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. Author manuscript; available in PMC: 2017 Feb 15.
Published in final edited form as: J Immunol. 2016 Jan 11;196(4):1617–1625. doi: 10.4049/jimmunol.1501737

Hepatic Stellate Cells Directly Inhibit B Cells via Programmed Death-Ligand 1

Yan Li 1, Lina Lu 1,2, Shiguang Qian 1,2, John J Fung 2, Feng Lin 1,*
PMCID: PMC4784705  NIHMSID: NIHMS744358  PMID: 26755818

Abstract

We previously demonstrated that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1), but whether HSCs exert any effects on B cells, the other component of the adaptive immune system, remains unknown. In this study, we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that (a) HSCs inhibited the upregulation of activation markers on activated B cells; (b) HSCs inhibited the proliferation of activated B cells and their cytokine/immunoglobulin production in vitro; and (c) pharmaceutically or genetically blocking the interaction of PD-L1 with programmed death-1 (PD-1) impaired the ability of HSCs to inhibit B cells. To test the newly discovered B-cell-inhibitory activity of HSCs in vivo, we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll, a T-cell-independent antigen, significantly suppressed antigen-specific IgM and IgG production in vivo, whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion, in addition to inhibiting T cells, mouse HSCs concurrently inhibit B cells via PD-L1. This direct B-cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver’s immune homeostasis.

Introduction

The liver is considered an immunoprivileged organ1. In rodents, almost all liver allografts are spontaneously accepted without rejection(2–4); in humans, patients who have received liver allograft transplantation generally require lower doses of immunosuppressive drugs to prevent rejection than patients who have received other transplanted organs, such as kidney or heart5. Also, approximately 20%–40% of patients with liver transplants can gradually be weaned off immunosuppressive drugs without rejecting their new liver(6, 7). In addition, the liver must maintain overall homeostasis while physiologically it is under chronic exposure to many foreign antigens (e.g., food and microbial antigens) and stimulants (e.g., lipopolysaccharides)(8, 9). Interestingly, while whole liver transplantation can be tolerated, transplanted hepatocytes are rejected10, suggesting that nonparenchymal cells in the liver are critical to maintaining the immunoprivileged status of the liver.

Hepatic stellate cells (HSCs) account for about one third of such nonparenchymal cells11. Upon activation, HSCs secrete a number of local growth factors (12, 13) and matrix metalloproteinases associated with liver repair and fibrosis, as well as systemic acute phase proteins (14, 15). Although the role of HSCs in liver injury and fibrosis has been extensively investigated, the potential role of HSCs in liver immunoregulation and their underlying mechanisms remain understudied. We previously reported that HSCs directly inhibit T cells through programmed death-ligand 1 (PD-L1) on the HSC cell surface16 and that HSCs also indirectly suppress the adaptive immune system by inducing the propagation of myeloid-derived suppressor cells from hematopoietic stem cells17. However, whether HSCs have any direct effect on B cells, and if this is the case, what the underlying mechanisms might be, remain unknown. B cells reside in the liver together with T cells under physiological conditions (18–20). In addition to producing antibodies, both in response to pathogens as well as being associated with autoimmune disorders, activated B cells are also found to be the major source of inflammatory cytokines including IL- 6 in the lymphoid organs 21, and serve as antigen-presenting cells to promote T-cell responses22. It is established that B cells play important roles not only in the traditional humoral immunity-mediated diseases, but also in many diseases conventionally believed to be mediated by T cells23. We report here that using isolated mouse primary HSCs, we found in vitro and in vivo evidence suggesting that HSCs directly inhibit B cells, in which the HSC-expressed PD-L1 is integrally involved.

Materials and Methods

Mice

C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME), and PD-L1 knockout (KO) mice (on C57BL/6 background) were kindly provided by Lieping Chen, MD, PhD, Yale University24. All mice were housed in Cleveland Clinic’s Biological Resources Unit in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and the animal experimental protocols have been approved by the Institutional Animal Care and Use Committee at Cleveland Clinic. Mice 8–16 weeks old were used in all experiments.

