Local accumulation and activation of regulatory Foxp3+ CD4 TR cells accompanies the appearance of activated CD8 T cells in the liver†
Article first published online: 10 NOV 2008
Copyright © 2008 American Association for the Study of Liver Diseases
Volume 48, Issue 6, pages 1954–1963, December 2008
How to Cite
Bochtler, P., Riedl, P., Gomez, I., Schirmbeck, R. and Reimann, J. (2008), Local accumulation and activation of regulatory Foxp3+ CD4 TR cells accompanies the appearance of activated CD8 T cells in the liver. Hepatology, 48: 1954–1963. doi: 10.1002/hep.22559
Potential conflict of interest: Nothing to report.
- Issue published online: 24 NOV 2008
- Article first published online: 10 NOV 2008
- Manuscript Accepted: 26 JUL 2008
- Manuscript Received: 14 MAR 2008
- Deutsche Forschungsgemeinschaft. Grant Number: Re 549/10-3
Only small populations of nonactivated, nonproliferating Foxp3+ CD4 regulatory T cell (TR) cells are found in the nonparenchymal cell compartment of the mouse liver while liver-draining celiac nodes contain expanded, activated TR cell populations (similar to other lymph nodes). Liver Foxp3+ CD4 TR cells suppress activation of T cell responses. Polyclonal, systemic T cell activation in vivo (via anti-CD3 antibody injection) is accompanied by intrahepatic accumulation of T blasts and a rapid but transient intrahepatic increase of activated, proliferating Foxp3+ CD4 TR cells. Following vaccination, the appearance of peripherally primed, specific CD8 T blasts in the liver is preceded by a transient rise of Foxp3+ CD4 TR cells in the liver. The adoptive transfer of immune CD8 T cells into congenic hosts that express the relevant antigen only in the liver leads to the accumulation of specific donor CD8 T cells and of host Foxp3+ CD4 TR cells in the liver. Conclusion: Although it contains only a small population of quiescent Foxp3+ CD4 TR cells, the liver can rapidly mobilize and/or recruit this T cell control in response to the intrahepatic appearance of peripherally or locally generated CD8 T blasts. (HEPATOLOGY 2008;48:1954-1963.)
Specific priming and recall of T cell immunity, potent induction of T cell tolerance, and elimination of activated CD8 T blasts are observed in the liver.1 Different mechanisms have been proposed to contribute to the control of immune responses in the liver including different types of regulatory T cells.2 In addition to interleukin (IL)-10–producing CD4 regulatory T cell (TR) cells and transforming growth factor β–producing TH3 cells, Foxp3+ CD4 TR cells are the best characterized regulatory T cell subset.2, 3 CD25hi Foxp3+ CD4 TR cells have been analyzed in patients with chronic hepatitis B and hepatitis C virus infection, autoimmune liver diseases, and hepatocellular carcinoma. High numbers of functional CD25hi CD4 TR cells were found in peripheral blood and livers of patients with chronic hepatitis B4, 5 and chronic hepatitis C.6, 7 Enhanced numbers of functional CD25hi Foxp3+ CD4 TR cells were also found in peripheral blood8 and locally in and around growing tumors in patients with hepatocellular carcinoma.9 Emergence of autoimmune complications (cryoglobulinemia and vasculitis) in chronic hepatitis C infection coincided with a reduction of CD25hi Foxp3+ CD4 TR cell numbers.10 Reduced Foxp3+ CD4 TR cell numbers were also found in patients with autoimmune liver diseases,11 including primary biliary cirrhosis.12 These clinical data indicate that local and systemic numbers of functional CD25hi Foxp3+ CD4 TR cells increase in chronic infection with hepatotropic viruses and in cancer but decrease in T cell–mediated autoimmune liver diseases. Murine models have confirmed the key role of CD4 Foxp3+ TR cells in controlling infectious diseases, tumor growth, allotolerance, or autoimmune diseases.13 In this report, we characterize the intrahepatic CD4 Foxp3+ TR cell population in wild-type B6 mice and investigate its response to local or systemic CD8 T cell activation.
