expansion cannot be ruled out. However, sinusoidal endothelium is disrupted for a considerable time, and KC as well as DC numbers decrease after reperfusion (76), emphasizing the potential significance of HSCs in liver allograft tolerance, especially during the initial period following graft reperfusion. The homing of Tregs has been shown to be critical for the development of peripheral tolerance and acceptance of heart allografts (77). HSC-expanded Tregs expressed CCR7 and especially CD62L, molecules implicated in homing of Tregs to secondary lymphoid tissues, at a high level (Fig. 7A). These findings are consistent with our in vivo observations in which adoptively transferred HSCs enhanced homing/recruitment as well as the expansion of Tregs in the lymph nodes (Fig. 8).

In conclusion, expansion of Tregs and concomitant apoptosis of naive conventional CD4+ T cells by HSCs could favor immunological tolerance after liver transplantation and also be of value for ex vivo expansion of Tregs for potential therapeutic applications.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Rachel Stewart, Jesse Ennis, and Mark Ross for excellent technical assistance. We also thank Dr. David M. Rothstein and Dr. Geetha Chalasni for providing GFP-B6 and CD45.1/CD45.2-B6 mice, respectively.

Footnotes

  • This work was supported by National Institutes of Health Grant PO1A1081678. G.R. is supported by a Junior Faculty Grant from the American Diabetes Association, a Joseph A. Patrick Research Fellowship in Transplantation, and a Clinical and Translational Science Institute Pilots to Expand Investigator Repertoire grant (funded through Grant UL1 RR024153 from the National Center for Research Resources). T.L.S. was the recipient of an American Society of Transplantation Basic Science Fellowship. S.K. is supported by National Institutes of Health Grant DK071753.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    7-AAD
    7-aminoactinomycin D
    B6
    C57BL/6 (H-2b)
    DC
    dendritic cell
    FasL
    Fas ligand
    HSC
    hepatic stellate cell
    I/R
    ischemic preservation and reperfusion
    KC
    Kupffer cell
    LSEC
    liver sinusoidal endothelial cell
    PD-1
    programmed death-1
    Treg
    regulatory T cell
    UW
    University of Wisconsin.

  • Received August 25, 2011.
  • Accepted February 12, 2012.

References

  1. ↵
    1. Crispe I. N.
    2009. The liver as a lymphoid organ. Annu. Rev. Immunol. 27: 147–163.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Thomson A. W.,
    2. P. A. Knolle
    . 2010. Antigen-presenting cell function in the tolerogenic liver environment. Nat. Rev. Immunol. 10: 753–766.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Calne R. Y.,
    2. R. A. Sells,
    3. J. R. Pena,
    4. D. R. Davis,
    5. P. R. Millard,
    6. B. M. Herbertson,
    7. R. M. Binns,
    8. D. A. Davies
    . 1969. Induction of immunological tolerance by porcine liver allografts. Nature 223: 472–476.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Sun J.,
    2. A. G. Sheil,
    3. C. Wang,
    4. L. Wang,
    5. K. Rokahr,
    6. A. Sharland,
    7. S. E. Jung,
    8. L. Li,
    9. G. W. McCaughan,
    10. G. A. Bishop
    . 1996. Tolerance to rat liver allografts: IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes. Transplantation 62: 1725–1730.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Pons J. A.,
    2. B. Revilla-Nuin,
    3. A. Baroja-Mazo,
    4. P. Ramírez,
    5. L. Martínez-Alarcón,
    6. F. Sánchez-Bueno,
    7. R. Robles,
    8. A. Rios,
    9. P. Aparicio,
    10. P. Parrilla
    . 2008. FoxP3 in peripheral blood is associated with operational tolerance in liver transplant patients during immunosuppression withdrawal. Transplantation 86: 1370–1378.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Castellaneta A.,
    2. A. W. Thomson,
    3. N. Nayyar,
    4. M. de Vera,
    5. G. V. Mazariegos
    . 2010. Monitoring the operationally tolerant liver allograft recipient. Curr. Opin. Organ Transplant. 15: 28–34.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Kreisel D.,
    2. H. Petrowsky,
    3. A. M. Krasinskas,
    4. A. S. Krupnick,
    5. W. Y. Szeto,
    6. A. D. McLean,
    7. S. H. Popma,
    8. A. E. Gelman,
    9. M. K. Traum,
    10. E. E. Furth,
    11. et al
    . 2002. The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts. Transplantation 73: 1501–1507.