Isolation of HSCs

HSCs were isolated from mouse liver and cultured in RPMI 1640 medium supplemented with 20% FBS (Life Technologies, Grand Island, NY) in 5% CO2 in air at 37°C for 14–21 days, following protocols well established in the laboratory, as previously described(16, 17, 25, 26). Purify of the isolated HSCs were generally > 95%, as assessed by using α-smooth muscle actin as a marker (Supplemental Fig. 1) followed by flow cytometry analysis. All the flow cytometry experiments in this reports were done using a BD FACSCalibur flow cytometer and Flowjo verrsion 7 software package.

B-cell activation assays

B cells (> 98% pure) were purified by negative selection (STEMCELL Technologies, Inc., Vancouver, BC, Canada) from splenocytes (Supplemental Fig. 2). The purified B cells were activated by incubation with either 10 µg/ml anti-IgM IgGs (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-CD40 IgGs (BioLegend, Inc., San Diego, CA) together with 100 U/ml of IL-4 (PeproTech, Inc., Rocky Hill, NJ), then co-cultured with different numbers of HSCs. After 24 hrs of incubation, B cells were assessed for the expression of activation markers CD69 and CD86 by flow cytometry after staining with 1 µg/ml PE-anti-mouse CD69 or FITC-anti-mouse CD86 monoclonal antibodies (mAbs; BioLegend).

B-cell proliferation assays

The proliferation of activated B cells was assessed by the carboxyfluorescein succinimidyl ester (CFSE) dilution assay and/or 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. For CFSE-based proliferation assays, purified B cells were first incubated with CFSE at 37°C for 10 min, then activated by incubation with either 10 µg/ml anti-IgM IgGs or anti-CD40 IgGs together with 100 U/ml of IL-4. After 72 hrs, proliferation of the activated B cells was assessed by flow cytometric analysis of the CSFE dilution on B cells. For BrdU incorporation-based proliferation assays, BrdU was added into the HSC:B-cell co-cultures 1 day before the assay, then suspended B cells were gently washed and collected to measure their proliferation (BrdU incorporation) using a BrdU ELISA kit (Roche Applied Science, Indianapolis, IN), following manufacturer protocols. At the same time, culture supernatants were collected to measure levels of IL-6, IgG and/or IgM by respective ELISAs, following manufacturer protocols.

Transwell experiments

HSCs were cultured at the bottom of the 24-well Transwell culture system (BD Biosciences, San Jose, CA) in 500 µl of media; anti-CD40/IL-4-activated and CFSE-labeled B cells were cultured in the inserts, which are separated from the bottom cells by a membrane of 0.1 µM pore size. After 72 hrs of culture, B cells were analyzed for proliferation by flow cytometry, and supernatants were collected to measure levels of IL-6 produced by the activated B cells.

Splenic artery injection of HSCs

Mice were anesthetized, and a transverse upper abdominal incision was used to expose the spleen. The splenic artery was visually identified and separated from the mesenteric adipose tissues. After closing off the proximal artery using a microvascular clamp clip, the artery was punctured by a sterile 32-Ga needle. Using the needle tip as a canal, a tip-modified 10-0 suture “guidewire” was inserted into the artery. Then using a wire catheter exchange technique, the modified catheter was placed into the lumen. After this step, 0.2 ×106 of wild-type (WT) or PD-L1-KO HSCs in 50 µl of sterile phosphate-buffered saline (PBS) was injected into the splenic artery. After injection, the proximal side of the injected artery was ligated, and 1 mL of warm 0.9% saline was injected into the abdominal cavity to replenish fluid losses and prevent dehydration. The abdomen and skin were then closed in layers with running 4/0 silk sutures or wound clips. Sham-operated mice that had not an injection of HSCs were included as controls. To demonstrate the distribution of the injected HSCs in the spleen, the same numbers of HSCs labeled with Vybrant® Dil Cell-Labeling Solution (Life Technologies, CA) were injected into a mouse; after sacrifice, the spleen was collected to make cryosections for examination under a fluorescence microscope (Leica Microsystems, Germany).