Materials and Methods
Wild-type (WT), interferon (IFN)-γ−/−, IFN-β−/−, type I IFN receptor (IFNAR)−/−, CD1d−/−, IL-10−/−, PD-1−/− and PD-L1−/−, HBs-tg, OT-I RAG1−/−, and Foxp3EGFP (B6.Cg-Foxp3tm2Tch/J, Jackson stock no.006772) mice, all on the C57BL/6 (B6) background, were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). PD-L1−/− mice were kindly provided by Dr. L. Chen (The Johns Hopkins University, Baltimore, MD). PD-1−/− mice were a generous gift of Dr. T. Honjo (Kyoto University, Kyoto, Japan). Germ-free B6 mice generated and bred in the animal facility of Ulm University were screened weekly for viral, bacterial, and fungal contamination. Female and male mice aged 8 to 10 weeks were used. All animal experiments were performed according to the guidelines of the local Animal Use and Care Committee and the National Animal Welfare Law.
Vaccination of Mice and Determination of Specific CD8 T Cell Frequencies.
Mice were immunized intramuscularly with the hepatitis B surface antigen (HBsAg)- or S-encoding pCI/S DNA vaccine (50 μg per mouse) or the antigenic/cationic S190-197(S2) VWLSVIWM-KKRRQRRR peptide (20 μg/mouse) complexed to oligodeoxynucleotide (ODN).14 OT-I RAG1−/− mice were immunized intramuscularly with 0.5 μg/mouse OVA257-264 peptide mixed with 10 μg ODN. Kb/S2 tetramer+ CD8 T cells and IFNγ+ CD8 T cells (induced by a 4-hour ex vivo specific peptide restimulation) were determined as described.14
Flow Cytometry Analysis.
Cells were stained with antibodies from BD Biosciences or eBioscience and analyzed with FACSCalibur (Becton & Dickinson, Mountain View, CA). Data were processed using FCS Express V3 (DeNovo) software. Fluorescence-activated cell sorting was performed on a FACSAria (BD Biosciences) by Dr. M. Rojewski (DRK, Ulm, Germany). FoxP3 staining was performed using a kit (eBioscience).
Isolation, Activation, Carboxyfluorescein Diacetate Succinimidyl Ester Labeling, and Coculture of T Cells.
T cells were activated in vivo via injection of 50 μg anti-CD3ϵ antibody 145-2C11 or isotype-matched hamster IgG1/κ control antibody (BD Biosciences). Spleen, lymph node (LNs), and nonparenchymal cells (NPCs) of the liver were prepared as described.15 CD4 and CD8 T cells were purified from spleens using the CD4 or CD8 T cell magnetic bead-activated cell sorting isolation kit (Miltenyi Biotec). The purity of the isolated CD8 or CD4 T cells was >98% as verified via flow cytometry. T cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) for 15 minutes at 37°C and the reaction was stopped with cold FCS. Purified CD4 or CD8 T cells (4 × 104/well) were cocultured with electronically sorted (CD25hi or GFP+) regulatory CD4 T cells (2 × 104/well) from spleen or liver for 96 hours. T cells were stimulated by anti-CD3/CD28 antibody-coupled MicroBeads (Invitrogen) at a bead/T cell ratio of 1:2.
Cytokine Determination via Enzyme-Linked Immunosorbent Assay.
Cytokines were detected in supernatants by conventional double-sandwich enzyme-linked immunosorbent assay. For detection and capture we used the following antibodies (BD Biosciences): JES6-1A12 and JES6-5H4 for IL-2; OptEIA ELISA Kit for IL-10; R4-6A2 and XMG1.2 for IFNγ. Extinction was analyzed at 405/490 nm on a TECAN microplate reader (TECAN, Crailsheim, Germany) using the EasyWin software (TECAN).
Spleen cells or purified splenic CD8 T cells pooled from 8 to 10 HBsAg-immune CD45.1 B6 donor mice were transferred intravenously into naïve, WT, or HBs-tg CD45.2 B6 hosts. The transferred cell numbers were adjusted so that the injected cell populations contained 3 × 105 specific (Kb/S2 tetramer+) CD8 T cells. Purified splenic CD45.1 CD4 T cells (5 × 106/mouse) were transferred intravenously into CD45.2 OT-I RAG1−/− B6 hosts.
Detection of In Vivo Proliferating Cells.