    OpenUrlCrossRefPubMed
    1. Bishop G. A.,
    2. C. Wang,
    3. A. F. Sharland,
    4. G. McCaughan
    . 2002. Spontaneous acceptance of liver transplants in rodents: evidence that liver leucocytes induce recipient T-cell death by neglect. Immunol. Cell Biol. 80: 93–100.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Araújo M. B.,
    2. L. S. Leonardi,
    3. M. I. Leonardi,
    4. I. F. Boin,
    5. L. A. Magna,
    6. E. A. Donadi,
    7. M. H. Kraemer
    . 2009. Prospective analysis between the therapy of immunosuppressive medication and allogeneic microchimerism after liver transplantation. Transpl. Immunol. 20: 195–198.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Knechtle S. J.,
    2. J. Kwun
    . 2009. Unique aspects of rejection and tolerance in liver transplantation. Semin. Liver Dis. 29: 91–101.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Tiegs G.,
    2. A. W. Lohse
    . 2010. Immune tolerance: what is unique about the liver. J. Autoimmun. 34: 1–6.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Friedman S. L.
    2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 275: 2247–2250.
    OpenUrlFREE Full Text
  13. ↵
    1. Geerts A.
    2001. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin. Liver Dis. 21: 311–335.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Gandhi C. R.
    2010. Stellate cells. In Molecular Pathology of Liver Diseases. Molecular Pathology Library, Part 1. Monga S. P., ed. Springer, New York, p. 53–80.
    1. Marra F.
    1999. Hepatic stellate cells and the regulation of liver inflammation. J. Hepatol. 31: 1120–1130.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Pinzani M.,
    2. F. Marra
    . 2001. Cytokine receptors and signaling in hepatic stellate cells. Semin. Liver Dis. 21: 397–416.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Yu M. C.,
    2. C. H. Chen,
    3. X. Liang,
    4. L. Wang,
    5. C. R. Gandhi,
    6. J. J. Fung,
    7. L. Lu,
    8. S. Qian
    . 2004. Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology 40: 1312–1321.
    OpenUrlCrossRefPubMed
    1. Winau F.,
    2. G. Hegasy,
    3. R. Weiskirchen,
    4. S. Weber,
    5. C. Cassan,
    6. P. A. Sieling,
    7. R. L. Modlin,
    8. R. S. Liblau,
    9. A. M. Gressner,
    10. S. H. Kaufmann
    . 2007. Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26: 117–129.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Jiang G.,
    2. H. R. Yang,
    3. L. Wang,
    4. G. M. Wildey,
    5. J. J. Fung,
    6. S. Qian,
    7. L. Lu
    . 2008. Hepatic stellate cells preferentially expand allogeneic CD4+ CD25+ FoxP3+ regulatory T cells in an IL-2-dependent manner. Transplantation 86: 1492–1502.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Schildberg F. A.,
    2. C. Kurts,
    3. P. A. Knolle
    . 2011. Prominent regulatory but weak antigen-presenting cell function of hepatic stellate cells. Hepatology 54: 1108.