NP-Ficoll immunization

Each HSC-injected or sham-operated mouse was immunized by intraperitoneal (i.p.) injection of 10 µg of 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll (Biosearch Technologies, Petaluma, CA). Serum samples were collected from tail bleeding, and NP-specific IgG and IgM levels in the sera were measured by ELISA using plates coated with NP- bovine serum albumin (BSA; Biosearch Technologies) following protocols described before.

NP-specific IgM and IgG ELISA

Serum samples were collected from immunized mice at Day 3, 9 and 14, then titers of NP-specific IgM and IgG were measured by ELISA, using methods described before. In brief, serum samples were 1:500 diluted in PBS and added into wells of a 96-well plate coated with 5 µg/ml of NP-BSA (Biosearch Technologies). After 2 hrs of incubation, horseradish peroxidase-conjugated rabbit anti-mouse IgG (1:4000) or anti-mouse IgM (1:4000) was added and incubated for another hour. The titers of NP-specific IgGs and IgMs in the sera were assessed by measuring the optical density (OD450) after development using tetramethylbenzidine (Thermo Scientific, Rockford, IL).

Statistical Analysis

All data were analyzed using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). To combine results from different proliferation or B cell activation (CD69 and CD86 upregulation) experiments, the following equation was used to normalize the data before combination: relative proliferation (or activated B cells) = [(A−B)/(C−B)] ×100%; where A represents the mean experimental proliferation or activated B cell percentages, B represents the basal cell proliferation or activation (resting B cells without stimuli), and C represents the maximum proliferation or activation (activated B cells alone, 100%). To determine whether groups were statistically different, results were compared using Student’s t test. A p value of <0.05 was considered significant.

Results

HSCs directly inhibit B cells in vitro

To determine whether HSCs have an effect on B cells, we first isolated HSCs from WT C57BL/6 mice and co-cultured different numbers of cells with 0.5 ×106 of purified B cells from WT C57BL/6 mice in the presence of anti-CD40/IL-4 (to activate the B cells). After 24 hrs of incubation, we assessed B-cell-activation status by analyzing the activation markers CD69 and CD86. These experiments showed that, consistent with previous reports (27, 28), expression of both CD69 and CD86 was significantly upregulated on B cells after activation. However, upregulation of both CD69 and CD86 (especially of CD86) was significantly suppressed in proportion to the numbers of HSCs in the co-cultures, suggesting that HSCs inhibited B-cell activation (Fig. 1).

Figure 1.

Figure 1

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HSCs inhibit the upregulation of CD69 and CD86 on activated B cells. B cells were purified from naïve mice by negative selection using magnetic beads and then incubated without (w/o) or with (w/a) anti-CD40 IgG and IL-4 for B cell activation. In some wells containing activating B cells, different numbers of HSCs were added (ratio, HSCs : B cells = 1:10, 1:20 or 1:40). Levels of CD69 and CD86 on B cells were assessed by flow cytometric analysis 24 hrs later. Panels A & B: Representative results from one experiment showing the inhibition of CD69 upregulation (A) and CD86 upregulation (B). Percentage numbers indicate CD69+ or CD86+ cells compared with isotype controls. Panels C & D show combined normalized CD69+ (C) and CD86+ (D) percentages from three independent experiments. Error bars represent SEM, * p < 0.05

