Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating...

Liver Sinusoidal Endothelial Cells Contributeto CD8 T Cell Tolerance Toward Circulating

Carcinoembryonic Antigen in MiceBastian H€ochst,1 Frank A. Schildberg,1 Jan B€ottcher,1 Christina Metzger,1 Sebastian Huss,2,3

Andreas Turler,4 Markus Overhaus,4 Andreas Knoblich,5 Berthold Schneider,5 Dimitrios Pantelis,6

Christian Kurts,1 J€org C. Kalff,6 Percy Knolle,1 and Linda Diehl1

Immunity against cancer is impeded by local mechanisms promoting development of tu-mor-specific T cell tolerance, such as regulatory T cells, myeloid-derived suppressor cells,or immunosuppressive factors in the tumor microenvironment. The release of soluble anti-gens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, hasbeen investigated for diagnostic purposes, but not for its immunological consequences.Here, we address the question of whether soluble CEA influences tumor-specific immunity.Mice were injected with soluble CEA protein, and CEA-specific CD8 T cells were analyzedfor their phenotype and functionality by means of restimulation ex vivo or antitumor effi-cacy in vivo. We furthermore characterized the CD8 T cell population in peripheral bloodmononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. Inmice, circulating CEA was preferentially taken up in a mannose receptor–dependent man-ner and cross-presented by liver sinusoidal endothelial cells, but not dendritic cells, toCD8 T cells. Such systemically circulating CEA promoted tolerization of CEA-specificCD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1.These CD8 T cells were not deleted but were rendered nonresponsive to antigen-specificstimulation and failed to control growth of CEA-expressing tumor cells. These nonrespon-sive CD8 T cells were phenotypically similar to central memory T cells beingCD44highCD62LhighCD25neg. We found T cells with a similar phenotype in PBMCs ofhealthy donors and at increased frequency also in patients with colorectal carcinoma.Conclusion: Our results provide evidence for the existence of an unrecognized tumorimmune escape involving cross-presentation of systemically circulating tumor antigensthat may influence immunotherapy of cancer. (HEPATOLOGY 2012;56:1924-1933)

The interaction of the immune system with can-cer cells is complex and may affect both cancerdevelopment and progression. The concept of

cancer immune surveillance has been refined andtermed ‘‘cancer immunoediting.’’1 This conceptualframework of the role of the immune system in cancerincludes three distinct phases: an elimination phase, inwhich tumor cells are killed; an equilibrium phase, inwhich the immune system controls growth of cancercells; and finally, the escape phase.1

Key to the understanding of immune control ofcancer was the identification of tumor antigens thatallow development of adaptive T cell immunity, whichis essential for the equilibrium phase of cancer immu-noediting. Presently, it is believed that the most criticalstep in cancer progression is the escape phase, wherenumerous mechanisms may cooperatively impedeimmune surveillance of cancer cells. These mechanismscan be divided into direct immune escape of cancercells becoming invisible to the immune system, e.g. by

Abbreviations: APC, antigen-presenting cell; CEA, carcinoembryonic antigen; CRC, colorectal carcinoma; CTL, cytotoxic T lymphocyte; DC, dendritic cell;IFNc, interferonc; IL, interleukin; LSEC, liver sinusoidal endothelial cell; MR, mannose receptor; OVA, ovalbumin; PBMC, peripheral blood mononuclear cell;PBS, phosphate-buffered saline.From the 1Institutes of Molecular Medicine and Experimental Immunology and the 2Institute of Pathology, University of Bonn, Germany; the 3Department of Pathology,

University Hospital Cologne, Cologne, Germany; the 4Department of General and Abdominal Surgery, Johanniter-Krankenhaus Bonn, Bonn, Germany; the 5Department ofGeneral, Abdominal and Trauma Surgery, St. Marien-Hospital Bonn, Bonn, Germany; and the 6Department of Surgery, University Hospital Bonn, Bonn, Germany.Received December 8, 2011; accepted May 8, 2012.Supported by a grant from the German Cancer Aid (to P. K. and L. D.).