    OpenUrlPubMed
  21. ↵
    1. Schildberg F. A.,
    2. A. Wojtalla,
    3. S. V. Siegmund,
    4. E. Endl,
    5. L. Diehl,
    6. Z. Abdullah,
    7. C. Kurts,
    8. P. A. Knolle
    . 2011. Murine hepatic stellate cells veto CD8 T cell activation by a CD54-dependent mechanism. Hepatology 54: 262–272.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Cheng F.,
    2. Y. Li,
    3. L. Feng,
    4. S. Li
    . 2008. Hepatic stellate cell activation and hepatic fibrosis induced by ischemia/reperfusion injury. Transplant. Proc. 40: 2167–2170.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Cheng Q.,
    2. K. T. Ng,
    3. S. T. Fan,
    4. Z. X. Lim,
    5. D. Y. Guo,
    6. X. B. Liu,
    7. Y. Liu,
    8. R. T. Poon,
    9. C. M. Lo,
    10. K. Man
    . 2010. Distinct mechanism of small-for-size fatty liver graft injury—Wnt4 signaling activates hepatic stellate cells. Am. J. Transplant. 10: 1178–1188.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Gao W.,
    2. R. C. Bentley,
    3. J. F. Madden,
    4. P. A. Clavien
    . 1998. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation. Hepatology 27: 1652–1660.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Stolz D. B.,
    2. M. A. Ross,
    3. A. Ikeda,
    4. K. Tomiyama,
    5. T. Kaizu,
    6. D. A. Geller,
    7. N. Murase
    . 2007. Sinusoidal endothelial cell repopulation following ischemia/reperfusion injury in rat liver transplantation. Hepatology 46: 1464–1475.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Uemura T.,
    2. C. R. Gandhi
    . 2001. Inhibition of DNA synthesis in cultured hepatocytes by endotoxin-conditioned medium of activated stellate cells is transforming growth factor-β and nitric oxide-independent. Br. J. Pharmacol. 133: 1125–1133.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Thirunavukkarasu C.,
    2. T. Uemura,
    3. L. F. Wang,
    4. S. C. Watkins,
    5. C. R. Gandhi
    . 2005. Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes. J. Cell. Physiol. 204: 654–665.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Thirunavukkarasu C.,
    2. S. C. Watkins,
    3. C. R. Gandhi
    . 2006. Mechanisms of endotoxin-induced NO, IL-6, and TNF-alpha production in activated rat hepatic stellate cells: role of p38 MAPK. Hepatology 44: 389–398.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Miyata T.,
    2. I. Yokoyama,
    3. S. Todo,
    4. A. Tzakis,
    5. R. Selby,
    6. T. E. Starzl
    . 1989. Endotoxaemia, pulmonary complications, and thrombocytopenia in liver transplantation. Lancet 2: 189–191.
    OpenUrlPubMed
  30. ↵
    1. Mehal W. Z.,
    2. A. E. Juedes,
    3. I. N. Crispe
    . 1999. Selective retention of activated CD8+ T cells by the normal liver. J. Immunol. 163: 3202–3210.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Liu W.,
    2. A. L. Putnam,
    3. Z. Xu-Yu,
    4. G. L. Szot,
    5. M. R. Lee,
    6. S. Zhu,
    7. P. A. Gottlieb,
    8. P. Kapranov,
    9. T. R. Gingeras,
    10. B. Fazekas de St Groth,
    11. et al
    . 2006. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 203: 1701–1711.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Raimondi G.,
    2. T. L. Sumpter,
    3. B. M. Matta,
    4. M. Pillai,
    5. N. Corbitt,
    6. Y. Vodovotz,
    7. Z. Wang,
    8. A. W. Thomson
    . 2010. Mammalian target of rapamycin inhibition and alloantigen-specific regulatory T cells synergize to promote long-term graft survival in immunocompetent recipients. J. Immunol. 184: 624–636.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Klugewitz K.,
    2. D. H. Adams,
    3. M. Emoto,
    4. K. Eulenburg,
    5. A. Hamann
    . 2004. The composition of intrahepatic lymphocytes: shaped by selective recruitment? Trends Immunol. 25: 590–594.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Bowen D. G.,
    2. G. W. McCaughan,
    3. P. Bertolino
    . 2005. Intrahepatic immunity: a tale of two sites? Trends Immunol. 26: 512–517.