We next tested whether HSCs inhibited the proliferation of, and immunoglobulin production from activated B cells, as well as their IL-6 secretion. We cultured different numbers of HSCs with CFSE-labeled B cells in the presence of anti-IgM IgG/IL-4 or anti-CD40 IgG/IL-4 (to activate B cells), then assessed the proliferation of the activated B cells by (a) measuring CFSE dilutions on B cells using flow cytometric analysis and (b) measuring BrdU incorporation in these B cells using a BrdU ELISA. From these experiments, we found that HSCs inhibited the proliferation of activated B cells in a dose-dependent manner, as assessed by both the CFSE- (Fig. 2 A, B, C, D) and BrdU-based (Fig. 2 E, F) B-cell proliferation assays. We also measured levels of IL-6, IgM and IgG in the culture supernatants by respective ELISAs. Because the anti-IgM antibodies we used to activate B cells interfere with sequential measurements of IgM produced by the activated B cells, and because this B-cell activation method leads to little IgG production, we measured levels of IgG/IgM only in co-cultures with B cells activated by the anti-CD40 IgG/IL-4. These ELISA experiments found that, in addition to diminished proliferation, activated B cells also showed reduced production of IL-6, IgM and IgG in proportion to the numbers of HSCs in the co-cultures (Fig. 2 G–J). These and the studies described above, taken together, show that HSCs inhibit B-cell activation, proliferation and IL-6/immunoglobulin production, demonstrating that HSCs have a previously unknown role in directly inhibiting B cells.

Figure 2.

Figure 2

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HSCs inhibit activated B-cell proliferation and IL-6/immunoglobulin production. HSCs were incubated with CFSE-labeled B cells in the absence (w/o) or presence (w/a) of anti-IgM IgG/IL-4 or anti-CD40 IgG/IL-4 at the ratios 1:10, 1:20 or 1:40 (HSCs : B cells). At 72 hrs, proliferation of the activated B cells was assessed by CFSE dilution using flow cytometric analysis or by BrdU incorporation using BrdU ELISA. Panels A&B show representative results of the CFSE-based B cell proliferation assays, panels C&D show combined CFSE-based B cell proliferation assay results from 3 independent experiments and panels E&F show combined BrdU incorporation-based B cell proliferation assay results from 3 independent experiments. Levels of IL-6 (G, H) and/or IgG (I) and IgM (J) in the culture supernatants were measured by respective ELISAs. Error bars represent SEM, *p < 0.05;

HSC cell-surface molecules are important for HSCs to directly inhibit B cells

To explore the mechanism by which HSCs directly inhibit B cells, we carried out transwell experiments in which HSCs were cultured in wells at the bottom, with anti-IgM/IL-4-activated B cells cultured in top inserts that allow only soluble factors to be exchanged, without direct cell-cell contact. We then measured B-cell proliferation and levels of IL-6 produced by the activated B cells in different wells at 72 hrs to assess the efficacy of B-cell inhibition. These assays found that without direct contact of HSCs with B cells, the inhibitory effect of the HSCs was significantly reduced, as indicated by more proliferation of the activated B cells (Fig. 3A&B) and higher levels of IL-6 produced (Fig. 3C) in transwells than in wells with direct cell-cell contact. These results suggest that HSC cell-surface molecules are important for the direct B-cell inhibitory activity of HSCs.

Figure 3.

Figure 3

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Cell-surface molecules are integrally involved in the B-cell-inhibitory activity of HSCs. HSCs and CFSE-labeled B cells were incubated together (cell-cell contact) or in a transwell system (Transwell) in the presence of anti-CD40 IgG/IL-4 to activate B cells. At 72 hrs, proliferation of the activated B cells was assessed by flow cytometric analysis. A, representative results from 3 independent experiments; B. combined results from these 3 independent experiments), and levels of IL-6 in the culture supernatants were measured by ELISA (B). Error bars represent SEM, *p < 0.05.