1924

mutational loss of major histocompatibility complexclass I molecules and tumor antigens,2 and into mech-anisms that establish an immunosuppressive statewithin the microenvironment of the tumor. Suchmechanisms include induction of regulatory mediatorssuch as transforming growth factor b, indoleamine2,3-dioxygenase, or galectin by tumor cells3–5 and alsorecruitment of immunosuppressive cells such as regula-tory T cells or myeloid-derived suppressor cells.6,7

So far, the escape mechanisms of cancer cells havebeen attributed to local modulation of immuneresponses in the direct vicinity of cancer cells. Therelease of tumor antigens from cancer cells into thebloodstream has been used for diagnostic purposes,such as carcinoembryonic antigen (CEA) for colorectal,gastric, and ovarian carcinoma.8–10 Systemic distribu-tion of such antigens in the absence of a strong innateimmune stimulus11 can lead to the elimination of anti-gen-specific CD8 T cells by cross-tolerance.12 How-ever, the relevance of circulating tumor antigens forthe modulation of tumor-specific immunity has notbeen addressed. Here, we demonstrate that circulatingCEA is preferentially taken up by liver sinusoidal en-dothelial cells (LSECs). CEA is cross-presented byLSECs to CD8 T cells and circulating CEA in vivoinduces a state of nonresponsiveness in antigen-specificCD8 T cells that in turn promotes immune escape ofCEA-expressing tumor cells.

Materials and Methods

Patients and Healthy Donors. Blood samples werecollected from patients with colorectal carcinoma(CRC) (n ¼ 15) prior to therapy start or healthy vol-unteers (n ¼ 14). The study was approved by theethics committee of the University of Bonn, and writ-ten consent was obtained from all patients beforeblood sampling. Peripheral blood mononuclear cells(PBMCs) were isolated from freshly obtained blood byway of density gradient centrifugation.

Mice. C57BL/6 mice were obtained from ElevageJanvier (Le Genest-Saint-Isle, France). B7H1�/� (a giftfrom L. Chen), MR�/� (a gift from S. Burgdorf ),RAG2�/�, TLR4�/�, and HLA-A*0201�C57BL/B6transgenic mice were bred under specific pathogen-free

conditions in the central animal facility in Bonnaccording to the Federation of European LaboratoryAnimal Science Association and were used for experi-ments at 6-10 weeks of age. All mouse experimentswere approved by the Animal Care Commission ofNorth Rhine–Westphalia.

Ex Vivo Cross-presentation Assay. HLA-A*0201�C57BL/6 and C57BL/6 or MR�/� micewere injected intravenously with either 100 lg CEA orovalbumin (OVA) protein or left untreated. After 15minutes, CD11bþ or CD11cþ cells were isolated fromthe spleen or liver nonparenchymal cells using CD11cor CD11b Macs beads, or LSECs were isolated fromliver nonparenchymal cells with CD146 Macs beadsaccording to the manufacturer’s protocol using anAuto-MACS. A total of 1 � 105 CD11bþ, CD11cþ,or CD146þ antigen-presenting cells (APCs) were incu-bated with 1 � 105 purified CD8 T cells frompGT64-CEA–vaccinated HLA-A*0201�C57BL/6mice and cocultured in the presence of brefeldin Aand monensin for 8 hours. As a positive control APCfrom nontreated HLA-A*0201�C57BL/6 mice wereloaded in vitro with 1.5 lg/mL of the CEA-derivedHLA-A*0201 binding peptide YLSGANLNL. APCsfrom C57BL/6 and MR�/� mice were loaded with theH-2Db binding CEA-derived peptide CGIQNSVSA.After 8 hours, T cells were stained intracellularly forIFNc and analyzed by way of flow cytometry.

Statistical Analysis. The Student t test was used todetermine statistical significance of the results. Dataare presented as the mean 6 SEM, and P � 0.05 wasconsidered significant.Additional Materials and Methods are presented in

the Supporting Information.