Mice were injected intraperitoneally with 1 mg bromodeoxyuridine (BrdU). Cells obtained 2 hours after injection were stained for incorporated BrdU using a kit (BD Bioscience).
Detection of Serum Alanine Aminotransferase.
Serum alanine aminotransferase (ALT) levels were measured using the Reflotron Plus analyzer (Roche Diagnostics, Germany).
Statistical analyses were performed with GraphPad Prism4 software. Data are exexpressed as the mean ± standard error of the mean. A P value of <0.05 was considered statistically significant (unpaired t test).
Activated CD8 T Blasts Accumulate in the Liver.
The liver selectively recruits activated T blasts from the circulating T cell population.1 Under steady state conditions, 30% to 50% of the CD8 T cells in the hepatic NPC population of young standard pathogen-free (SPF) B6 mice showed the CD69hi CD44hi CD62Llo surface phenotype (Fig. 1A). Following intramuscular immunization (into the leg), tetramer+ CD8 T blasts transiently appeared in the liver at day 10 to 14 after vaccination (Fig. 1B). At the peak of this response, Kb/S2 epitope–specific CD8 T blasts often represented 15% to 20% of the intrahepatic CD8 T cell population.16 Bystander hepatitis (collateral injury of hepatocytes during intrahepatic accumulation of specific CD8 T cells after extrahepatic virus infection) has been described.17 This raises the question of what controls potentially aggressive CD8 T blast populations that accumulate in this organ under steady state conditions, but especially after vaccination or infection.
Only Few Regulatory Foxp3+ CD4 TR Cells Are Found in the Liver NPC Population.
We found only low numbers of Foxp3+ CD4 TR cells in liver NPC populations. Whereas 10% to 15% of the CD4 T cells in spleen and LNs were Foxp3+, only 2% to 4% of the cells in intrahepatic, conventional CD4 T cell populations (excluding CD1d/αGalCer dimer+ CD4int natural killer T cells) expressed Foxp3 (Fig. 2A). Liver-draining celiac LNs (cLNs) contained 10% to 15% Foxp3+ CD4 TR cells typical for other LNs (Fig. 2A). Only ≈2 × 104 Foxp3+ CD4 TR cells were found in the individual mouse liver while ≈5 × 104 Foxp3+ CD4 TR cells were isolated from liver-draining cLNs (Table 1). In contrast, spleens contained 100-fold higher numbers (≈2 × 106 Foxp3+ CD4 TR cells) (Table 1). The Foxp3+ CD4 TR cell population in the liver is thus unexpectedly small.
|Tissue*||CD4 T Cells per Organ (×104) ± SEM†||Foxp3+ CD4 TR Cells‡|
|Per Organ (×104) ± SEM||Percentage of CD4 T Cells ± SEM|
|NPCs||46.54 ± 22.00||1.81 ± 1.14||3.05 ± 1.25|
|cLNs||40.88 ± 10.38||5.01 ± 2.60||12.30 ± 1.77|
|Secondary lymphoid tissues|
|Spleen||1603.69 ± 363.21||191.53 ± 62.17||11.94 ± 3.06|
|Inguinal LNs||124.41 ± 4.80||14.62 ± 1.63||11.73 ± 0.86|
Compared with splenic and LN Foxp3+ CD4 TR cells, 90% of the intrahepatic Foxp3+ CD4 TR cells expressed low levels of Foxp3 (Fig. 2A). Intrahepatic Foxp3+ CD4 TR cells are less activated than their counterparts in spleen or LN (Fig. 2B). Low surface expression of the IL-2 receptor α-chain (CD25) is unusual for Foxp3+ CD4 TR cells but (together with low surface expression of CD69) indicates low activation under steady state conditions. The small Foxp3hi subset showed higher surface expression of CD69 and CD25 (data not shown), suggesting that activation up-regulates Foxp3 expression. Fairly high CD103 (αE integrin) surface expression may be of interest, because this adhesion molecule binds E-cadherin expressed on the surface of most hepatocytes.18 Expression of CD127 (IL-7 receptor) by hepatic Foxp3+ CD4 TR cells was low, a phenotype characteristic of these regulatory T cells.19 Foxp3+ CD4 TR cells in liver-draining cLNs had an activated surface phenotype similar to that of splenic Foxp3+ CD4 TR cells. Enhanced constitutive activation of liver Foxp3+ CD4 TR cells is hence not observed.