    OpenUrlCrossRefPubMed
  35. ↵
    1. McAleer J. P.,
    2. R. J. Rossi,
    3. A. T. Vella
    . 2009. Lipopolysaccharide potentiates effector T cell accumulation into nonlymphoid tissues through TRIF. J. Immunol. 182: 5322–5330.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Maksimow M.,
    2. T. S. Söderström,
    3. S. Jalkanen,
    4. J. E. Eriksson,
    5. A. Hänninen
    . 2006. Fas costimulation of naive CD4 T cells is controlled by NF-kappaB signaling and caspase activity. J. Leukoc. Biol. 79: 369–377.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Francisco L. M.,
    2. V. H. Salinas,
    3. K. E. Brown,
    4. V. K. Vanguri,
    5. G. J. Freeman,
    6. V. K. Kuchroo,
    7. A. H. Sharpe
    . 2009. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J. Exp. Med. 206: 3015–3029.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Keir M. E.,
    2. M. J. Butte,
    3. G. J. Freeman,
    4. A. H. Sharpe
    . 2008. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26: 677–704.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Kinter A. L.,
    2. E. J. Godbout,
    3. J. P. McNally,
    4. I. Sereti,
    5. G. A. Roby,
    6. M. A. O’Shea,
    7. A. S. Fauci
    . 2008. The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J. Immunol. 181: 6738–6746.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Thornton A. M.,
    2. P. E. Korty,
    3. D. Q. Tran,
    4. E. A. Wohlfert,
    5. P. E. Murray,
    6. Y. Belkaid,
    7. E. M. Shevach
    . 2010. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J. Immunol. 184: 3433–3441.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Mucida D.,
    2. Y. Park,
    3. G. Kim,
    4. O. Turovskaya,
    5. I. Scott,
    6. M. Kronenberg,
    7. H. Cheroutre
    . 2007. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 317: 256–260.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Lau A. W.,
    2. S. Biester,
    3. R. J. Cornall,
    4. J. V. Forrester
    . 2008. Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells. J. Immunol. 180: 3889–3899.
    OpenUrlAbstract/FREE Full Text
    1. Darrasse-Jèze G.,
    2. S. Deroubaix,
    3. H. Mouquet,
    4. G. D. Victora,
    5. T. Eisenreich,
    6. K. H. Yao,
    7. R. F. Masilamani,
    8. M. L. Dustin,
    9. A. Rudensky,
    10. K. Liu,
    11. M. C. Nussenzweig
    . 2009. Feedback control of regulatory T cell homeostasis by dendritic cells in vivo. J. Exp. Med. 206: 1853–1862.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Zou T.,
    2. A. J. Caton,
    3. G. A. Koretzky,
    4. T. Kambayashi
    . 2010. Dendritic cells induce regulatory T cell proliferation through antigen-dependent and -independent interactions. J. Immunol. 185: 2790–2799.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Liao G.,
    2. S. Nayak,
    3. J. R. Regueiro,
    4. S. B. Berger,
    5. C. Detre,
    6. X. Romero,
    7. R. de Waal Malefyt,
    8. T. A. Chatila,
    9. R. W. Herzog,
    10. C. Terhorst
    . 2010. GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells. Int. Immunol. 22: 259–270.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Read S.,
    2. R. Greenwald,
    3. A. Izcue,
    4. N. Robinson,
    5. D. Mandelbrot,
    6. L. Francisco,
    7. A. H. Sharpe,
    8. F. Powrie
    . 2006. Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo. J. Immunol. 177: 4376–4383.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Huang C. T.,
    2. C. J. Workman,
    3. D. Flies,
    4. X. Pan,
    5. A. L. Marson,
    6. G. Zhou,
    7. E. L. Hipkiss,
    8. S. Ravi,
    9. J. Kowalski,
    10. H. I. Levitsky,
    11. et al
    . 2004. Role of LAG-3 in regulatory T cells. Immunity 21: 503–513.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Huehn J.,
    2. K. Siegmund,
    3. J. C. Lehmann,
    4. C. Siewert,
    5. U. Haubold,
    6. M. Feuerer,
    7. G. F. Debes,
    8. J. Lauber,
    9. O. Frey,
    10. G. K. Przybylski,
    11. et al
    . 2004. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J. Exp. Med. 199: 303–313.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Rudensky A. Y.