Results

LSECs Take Up the Tumor Antigen CEA in aMannose Receptor–Dependent Fashion. We haveshown that LSECs efficiently scavenge the soluble pro-tein ovalbumin from the circulation and cross-presentit to CD8 T cells.13 Because CEA, when released fromtumor cells, gains access to the systemic circulation, weinvestigated whether LSECs and other hepatic andsplenic APCs were capable of taking up CEA from the

Address reprint requests to: Linda Diehl, Ph.D., Institute of Molecular Medicine and Experimental Immunology, University Hospital Bonn, Sigmund-Freud Str.25, 53127 Bonn, Germany. E-mail: [email protected]; fax: (49)-228-28711052.CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25844Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

HEPATOLOGY, Vol. 56, No. 5, 2012 HOCHST ET AL. 1925

circulation. To this end, C57BL/6 mice were injectedintravenously with fluorescently labeled CEA. Within15 minutes, CD146þ LSECs internalized largeamounts of CEA. No significant uptake of CEA by he-patic or splenic CD11cþ dendritic cells (DCs) wasobserved (Fig. 1A and C, Suppl. Fig. 1A). Comparedwith LSECs, CEA uptake by hepatic CD11bþ or F4/80þ cells occurred to a lesser extent (Fig. 1A). BecauseCEA is heavily glycosylated, we analyzed the involve-ment of the mannose receptor (MR), which partici-pates in uptake of glycosylated antigens, for endocyto-sis of CEA by LSECs. Intravenous injection ofCEA�Alexa647 into wild-type C57BL/6 or MR�/� miceshowed that CEA internalization into LSECs mainlydepended on the MR (Fig. 1B,C), whereas uptake intoDCs or Kupffer cells (Fig. 1C) was not altered inMR�/� mice. This is different from the uptake ofOVA by LSECs, which was not influenced by the ab-sence of the MR, whereas efficient ovalbumin uptakeby hepatic macrophages and DCs requires the MR(Fig. 1D). Furthermore, we detected uptake of neitherCEA nor OVA into splenic CD11cþ or CD11bþ cells(Supporting Fig. 1). Taken together, these data showthat LSECs are proficient in uptake of circulatingCEA via the MR.

CEA Is Efficiently Cross-presented by LSECs InVivo to CD8 T Cells. Because LSECs are capable ofcross-presenting soluble antigens to antigen-specific

naıve CD8 T cells in vivo,13–15 we analyzed whetherthe CEA protein is cross-presented to CD8 T cells af-ter uptake by LSECs. Because there are no CEA-spe-cific TCR transgenic animals available, we generatedCEA-specific cytotoxic T lymphocytes (CTLs) by wayof DNA vaccination of C57BL/6 mice, which werethen incubated in vitro with CEA and OVA protein orCEA and OVA peptide–pulsed LSECs. Both CEA pro-tein and CEA peptide–loaded LSECs induced IFNcand interleukin (IL)-2 release by the CEA-specificCTLs (Fig. 2A,B). No IFNc or IL-2 was producedwhen an irrelevant antigen (OVA or SIINFEKL pep-tide) was presented by LSECs. We excluded the possi-bility that our CEA protein preparation altered LSECfunction due to contamination with lipopolysaccharideor other Toll-like receptor ligands, because LSECs didnot produce any IL-6 after incubation with CEA pro-tein (Supporting Fig. 2).We next investigated whether cross-presentation of

CEA was dependent on the MR. Indeed, only LSECsthat were isolated from C57BL/6 but not MR�/�

mice that were intravenously injected with CEA pro-tein induced IFNc production in CEA-specific CTLsin vitro (Fig. 2C). MR�/� LSECs were in principlecapable of stimulating CD8 T cells, because CEApeptide–loaded MR�/� LSECs induced amounts ofIFNcþ CD8 T cells similar to that of CEA peptide–loaded C57BL/6 LSECs. More importantly, we

Fig. 1. Uptake of CEA by LSECs is MR-dependent. Alexa647-labeled CEA (A-C), PBS (B), or OVA (D) protein was injected intravenously intoC57BL/6 (A-D) or MR�/� (B-D) mice. Fifteen minutes after injection, liver nonparenchymal cells were isolated and stained for CD146, CD11b,CD11c, or F4/80 and analyzed using flow cytometry. (A) Dot plots of viable cells. Numbers indicate percentages of Alexa647þ cells within thegated cell population. (B) Histograms show CD1466 C57BL/6 LSECs (black line) or MR�/� LSECs (dotted line) from CEAAlexa647-injected miceor LSECs from PBS-injected mice (filled gray line). (C, D) Mean fluorescence intensity of liver CD146þ, CD11cþ, CD11bþ or F4/80þ cells fromC57BL/6 or MR�/� mice previously injected with CEAAlexa647 (C) or OVAAlexa647 (D). The cumulative results of three independent experiments areshown. *P � 0.05.