Germ-Free and SPF Mice Contain Comparable Populations of Foxp3+ CD4 TR Cells in the Liver.
In SPF but not germ-free (GF) mice, the liver is continuously challenged by gut flora-derived constituents delivered through the portal vein. The number and surface phenotype of Foxp3+ CD4 TR cells in splenic and intrahepatic CD4 T cell populations were similar in age- and sex-matched GF and SPF mice (Fig. 3). The number of conventional CD4 T cells in the liver (but not in the spleen) of GF mice is reduced by 50% (data not shown). Hence, the numbers of intrahepatic Foxp3+ CD4 TR cells were lower in GF than SPF mice. Gut flora-derived constituents thus do not selectively regulate the size or activation status of the liver Foxp3+ CD4 TR cell population.
Enhanced Numbers of Intrahepatic Foxp3+ CD4 TR Cells in PD-1−/−/PD-L1−/− B6 Mice.
We determined intrahepatic Foxp3+ CD4 TR cell numbers and their phenotype in (age- and sex-matched) B6 mice deficient in IFN (IFNγ−/−, IFNβ−/−, IFNAR−/−), IL-10, PD-1, PD-L1, or CD1d (Fig. 4). Deficiency in interferons or IL-10 had no effect on intrahepatic Foxp3+ CD4 TR cell numbers. Natural killer T cell–deficient CD1d−/− B6 mice have normal numbers of intrahepatic Foxp3+ TR cells with a similar surface phenotype as WT mice. Foxp3+ CD4 TR cells numbers were increased in liver NPC but not spleens of PD-1−/− and PD-L1−/− mice (Fig. 4). PD-1 and PD-L1 deficiency leads to increased numbers of T blasts that accumulate in the liver.20, 21 The intrahepatic increase of Foxp3+ CD4 TR cells may be a response to this T blast accumulation in the liver. Alternatively, PD-1 or PD-L1 deficiency of Foxp3+ CD4 TR cells themselves may drive their activation and expansion.
Polyclonal, Systemic T Cell Activation Increases the Number of Activated, Proliferating Foxp3+ CD4 TR Cells in the Liver.
Polyclonal T cell activation triggered by injection of mitogen, anti-CD3 antibody, superantigens, or antigenic peptides (into TCR transgenic mice) leads to acute and extensive accumulation of T blasts in the liver, often associated with liver injury.1 We tested if activation of resident T cells and/or influx of activated T blasts in the liver are associated with intrahepatic activation and proliferation of Foxp3+ CD4 TR cells. The numbers of intrahepatic CD4 T cells increased two- to three-fold, and the numbers of Foxp3+ CD4 TR cells increased eight- to ten-fold following injection of anti-CD3 antibody by day 3 (Fig. 5A). Foxp3+ CD4 TR cell numbers in the spleen and in cLNs did not increase (Fig. 5A, data not shown). Antibody-injected, splenectomized mice showed a similar increase in intrahepatic Foxp3+ CD4 TR cell numbers (data not shown). Hence, T cell traffic from the spleen to the liver is not a major source of intrahepatic T blast accumulation. This systemic, polyclonal T cell stimulation also activated intrahepatic Foxp3+ CD4 TR cells, which was apparent by the up-regulated surface expression of CD69, CD25, and PD-L1 (Fig. 5B). A 2-hour BrdU pulse in vivo 48 hours after anti-CD3 antibody injection labeled 10% of intrahepatic (regulatory) Foxp3+ and (nonregulatory) Foxp3− CD4 T cells but 25% to 30% of intrahepatic CD8 T cells (Fig. 5C). Hence, proliferation seems to contribute to the intrahepatic expansion of the Foxp3+ CD4 TR cell population. Transient but minor liver injury (apparent as a rise in serum aminotransferase levels) was apparent after antibody injection (Fig. 5D). Intrahepatic Foxp3+ CD4 TR cells can thus be activated and expanded, but it is uncertain if this results from direct stimulation by anti-CD3 antibody or indirectly in response to polyclonal T cell activation. The following experiments tested if intrahepatic Foxp3+ CD4 TR cells also respond to the influx of recently activated T blasts generated in vaccine-induced CD8 T cell responses in the periphery.