Blocking PD-L1/PD-1 interactions reduces the B-cell-inhibitory activity of HSCs

We have demonstrated that activated HSCs have elevated levels of PD-L1 on their surface and that these molecules are important for HSCs to suppress T cells via interactions with its ligand, programmed cell death protein 1 (PD-1), on activated T cells14. It has also been reported that PD-1 is present on activated B cells in both mice29 and humans30. In light of this previous work, and the above results that HSC cell-surface molecules are crucial for HSCs to directly inhibit B cells, we first tested a putative role of PD-L1 on HSCs to directly inhibit B-cell activity by blocking PD-L1 on HSCs using a PD-L1 function neutralizing antibody (eBioscience, San Diego, CA). We co-cultured HSCs with different numbers of anti-CD40/IL-4-activated or anti-IgM/IL-4-activated B cells in the presence of either a PD-L1-blocking mAb or control IgGs, then assessed the efficacy of HSCs in inhibiting the activated B cells by measuring the proliferation of the activated B cells and levels of IL-6 produced by the activated B cells in the culture supernatants. From these experiments, we found that under both different B-cell activation protocols, blocking PD-L1 reduced the efficacy of HSCs in inhibiting both the proliferation of (Fig. 4A, B, C & D) and IL-6 production from (Fig. 4E,F) the activated B cells; this result suggests that PD-L1 is required for HSCs to directly inhibit B cells.

Figure 4.

Figure 4

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Blocking PD-L1/PD-1 interactions reduces the potency of HSCs in inhibiting B cells. HSCs were incubated with CFSE-labeled B cells in the absence (w/o) or presence (w/a) of anti-IgM IgG/IL-4 or anti-CD40 IgG/IL-4 at a 1:10 ratio. In some wells containing activated B cells, 10 µg/ml of anti-PD-L1 IgG or control IgG was added. At 72 hrs, proliferation of the activated B cells was assessed by flow cytometry (A & B, representative results from 3 independent experiments, C & D, combined results from 3 independent experiments). Levels of IL-6 in the culture supernatants were measured by ELISA (E, F). In another set of experiments with the same settings, the same concentration of anti-PD-1 IgG or control IgG were added into the co-cultures, and the proliferation of B cells were assessed using the same method. G. representative results from 2 independent experiments, H. combined results from these 2 independent experiments. Error bars represent SEM, *p < 0.05.

We next tested the effect of blocking PD-1 in the HSC-mediated B-cell inhibiting experiments. We incubated CFSE-labeled- and anti-CD40/IL-4-activated B cells with WT HSCs in the presence of 10 µg/ml PD-1-blocking IgGs (BioLegend) or control IgGs for 72 hrs, then analyzed the proliferation of activated B cells by measuring CFSE dilution using flow cytometry. These experiments found that blocking PD-1 significantly improved the proliferation of the activated B cells in the presence of HSCs (Fig. 4G, H). These results, taken together, demonstrated that blocking PD-1/PD-L1 interactions either by an anti-PD-L1 or an anti-PD-1 mAb diminished the newly discovered B-cell-inhibitory activity of HSCs.

Deficiency of PD-L1 on HSCs reduces their B-cell-inhibitory activity

In addition to the above-described PD-L1 and PD-1 blocking experiments, we explored the importance of HSCs in B cell inhibition using a genetic approach by comparing HSCs isolated from WT mice and PD-L1 KO mice (both on the C57BL/6 background) in the same B-cell activation marker upregulation and IL-6/immunoglobulin production assays. These experiments showed that in accordance with the antibody-blocking experiments, comparing with the same numbers of WT HSCs, PD-L1-KO HSCs showed reduced efficacy in inhibiting the upregulation of both CD69 and CD86 on activated B cells (Fig. 5A, B) and in reducing the production of IL-6 and/or immunoglobulins from the activated B cells (Fig.5C–F)), further confirming that PD-L1 on HSCs is required for the efficient inhibition of B cells.

Figure 5.

Figure 5

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Deficiency of PD-L1 on HSCs reduces their ability to inhibit B cells. B cells were incubated without (w/o) or with (w/a) anti-IgM IgG/IL-4 or anti-CD40 IgG/IL-4 for activation. In some wells containing activating B cells, WT or PD-L1-deficient HSCs were added (ratio, HSCs : B cells, 1:10 or 1:20). Levels of CD69 and CD86 on B cells were assessed by flow cytometric analysis 24 hrs later. Percentage numbers indicate CD69+ (A) or CD86+ cells (B) compared with isotype controls. The same experiments were repeated and at 72 hrs, levels of IL-6 (C,D), IgG (E), IgM (F) in the culture supernatants were measured by respective ELISAs. Combined results from 2 independent experiments. Error bars represent SEM, *p < 0.05.