1926 HOCHST ET AL. HEPATOLOGY, November 2012

assessed whether other APC populations from the liverand spleen were capable of cross-presenting CEA pro-tein when administered in vivo. To this end, we iso-lated various APC populations from CEA of OVAprotein or phosphate-buffered saline (PBS)-injectedHLA-A*0201�C57BL/6 transgenic mice, and co-cultured them ex vivo together with CEA-specific

CTLs generated by DNA vaccination in HLA-A*0201�C57BL/6 transgenic mice. All APCs were ca-pable of inducing IFNc production by these CTLswhen they were loaded in vitro with the CEA-derivedpeptide YLSGANLNL, indicating that the cells wereviable after isolation (data not shown). However, onlyCD146þ LSECs from CEA-injected mice also induced

Fig. 2. LSECs cross-present CEA protein to CTLs in vitro and in vivo. CEA571-579 peptide–specific CTLs were generated as described. C57BL/6LSECs were incubated with 100 lg/mL CEA protein, 100 lg/mL OVA protein, 1.5 lg/mL CEA571-579 peptide (CGIQNSVSA), or 1.5 lg/mLOVA257-264 peptide (SIINFEKL) for 2 hours. After washing, 1 � 106 CEA-specific CTLs were added, and after 24 hours IFNc (A) and IL-2 (B)secretion into the supernatant was measured via enzyme-linked immunosorbent assay. The cumulative results of three independent experimentsare shown. (C) C57BL/6 or MR�/� mice were injected intravenously with 100 lg CEA protein or were left untreated. After 15 minutes, LSECswere isolated and incubated with CEA-specific CTLs. LSECs from noninjected C57BL/6 or MR�/� mice were loaded with 1.5 lg/mL CEA571-579peptide (CGIQNSVSA). The amount of IFNc-producing CD8 T cells was determined by intracellular staining. (D) HLA-A*0201�C57BL/6 micewere injected intravenously with 100 lg CEA or OVA protein or PBS. After 15 minutes, various APC populations were isolated. APCs from PBS-injected mice were loaded with CEA peptide as a positive control. CD11bþ, CD11cþ, or CD146þ APCs were then cocultured with CEA-specificCTLs from HLA-A*0201�C57BL/6 mice in the presence of brefeldin A/monensin and stained for intracellular IFNc 8 hours later. The percentageof IFNc-producing CEA-specific CD8 T cells within the total CD8 population is shown. The results are representative of two independent experi-ments. *P � 0.05; **P � 0.01.


Fig. 3. CEA-specific CD8 T cells in HLA-A*0201�C57BL/6 mice after systemic CEA administration are phenotypically and functionally toler-ant. (A, C, D, E) HLA-A*0201�C57BL/6 mice were injected intravenously with CEA protein, vaccinated with pGT64 CEA plasmid, or were leftuntreated. Splenic and hepatic lymphocytes were isolated, stained with CD8a, a CEA-peptide YLSGANLNL-loaded HLA-A*0201 dextramer, or acontrol peptide-loaded dextramer, and analyzed by flow cytometry. The percentage of dextramerpos cells in total CD8 T cells is shown. (B) NaıveOT-1 T cells were transferred into C57BL/6 mice that subsequently received OVA protein intraperitoneally (tolerized OT-1 T cells) or were injectedsubcutaneously with 200 lg OVA in incomplete freundßs adjuvant (IFA) (activated OT-1 T cells). Splenic OT-1 T cells were analyzed via flowcytometry. (C) Histograms show the expression levels of CD44, CD62L, and CD25 on CD8a pos and YLSGANLNL-dextramerpos T cells from the liv-ers and spleens of pGT64-CEA, CEA-injected, or control HLA-A*0201�C57BL/6 transgenic mice. (D) Splenocytes were restimulated with PMA/ionomycin, stained with CD8a, YLSGANLNL-dextramer, and intracellularly with IFNc or control antibody. Dot plots are gated on CD8pos cells, andnumbers are the percentage of cells in each quadrant. (E) Enumeration of CEA-specific IFNc-producing cells in the liver and spleen of plasmid-vaccinated or CEA protein–treated animals. Data are representative of three independent experiments. *P � 0.05; **P � 0.01.