Intrahepatic Foxp3+ CD4 TR Cells Transiently Respond to Specific CD8 T Cell Accumulation in the Liver After Vaccination.
In individual mice vaccinated intramuscularly (into the leg) with plasmid DNA encoding HBsAg, we followed the appearance of Kb/S2-specific CD8 T cells in the spleen and the liver, as well as the numbers of splenic and intrahepatic Foxp3+ CD4 TR cells. A 10% to 15% increase in Foxp3+ CD4 TR cell numbers was observed in spleens, and a 30% to 40% increase in Foxp3+ CD4 TR cell numbers was observed in livers during the influx of recently activated, specific CD8 T blasts into these tissues (Fig. 6A). Interestingly, the increase in Foxp3+ CD4 TR cell numbers preceded the rise in specific CD8 T cell numbers. It was already apparent at a time point at which only low numbers of specific CD8 T cells were detectable in the spleen or liver. Similar data were obtained when we primed a monospecific CD8 T cell response (with the same restriction/epitope specificity) using a cationic peptide vaccine14 (Fig. 6B). A peptide-primed, monospecific CD8 T cell response in TCR-tg OT-I RAG1−/− mice recruits adoptively transferred Foxp3+ CD4 TR cells into the liver (Fig. 6C). Liver Foxp3+ CD4 TR cells thus are recruited and/or increased in number in response to the influx of recently primed CD8 T blasts into this organ.
Intrahepatic Foxp3+ CD4 TR Cells Respond to Specific Activation of CD8 T Cells in the Liver.
We primed and boosted CD8 T cell responses to HBsAg in CD45.1 B6 mice (Fig. 7A). Splenic CD8 T cells containing 3 × 105 tetramer+ CD8 T cells were adoptively transferred from immune donors into HBs-tg CD45.2 B6 hosts that express HBsAg selectively in the liver. Tetramer+ CD45.1 CD8 T cells were found after transfer in the liver but not the spleen of the adoptive host (Fig. 7B). Immune but not nonimmune CD8 T cells induced transient but severe hepatocyte injury in transgenic hosts (Fig. 7C). The numbers of intrahepatic, host-derived CD45.2 Foxp3+ CD4 TR cells increased seven-fold following the appearance of Kb/S-specific CD8 T cells in the antigen-presenting liver (Fig. 7D), expressed a CD69hi CD25hi surface phenotype (data not shown) and remained high for more than 7 days after transfer (Fig. 7D). Conventional CD4 T cells increased only four-fold after transfer (Fig. 7D). Antigen-driven, local CD8 T cell activation thus triggers an increase in number and activation of the Foxp3+ CD4 TR cell population in the liver.
Hepatic Foxp3+ CD4 TR Cells Are Suppressive.
Foxp3+ CD4 TR cells were purified from spleen or liver of Foxp3EGFP B6 mice (that carry an EGFP knock-in into the Foxp3 transcription factor-encoding locus) (Fig. 8A). Alternatively, liver Foxp3+ CD4 TR cells were activated in vivo by anti-CD3 antibody injection to allow sorting of CD25hi liver TR cells (Fig. 8B). Sorter-purified, splenic, or hepatic (GFP+ or CD25hi) Foxp3+ CD4 TR cells were mixed with purified, naïve, CFSE-labeled CD4 or CD8 T cells and stimulated with anti-CD3/CD28 antibody-coated beads. Stimulated CD4 and CD8 T cells produced IL-2 and IFNγ (but no IL-10). Both splenic and hepatic Foxp3+ CD4 TR cell populations suppressed production of IL-2 and IFNγ of cocultured CD4 or CD8 T cells and down-regulated their proliferative response (Fig. 8A). Splenic and hepatic CD25hi Foxp3+ CD4 TR cells produced IL-10 but not IL-2 in response to stimulation (Fig. 8B). Liver Foxp3+ CD4 TR cells hence suppress IL-2 and IFNγ production and proliferation of activated CD4 and CD8 T cells.