Local delivery of HSCs into mouse spleen by intrasplenic artery injection

To determine whether HSCs directly inhibit B-cell responses in vivo, we used a model in which mice were immunized by i.p. injection with the T-cell-independent antigen NP-Ficoll31. We first tried to deliver HSCs into the mice by intravenous (i.v.) injection into the tail vein after NP-Ficoll immunization, then to evaluate the effect of HSCs in suppressing the in vivo production of NP-specific IgG/IgM. However, most mice died of pulmonary embolism immediately after tail vein i.v. injection of HSCs (not shown), presumably as a result of the large size of HSCs. To solve this problem, in view of previous reports that after i.p. immunization of NP-Ficoll, most of the reactive, IgM/IgG-producing B cells are located within the marginal zone of the spleen(32, 33), we first developed a protocol to locally deliver HSCs into the spleen by intrasplenic artery injection. In brief, we anesthetized the mice and performed laparotomy to identify the splenic artery, then injected 0.2 × 106 of red Vybrant Dil-labeled HSCs in 50 µl of sterile PBS into the spleen through the splenic artery using a wire-guided catheter exchange technique. To demonstrate that the HSCs had successfully been delivered into the spleen, at sacrifice, we took out the injected spleen to prepare cryosections and examined them for the presence of HSCs (red cells) under a fluorescence microscope. These experiments found that red HSCs were distributed throughout the spleen in the red pup after intrasplenic artery injection (Fig. 6), suggesting that HSCs can be locally delivered into the spleen by this approach without inducing lethal pulmonary embolism in mice.

Figure 6.

Figure 6

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Local delivery of HSCs into mouse spleen by intrasplenic artery injection. For each mouse, the splenic artery and vein (arrows) were visually identified and separated from the mesenteric adipose tissues (A, B). Red Vybrant Dil-labeled HSCs (0.2 × 106) in 50 µl of sterile PBS were injected into the spleen through the splenic artery using a wire-guided catheter exchange technique. At sacrifice, splenic cryosections were prepared to examine the distribution of the injected red HSCs under a fluorescence microscope at 100× (C), 200× (D) and 400× (E). Bright field pictures of the same area were also taken at the same time (F,G & H).

HSCs inhibit the in vivo production of NP-specific antibodies after NP-Ficoll immunization

After establishing the protocol for local delivery of HSCs into the spleen through intrasplenic artery injection, we next tested the efficacy of HSCs in inhibiting B cells in vivo and the role of PD-L1 in this process. We first injected each mouse with 0.2 × 106 of WT or PD-L1-KO HSCs using our established intrasplenic artery injection method; sham-operated mice without HSC injection were included as controls. At 24 hrs after the injection, we immunized the mice with NP-Ficoll by i.p. injection, then collected blood from the tail vein on Day 3, 9 and 14. We measured serum levels of NP-specific IgM and IgG using respective ELISAs. These experiments showed that, for IgM measurements, on Day 3, NP-specific IgMs were barely detectable (data not shown). On Day 9 after local delivery of HSCs, compared with NP-immunized mice with sham operations, immunized mice with injected WT HSCs (but not PD-L1-KO HSCs) showed significantly reduced titers of NP-specific IgMs (Fig. 7A). By Day 14, there was no difference of NP-specific IgM titers among the three groups (Fig. 7B). For IgG measurements, again, there were no detectable NP-specific IgGs on Day 3 (data not shown). On Day 9, NP-specific IgGs started to be measurable in the sera, and compared with the NP-immunized sham mice, NP-immunized mice injected with WT HSCs (but not PD-L1-KO HSCs) showed significantly reduced titers of NP-specific IgGs (Fig. 7C). The differences of NP-specific IgG titers among the groups of mice were even more significant on Day 14 (Fig. 7D), demonstrating that HSCs directly inhibited B cells in vivo and that PD-L1 on HSCs are required in this process.

Figure 7.