IFNc production in CEA-specific CTLs directly exvivo (Fig. 2D). In contrast, both splenic and hepaticCD11cþ and CD11bþ APCs did not cross-present cir-culating CEA ex vivo, indicating that CEA is not onlypreferentially taken up but also preferentially cross-pre-sented by LSECs in the liver and not by DCs or mac-rophages in the liver or spleen.

Cross-Presentation of Systemically CirculatingSoluble CEA Leads to the Development of TolerantCEA-Specific CD8 T Cells in HLA-A*02013C57BL/6 Transgenic Mice. Next, we investigated whether cir-culating CEA would lead to priming of naıve CD8 Tcells. To this end, CEA-specific CD8 T cells from theendogenous naıve CD8 T cell pool in HLA-A*0201�C57BL/6 transgenic mice were analyzed 2weeks after injection of CEA protein or after pGT64-CEA plasmid DNA vaccination. After DNA vaccina-tion, we detected between 7% and 11% of CEA-spe-cific CTLs among total CD8 T cells in the liver andspleen by staining with HLA-A*0201YLSGANLNL dex-tramers (Fig. 3A). Importantly, after intravenous injec-tion of purified CEA protein, we detected CEA-specificCD8 T cells in the liver and spleen that accounted forapproximately 4% of total CD8 T cells (Fig. 3A). Wehave reported that stimulation of naıve CD8 T cells bycross-presenting LSECs does not cause clonal deletionbut leads to the induction of a population of CD8 Tcells with a surface marker phenotype clearly distinctfrom both naıve and DC-activated CD8 T cells (Fig.3B; von Oppen et al.13 and Diehl et al.15). SuchLSEC-stimulated CD8 T cells are CD44high,CD62Lhigh, and CD25neg. Interestingly, the expressionpattern of the CEA dextramer–positive T cells in theCEA protein–treated mice matched that of LSEC-toler-ized T cells being CD44high, CD62Lhigh, and CD25neg,whereas the CEA tetramer–positive T cells generated by

DNA vaccination showed an activated phenotype beingCD44high, CD62Llow, and CD25pos (Fig. 3C). Thus,systemically circulating CEA protein induces CEA-specific CD8 T cells with a surface marker expressionprofile similar to that of CD8 T cells primed by tolero-genic cross-presenting LSECs. Furthermore, CEA dex-tramer–positive CD8 T cells from mice that receivedCEA protein intravenously did not produce IFNc uponrestimulation, whereas CD8 T cells from pGT64-CEA–vaccinated animals did (Fig. 3D,E). Taken together,these results suggest that cross-presentation of systemi-cally circulating CEA protein in vivo does not lead toclonal deletion but induces phenotypically and func-tionally tolerized CD8 T cells.

CD8 T Cells Fail to Control Growth of TumorsExpressing CEA in the Presence of Systemically Cir-culating CEA. To further characterize the functionalcapacity of CEA-specific CD8 T cells generated as aconsequence of circulating CEA, we investigated theability of these cells to control the growth of a CEAexpression tumor in vivo. To this end, Rag2�/� micereceived purified splenic CD8 T cells isolated eitherfrom C57BL/6 mice that were injected every secondday with 2 mg CEA intravenously for 2 weeks or thatwere vaccinated with pGT64-CEA DNA or fromuntreated mice. Three days after CD8 T cell transfer,Rag2�/� mice were injected subcutaneously with themurine tumor cell lines MC38 and MC38-CEA ondifferent flanks. The mice that received CD8 T cellsfrom DNA-vaccinated animals controlled MC38-CEAgrowth compared with mice that received naıve CD8T cells (Fig. 4A,D). However, Rag2�/� mice thatreceived CD8 T cells from CEA protein–treated micefailed to control MC38-CEA tumor growth; instead,tumors grew even faster than in Rag2�/� mice thatreceived naıve CD8 T cells (Fig. 4A,D). This indicates