Only small populations of potentially functional Foxp3+ CD4 TR cells are found in the liver NPC compartment. The intrahepatic Foxp3+ TR cell population is readily activated and expanded in response to polyclonal, systemic T cell activation (triggered by anti-CD3 antibody injection) or the appearance of specific CD8 T cell blasts in the liver (following their peripheral, vaccine-induced oligoclonal activation). Intrahepatic Foxp3+ CD4 TR cells are increased in number when CD8 T cells respond locally to their cognate antigen presented by hepatocytes. Though small and quiescent under steady state conditions, intrahepatic Foxp3+ CD4 TR cell populations thus rapidly respond to the local appearance of CD8 T blasts.
The total number of Foxp3+ CD4 TR cells per liver is 100-fold lower than the total number of Foxp3+ CD4 TR cells in the spleen of young adult mice (Table 1). Under steady state conditions, most intrahepatic Foxp3+ CD4 TR cells show low surface expression of CD69, CD25, and PD-1, and low expression of the Foxp3 transcription factor. The small Foxp3hi subset in the liver Foxp3+ CD4 TR cell population showed higher CD69 CD25 surface expression indicating that Foxp3 expression levels correlate with activation. Factors that activate a response of intrahepatic Foxp3+ CD4 TR cells are unknown. In polyclonal, systemic T cell activation by anti-CD3 antibody, direct activation of intrahepatic Foxp3+ TR cells in situ by CD3ϵ ligation is likely. Gut flora-derived constituents do not seem to activate or expand the intrahepatic Foxp3+ CD4 TR cell populations. The intrahepatic Foxp3+ TR cell response was triggered equally well by a DNA vaccine encoding HBsAg that primes CD8 and CD4 T cell responses and a cationic/antigenic peptide vaccine that primes monospecific CD8 T cell responses.14 Hence, this response can be elicited by CD8 T cell responses. Foxp3+ CD4 TR cells were not specifically activated by the antigenic peptide used as vaccine to prime monospecific CD8 T cells responses (data not shown). Foxp3+ CD4 TR cell expansion occurred in spleen and liver days before the numbers of specifically primed CD8 T cells peaked. A systemic signal of unknown identity generated early in CD8 T cell responses may mobilize Foxp3+ CD4 TR cell populations in spleen and liver—that is, the regulatory T cell compartment may sense the unfolding of a peripheral CD8 T cell response similar to what has been described for natural killer T cells.22 Alternatively, low numbers of CD8 T blasts migrating at an early time point from the site of vaccine injection may elicit the Foxp3+ CD4 TR cell responses. The available data do not allow us to distinguish these possibilities.
The most extensive response of intrahepatic Foxp3+ TR cells was induced when CD8 T cells recognized their cognate antigen selectively presented by hepatocytes. In this system, primed tetramer+ CD45.1 CD8 T cells were intravenously injected into syngeneic CD45.2 hosts that expressed the relevant antigen (HBsAg) in hepatocytes. Tetramer+ CD8 T cells of donor origin were found only in the liver of the adoptive host indicating efficient and selective trapping of specific CD8 T cells by the target organ. Because the host could not sense the developing CD8 T cell response, the intrahepatic attack ran largely uncontrolled for the first days, as evident by rising serum ALT levels (Fig. 7C). The intrahepatic Foxp3+ CD4 TR cell numbers increased seven-fold within 3 days after transfer and developed an activated surface phenotype. As expected, all Foxp3+ CD4 TR cells in the liver were of host origin. Serum ALT levels, the number of CD4 and CD8 T cells, and the number of specific CD8 T cells declined in the liver starting at day 3 after transfer. In contrast, the number of intrahepatic Foxp3+ CD4 TR cells remained high for more than 7 days but normalized in the second and third week after transfer (data not shown). The intrahepatic regulatory Foxp3+ CD4 TR cell compartment thus responds to CD8 T cell responses primed either in the periphery or triggered in situ. The correlation between CD8 T blasts appearing in the liver and the intrahepatic increase in Foxp3+ CD4 TR cells is intriguing, but further work is needed to identify the mediators involved in the local cross talk of Foxp3+ CD4 TR cells with CD8 effector cells (that they presumably control).
We greatly appreciate the expert technical assistance of Ellen Allmendinger and Ina Sebald. We thank Prof. G. Adler for continuous support.