Figure 7

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WT but not PD-L1-deficient HSCs inhibit NP-specific IgM and IgG production in vivo. WT or PD-L1-deficient HSCs (0.2 × 106 per mouse) were injected into the spleen by intrasplenic artery injection, and sham-operated mice without cell injection were included as controls. After injection, mice were immunized with NP-Ficoll (10 µg/mouse), and serum samples were collected at different time points. ELISA using NP-BSA-coated plates were used to measure the titers of NP-specific IgM on Day 3 (not shown), Day 9 (A) and Day 14 (B) and of NP-specific IgG on Day 3 (not shown), Day 9 (C) and Day 14 (D). Each dot represents one mouse. *p < 0.05.

Discussion

To examine whether HSCs concurrently inhibit B cells as well as T cells, we developed an intrasplenic artery injection approach to locally deliver HSCs into the spleen to test the effects of HSCs on B cells in vivo. We found that HSCs directly inhibit B cells both in vitro and in vivo and that PD-L1 expressed by HSCs is integrally involved in these progresses. These new results, together with our previous report showing that HSCs inhibit T cells through PD-L1, suggest that PD-L1 is needed for HSCs to concurrently inhibit both T and B cells in the adaptive immune system, which could be an important mechanism by which HSCs help to maintain liver homeostasis.

Although T cells have been thought to be the major mechanism underlying allograft rejection, recently, an increasing number of studies have suggested that B cells also play important roles not only in allograft rejection34, but also in many other pathological situations (e.g., multiple sclerosis and autoimmune uveitis) in which T cells were thought to be the major player in the pathogenesis35. B cells can induce tissue damage by secreting pathological antibodies or by producing cytotoxic cytokines, including IL-6. They can also shape the splenic architecture36 and serve as antigen-presenting cells to stimulate T cells 37, mechanisms that amplify T-cell-mediated tissue damage. Under normal conditions, B cells reside in the liver together with T cells (19, 20). Upon stimulation with lipopolysaccharides, these hepatic B cells produce massive amounts of inflammatory cytokines, including IL-6, IFNγ and TNFα, but little IL-10, which could initiate liver inflammation in situ 18. In addition, B-cell infiltration has been found in transplanted renal and liver allografts during acute rejection, and consequently, depletion of B cells with rituximab ameliorates renal graft rejection38. This B-cell depletion therapy is emerging as a new and effective approach for treating many autoimmune diseases that were originally thought to be mediated by T cells39. Our discovery that HSCs directly inhibit B cells adds a new mechanism to explain how HSCs regulate immune reactions in the liver, which could contribute to the importance of HSCs in maintaining the immune tolerance status of the liver.

In transwell experiments, when HSCs were cultured separately from activated B cells, their power to inhibit B cells was significantly impaired, suggesting that direct cell-cell contact is required for HSCs to efficiently inhibit B cells. This finding led us to investigate the role of PD-L1, expressed on the surface of HSCs, in this process. However, in the presence of large numbers of HSCs (at a 1:10 ratio), even without direct contact of HSCs with B cells in the transwell culture system, B cells were still inhibited by the HSCs to some degree, suggesting that some soluble factors produced by the HSCs still play a role in the HSC-mediated, direct B-cell inhibition. These soluble factors remain to be fully characterized.

PD-L1 is a member of the B7 family, serving as a major ligand of PD-1, which is a strong, inducible, negative immune modulatory receptor present on activated T and B cells. PD-L1 is expressed on the cells of many different tissues, including many tumor cells, retinal pigment epithelial cells and HSCs, and PD-L1 expression is also significantly upregulated in response to inflammation. Interactions between PD-L1 on these peripheral tissue cells and PD-1 on activated T cells potently suppress T-cell responses, which in turn enables tumors to escape immunosurveillance and help organs like the eye or liver to maintain their immunoprivileged status. Reagents targeting PD-L1 or PD-1 are under intensive study in clinical trials for cancer treatment, with positive preliminary results. Pembrolizumab and nivolumab, humanized anti-PD-1 mAbs, have recently been approved for treating unresponsive melanoma 40 and non-small cell lung cancer41, further confirming that the PD-L1/PD-1 interaction is a potent T-cell inhibitory mechanism.