    2011. Regulatory T cells and Foxp3. Immunol. Rev. 241: 260–268.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Li W.,
    2. C. S. Kuhr,
    3. X. X. Zheng,
    4. K. Carper,
    5. A. W. Thomson,
    6. J. D. Reyes,
    7. J. D. Perkins
    . 2008. New insights into mechanisms of spontaneous liver transplant tolerance: the role of Foxp3-expressing CD25+CD4+ regulatory T cells. Am. J. Transplant. 8: 1639–1651.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Bertolino P.,
    2. M. C. Trescol-Biémont,
    3. J. Thomas,
    4. B. Fazekas de St Groth,
    5. M. Pihlgren,
    6. J. Marvel,
    7. C. Rabourdin-Combe
    . 1999. Death by neglect as a deletional mechanism of peripheral tolerance. Int. Immunol. 11: 1225–1238.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Hellerbrand S. C.,
    2. S. C. Wang,
    3. H. Tsukamoto,
    4. D. A. Brenner,
    5. R. A. Rippe
    . 1996. Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells. Hepatology 24: 670–676.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Knittel T.,
    2. C. Dinter,
    3. D. Kobold,
    4. K. Neubauer,
    5. M. Mehde,
    6. S. Eichhorst,
    7. G. Ramadori
    . 1999. Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair. Am. J. Pathol. 154: 153–167.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Aziz-Seible R. S.,
    2. C. A. Casey
    . 2011. Fibronectin: functional character and role in alcoholic liver disease. World J. Gastroenterol. 17: 2482–2499.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Moore C.,
    2. X. D. Shen,
    3. C. Fondevila,
    4. F. Gao,
    5. A. J. Coito
    . 2005. Blockade of fibronectin-alpha4beta1 adhesive interactions down-regulates cyclooxygenase-2 inducible nitric oxide synthase and prolongs recipient survival in a 24-hour model of cold hepatic ischemia-reperfusion injury. Transplant. Proc. 37: 1682–1683.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Hill J. A.,
    2. J. A. Hall,
    3. C. M. Sun,
    4. Q. Cai,
    5. N. Ghyselinck,
    6. P. Chambon,
    7. Y. Belkaid,
    8. D. Mathis,
    9. C. Benoist
    . 2008. Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+CD44hi Cells. Immunity 29: 758–770.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Zhai Y.,
    2. X. D. Shen,
    3. W. W. Hancock,
    4. F. Gao,
    5. B. Qiao,
    6. C. Lassman,
    7. J. A. Belperio,
    8. R. M. Strieter,
    9. R. W. Busuttil,
    10. J. W. Kupiec-Weglinski
    . 2006. CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury. J. Immunol. 176: 6313–6322.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Chen C. H.,
    2. L. M. Kuo,
    3. Y. Chang,
    4. W. Wu,
    5. C. Goldbach,
    6. M. A. Ross,
    7. D. B. Stolz,
    8. L. Chen,
    9. J. J. Fung,
    10. L. Lu,
    11. S. Qian
    . 2006. In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology 44: 1171–1181.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Benten D.,
    2. V. Kumaran,
    3. B. Joseph,
    4. J. Schattenberg,
    5. Y. Popov,
    6. D. Schuppan,
    7. S. Gupta
    . 2005. Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment in the rat. Hepatology 42: 1072–1081.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Wiegard C.,
    2. C. Frenzel,
    3. J. Herkel,
    4. K. J. Kallen,
    5. E. Schmitt,
    6. A. W. Lohse
    . 2005. Murine liver antigen presenting cells control suppressor activity of CD4+CD25+ regulatory T cells. Hepatology 42: 193–199.
    OpenUrlCrossRefPubMed
  61. ↵
    1. Paulsen M.,
    2. B. Mathew,
    3. J. Qian,
    4. M. Lettau,
    5. D. Kabelitz,
    6. O. Janssen
    . 2009. FasL cross-linking inhibits activation of human peripheral T cells. Int. Immunol. 21: 587–598.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Pender M. P.
    1999. Activation-induced apoptosis of autoreactive and alloreactive T lymphocytes in the target organ as a major mechanism of tolerance. Immunol. Cell Biol. 77: 216–223.
    OpenUrlCrossRefPubMed
  63. ↵
    1. Egawa T.,
    2. R. E. Tillman,
    3. Y. Naoe,
    4. I. Taniuchi,
    5. D. R. Littman
    . 2007. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells. J. Exp. Med. 204: 1945–1957.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Wan Y. Y.,
    2. R. A. Flavell
    . 2007. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 445: 766–770.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Knolle P.,
    2. J. Schlaak,
    3. A. Uhrig,
    4. P. Kempf,
    5. K. H. Meyer zum Büschenfelde,
    6. G. Gerken
    . 1995. Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge. J. Hepatol. 22: 226–229.