Fig. 4. CD8 T cells from mice that received CEA protein systemically are incapable of controlling CEA-expressing tumor cells. C57BL/6 miceand B7H1- and MR-deficient mice received 2 mg of CEA protein intravenously every second day, were immunized with pGT64 CEA plasmid, orwere left untreated. A total of 5 � 106 purified splenic CD8 T cells from C57BL/6 (A, D), MR�/� (B, D), or B7H1�/� (C, D) were adoptivelytransferred into Rag2-deficient mice. Three days later, 5 � 106 MC-38-CEA or MC-38 cells were injected into the right flank or left flank, respec-tively. Graphs show tumor growth of MC38-CEA in Rag2�/� mice that received CD8 T cells from C57BL/6 (A, D), MR�/� (B, D), or B7H1�/�

mice (C, D). D: Specific MC38-CEA tumor volumes at day 15 of mice receiving CD8 T cells from C57BL/6, MR�/� and B7H1�/� mice. *P �0.05; ***P � 0.001; ns, not significant.


that CEA-specific CD8 T cells generated after circulat-ing CEA were tolerized and did not have a protectivefunction in antitumor defense. The tolerization of Tcells was antigen-specific, because the MC38 tumorsnot expressing CEA grew equally fast in all threegroups (Supporting Fig. 3), suggesting that there is nobystander effect of tolerized T cells on T cells with dif-ferent antigen specificity. Because we found the uptakeof CEA to be MR-dependent (Fig. 1B), we furthertested whether the lack of uptake of soluble CEA byLSECs in MR�/� would result in the lack of toleriza-tion of CEA-specific CD8 T cells in the naıve endoge-nous T cell repertoire. Indeed, as shown in Fig. 4C,tumor growth in Rag2�/� mice receiving CD8 T cellsfrom CEA-injected MR�/� mice did not show acceler-ated growth, as was the case with CD8 T cells fromC57BL/6 mice (Fig. 4A), but instead had similarkinetics as CD8 T cells from naıve C57BL/6 (Fig. 4A)and MR�/� (Fig. 4C) mice.LSEC-induced tolerization of OVA-specific CD8 T

cells is B7H1-dependent in vitro.15 To investigatewhether tolerance induction in endogenous CEA-spe-cific CD8 T cells in vivo in response to circulatingCEA-protein is also B7H1-dependent, we adoptively

transferred CD8 T cells (which were generated inB7H1-deficient mice after CEA protein injection orDNA vaccination as described, into Rag2�/�) thatwere subsequently challenged with MC38 or MC38-CEA tumor cells (Fig. 4C,D). The CEA-specific CD8T cells generated in B7H1�/� mice after systemicCEA were better at controlling MC38-CEA tumorgrowth (Fig. 4C) than T cells from untreated B7H1�/�

mice and T cells from CEA protein–treated C57BL/6mice. Taken together, these data show that systemicallycirculating CEA protein results in a B7H1-mediatedtolerization of antigen-specific CD8 T cells that fail toeliminate CEA-expressing tumors.

CD8 T Cells with Phenotypic and FunctionalCharacteristics of LSEC-Tolerized CD8 T Cells AreDetected in Healthy Individuals and Are Increasedin Patients with Colorectal Carcinoma. Given theability of LSECs to cross-present circulating antigensin mice, we reasoned that LSECs in humans mightsimilarly function as APCs that tolerize CD8 T cells.This should lead to a population of CD8 T cells withsimilar phenotypic and functional characteristics.Indeed, we identified a sizeable population of CD8pos,CD45ROpos antigen-experienced T cells in PBMCs