Although the PD-L1/PD-1 pathway in inhibiting T cells has been extensively investigated, the significance of this pathway in regulating B-cell responses remains understudied. Since early reports in the 1990s showing that PD-1 is present on activated B cells29 and that PD-1-KO mice develop elevated levels of autoantibodies42, there has been only one follow-up report in 2013 showing that PD-1 is important in regulating B-cell function30. Practically all investigative efforts had focused on the role of PD-1 on T cells in mediating T-cell suppression. The impact of PD-1 on B cells and its interaction with PD-L1 on other cells has been almost neglected. By flow cytometric analysis, we found that, consistent with previous reports29, PD-1 is present on activated B cells (data not shown), and our results, described above, indicate that HSCs directly suppress B cells through PD-L1, which interacts with PD-1 on B cells, to inhibit the proliferation of and antibody/cytokine production from activated B cells. These results provide further evidence suggesting that the PD-L1/PD-1 pathway is an important pathway regulating B-cell function.

Systemic i.v. injection of cells is the most common approach for cell-based therapy studies in both animals and humans. It is established that immediately after i.v. injection, most cells are first trapped in the lung; some of them then migrate out and into other organs. Although in mice, tail vein i.v. injection has been successfully employed to systemically deliver many cell types (e.g., dendritic cells43, myeloid-derived suppressor cells44, and mesenchymal stem cells45), we found that tail vein i.v. injection of primary HSCs led to lethal pulmonary embolisms, potentially due to the large size of HSCs, which made our in vivo studies to test the effect of HSCs in inhibiting B-cell responses impossible. Since it has been demonstrated that after i.p. NP-Ficoll immunization, most of the reactive B cells are located in the marginal zone of the spleen, we hypothesized that we could locally deliver HSCs into the spleen to suppress the NP-Ficoll-responding B cells without causing pulmonary embolisms. Although direct intrasplenic injection has been employed to deliver cells including hepatocytes46 and tumor cells47 into the spleen in experimental studies, this approach was not useful for our purposes, as it would likely cause splenic injury and uneven distribution of the injected cells in the spleen. Given the sinusoidal structure of the spleen, we speculated that intrasplenic artery injection of the HSCs would be a better approach to locally delivering HSCs, thus enabling them to directly interact with marginal zone B cells. Indeed, we found that intrasplenic artery injection was practical.

HSCs are one of the major components of nonparenchymal cells in the liver11, and the significance of HSCs in liver fibrosis has been the focus of intensive studies. Emerging evidence also suggests that HSCs are an important group of resident cells that regulate immune responses in the liver. We hypothesized that HSCs are essential for the liver to maintain its homeostasis. In support of this concept, we and others have found that HSCs are strongly immunosuppressive(16, 48). We further demonstrated that HSCs inhibit T cells through PD-L116 and that HSCs induce the propagation of myeloid-derived suppressor cells, which concurrently inhibit both T and B cells44. The results presented in this report establish that in addition to the previous discoveries, HSCs also directly inhibit B cells and that the PD-L1 expressed on the surface of HSCs is integrally involved in the underlying mechanism.

In summary, we examined the potential role of HSCs in regulating B cells, the second major component of the adaptive immune system. We found that HSCs directly inhibited B cells through PD-L1/PD-1 interactions in vitro. By establishing a protocol to locally deliver HSCs into the spleen through intrasplenic artery injection, we further confirmed the direct B-cell-inhibiting activity of HSCs and the importance of the PD-L1/PD-1 interaction in vivo. These results established a new approach to locally deliver HSCs and other large cells into the spleen without causing pulmonary embolism and demonstrated a novel mechanism by which HSCs regulate immune responses in the liver.

Supplementary Material

1

Acknowledgments

Supported in part by Muscular Dystrophy Association Grant 234458 (FL) and NIH grant AR061564 (FL)

References

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