    OpenUrlCrossRefPubMed
  66. ↵
    1. Groux H.,
    2. M. Bigler,
    3. J. E. de Vries,
    4. M. G. Roncarolo
    . 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J. Exp. Med. 184: 19–29.
    OpenUrlAbstract/FREE Full Text
  67. ↵
    1. Knolle P. A.,
    2. T. Germann,
    3. U. Treichel,
    4. A. Uhrig,
    5. E. Schmitt,
    6. S. Hegenbarth,
    7. A. W. Lohse,
    8. G. Gerken
    . 1999. Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells. J. Immunol. 162: 1401–1407.
    OpenUrlAbstract/FREE Full Text
  68. ↵
    1. Bamboat Z. M.,
    2. J. A. Stableford,
    3. G. Plitas,
    4. B. M. Burt,
    5. H. M. Nguyen,
    6. A. P. Welles,
    7. M. Gonen,
    8. J. W. Young,
    9. R. P. DeMatteo
    . 2009. Human liver dendritic cells promote T cell hyporesponsiveness. J. Immunol. 182: 1901–1911.
    OpenUrlAbstract/FREE Full Text
  69. ↵
    1. Pasare C.,
    2. R. Medzhitov
    . 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299: 1033–1036.
    OpenUrlAbstract/FREE Full Text
  70. ↵
    1. Erhardt A.,
    2. M. Biburger,
    3. T. Papadopoulos,
    4. G. Tiegs
    . 2007. IL-10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice. Hepatology 45: 475–485.
    OpenUrlCrossRefPubMed
  71. ↵
    1. Knolle P. A.,
    2. E. Löser,
    3. U. Protzer,
    4. R. Duchmann,
    5. E. Schmitt,
    6. K. H. M. zum Büschenfelde,
    7. S. Rose-John,
    8. G. Gerken
    . 1997. Regulation of endotoxin-induced IL-6 production in liver sinusoidal endothelial cells and Kupffer cells by IL-10. Clin. Exp. Immunol. 107: 555–561.
    OpenUrlCrossRefPubMed
  72. ↵
    1. Irla M.,
    2. N. Küpfer,
    3. T. Suter,
    4. R. Lissilaa,
    5. M. Benkhoucha,
    6. J. Skupsky,
    7. P. H. Lalive,
    8. A. Fontana,
    9. W. Reith,
    10. S. Hugues
    . 2010. MHC class II-restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell-mediated autoimmunity. J. Exp. Med. 207: 1891–1905.
    OpenUrlAbstract/FREE Full Text
  73. ↵
    1. Kambayashi T.,
    2. E. J. Allenspach,
    3. J. T. Chang,
    4. T. Zou,
    5. J. E. Shoag,
    6. S. L. Reiner,
    7. A. J. Caton,
    8. G. A. Koretzky
    . 2009. Inducible MHC class II expression by mast cells supports effector and regulatory T cell activation. J. Immunol. 182: 4686–4695.
    OpenUrlAbstract/FREE Full Text
  74. ↵
    1. Hombach A. A.,
    2. D. Kofler,
    3. A. Hombach,
    4. G. Rappl,
    5. H. Abken
    . 2007. Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2. J. Immunol. 179: 7924–7931.
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Chen X.,
    2. P. E. Jensen
    . 2007. Cutting edge: primary B lymphocytes preferentially expand allogeneic FoxP3+ CD4 T cells. J. Immunol. 179: 2046–2050.
    OpenUrlAbstract/FREE Full Text
  76. ↵
    1. Bamboat Z. M.,
    2. L. M. Ocuin,
    3. V. P. Balachandran,
    4. H. Obaid,
    5. G. Plitas,
    6. R. P. DeMatteo
    . 2010. Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion. J. Clin. Invest. 120: 559–569.
    OpenUrlCrossRefPubMed
  77. ↵
    1. Ochando J. C.,
    2. A. C. Yopp,
    3. Y. Yang,
    4. A. Garin,
    5. Y. Li,
    6. P. Boros,
    7. J. Llodra,
    8. Y. Ding,
    9. S. A. Lira,
    10. N. R. Krieger,
    11. J. S. Bromberg
    . 2005. Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells. J. Immunol. 174: 6993–7005.
    OpenUrlAbstract/FREE Full Text
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