Fig. 5. Detection of phenotypically tolerant CD8 T cells in healthy individuals and CRC patients. PBMCs from healthy individuals (A, B, C, D)or CRC patients (D) were stained for CD8, CD45RO, CD62L, and CD25. (A) Dot plot shows CD62L and CD25 expression on viableCD8þCD45ROþ T cells of a healthy donor. Numbers indicate the percentage of cells in each quadrant. (B) Flow cytometric analysis of Foxp3, IL-10, PD-1, granzyme B, and perforin expression in CD8posCD45ROposCD62Lhigh and CD25neg (solid line) or CD62Lhigh and CD25pos (dotted line) Tcells from healthy donors. (C) CD8posCD44highCD45ROpos T cells were sorted into CD62Lhigh and CD25pos or CD62Lhigh and CD25neg fractions. Atotal of 10,000 cells were stimulated as indicated or were left untreated. IFNc secretion was analyzed via enzyme-linked immunosorbent assay.The data for two individual donors are shown. (D) Enumeration of CD8posCD45ROposCD25neg and CD62Lhigh T cells in PBMCs from healthydonors (n ¼ 14) and CRC patients (n ¼ 15). ***P � 0.001.


from healthy individuals that were CD25neg andCD62Lhigh (Fig. 5A). These cells did not differ fromCD25pos T cells with respect to expression of Foxp3,IL-10, granzyme B, perforin, and PD-1, indicating thatno regulatory or exhausted T cells were present in thiscell population (Fig. 5B). Murine LSEC-tolerizedCD25neg CD8 T cells do not respond to secondarystimulation through the T cell receptor.14,15 To investi-gate whether the human CD25neg CD8 T cells were re-sponsive to restimulation, we sorted CD62LhighCD25pos

or CD62LhighCD25neg T cells from CD8pos, CD45ROpos

PBMCs and restimulated the T cells with either anti-CD3/CD28 beads or PMA/ionomycin or left themuntreated. CD62LhighCD25pos CD8 T cells producedIFNc upon stimulation (Fig. 5C). However,CD62LhighCD25neg T cells did not secrete IFNc (Fig.5C). The CD25neg population neither proliferated nordegranulated (Supporting Fig. 4), whereas theirCD25pos counterpart did, suggesting that bothphenotypically and functionally CD45ROposCD62L-highCD25neg CD8 T cells resemble tolerized murineCD44highCD62LhighCD25neg CD8 T cells. Impor-tantly, when we analyzed CRC patients (Supporting Ta-ble 1) for the frequency of this particular population ofCD8 T cells in peripheral blood, we found that it wassignificantly increased (Fig. 5D). From these 15 patients,who did not receive prior treatment, seven were HLA-A2–positive by way of flow cytometry, and one of thesepatients had an elevated serum CEA value (>2,400 mg/L). In PBMCs of this patient, 3.6% of the CD8pos,CD45ROpos, CD62Lhigh, and CD25neg T cells wereCEA-dextramer positive (data not shown), indicatingthat circulating CEA protein can generate tolerized CD8T cells in CRC patients. In conclusion, in peripheralblood of healthy individuals, CD8 T cells with a pheno-type and functional repertoire similar to that of murineCD8 T cells tolerized to systemically circulating antigensare present, and this CD8 T cell population was foundin increased frequency in cancer patients. This findingsuggests that this novel immunosuppressive mechanismmay hamper anticancer immunotherapy strategies.

Discussion

Several immunoregulatory mechanisms have beenidentified that impede the development of antitumorimmunity and can impair the success of cancer immu-notherapy.6,7,16 Certain tumors such as CRC releasesubstantial amounts of antigens such as CEA into thecirculation. The significance of the release of such tu-mor antigens for tumor-specific immunity is not clear.Here, we describe a novel mechanism by which the

presence of systemically circulating tumor antigens canadditionally circumvent the development of tumor-spe-cific CD8 T cell immunity. LSECs efficiently scavengecirculating antigens, cross-present them to naıve CD8T cells, and induce nondeletional T cell tolerance17 andmay thus skew tumor-specific CD8 T cell responses.18

We show that systemically circulating CEA proteinwas efficiently taken up by LSECs, whereas there wasvery little uptake of CEA by DCs and macrophages.In LSECs, but not DCs or macrophages, uptakedepended on MR expression. MR-mediated uptake ofantigens is essential for efficient cross-presentation inDCs because of routing of this receptor into endocyticcompartments competent for cross-presentation.19

Thus, circulating CEA was efficiently cross-presentedto CEA-specific CD8 T cells by LSECs in vivo. DCsand macrophages from both the liver and spleen didnot cross-present CEA, which might be attributable tothe modest uptake of CEA combined with the lack ofMR dependency.It is therefore likely that circulating CEA is pre-

dominantly cross-presented by LSECs for presenta-tion to circulating naıve CEA-specific CD8 T cells.LSECs form a large population of cells (>1 � 1010

in human liver) with a substantial cumulative surfacewithin the hepatic sinusoids where circulating T cellscan easily establish cognate interaction because ofslow blood flow and narrow vessel diameter.20 It isthus conceivable that LSECs can compete for func-tional differentiation of naıve CD8 T cells with pro-fessional APCs that exert their function in a distinctanatomic location (i.e., secondary lymphatic tissue).Furthermore, we provide evidence that the conse-quence of cross-presentation of circulating CEA isinduction of tolerant CEA-specific CD8 T cells thatcannot execute CTL effector function in response toantigen-specific restimulation. Such CEA-specific yettolerant CD8 T cells fail to control the growth of aCEA-expressing CRC cell line in vivo. This suggeststhat tolerogenic functional differentiation of naıveCEA-specific CD8 T cells in the context of systemicCEA protein distribution determines their subsequentnonresponsiveness when encountering their cognateantigen on tumor cells, in other words establishing anadditional form of tumor immune escape. Many tu-mor-associated antigens (e.g., a-fetoprotein, CA-19-9,CA-125) are present in the circulation of tumorpatients.21,22 Therefore, the immunotolerizing effectsof circulating tumor antigens on the endogenousCD8 T cell repertoire may be relevant for additionaltypes of tumor antigens as well as other types of can-cer in addition to CRC.


The key coinhibitory molecule B7H1 involved inLSEC tolerance induction in vitro15 is also relevant fordevelopment of CEA-specific tolerant CD8 T cellsupon systemic administration of CEA in vivo. Clearly,CEA-specific CD8 T cells generated in B7H1�/� butnot C57BL/6 mice after cross-presentation of circulat-ing CEA protein showed potent antitumor activity,demonstrating the essential role of B7H1 for toleranceinduction toward CEA (Fig. 4). This finding demon-strates the essential role of B7H1 in this novel form oftumor immune escape and reiterates the similaritybetween LSEC-mediated tolerance induction and CD8T cell tolerance developed in the presence of circulat-ing CEA. In addition, CEA-specific CD8 T cellsrendered tolerant after systemic application of CEAprotein showed phenotypic characteristics similar tothose of LSEC-tolerized CD8 T cells that wereCD44highCD62LhighCD25neg and nonresponsive toPMA/ionomycin stimulation.Importantly, we detect this particular antigen-experi-

enced yet tolerant CD8 T cell population in normalhealthy human individuals indicating that induction ofCD8 T cells nonresponsive to circulating antigens maybe occurring normally in a physiological situation.This particular surface phenotype is also found in cen-tral memory CD8 T cells.23 However, these memoryT cells are easily activated upon re-encounter withtheir specific antigen.23 Intriguingly, in patients withCRC, we found increased frequencies of this antigen-experienced but nonresponsive CD8 T cell populationand detected CEA-specific CD8 T cells with this par-ticular phenotypical population, which suggests thattumor cells may have initiated CTL immune escape bythe mechanism observed in the experimental murinetumor model.The blockade of B7H1/PD-1 interactions has pro-

ven successful in re-activating PD-1–positive exhaustedT cells in chronic viral infections.24 Studies in micehave shown that PD-1 blockade can also have benefi-cial effects in antitumor therapy25 and first studies incancer patients have yielded positive results.26 Theblockade of coinhibition may allow naıve precursorCD8 T cells with tumor specificity to be stimulatedby normally tolerizing APCs, but because of the lowfrequency of such T cells, the generation of a sizeablepopulation of tumor-reactive CTLs will require time.The blockade of coinhibitory molecules will likely notreinvigorate the already tolerized CD8 T cells toincrease tumor-specific immunity, because these cellsalready resist stimulation via the TCR. The conditionsnecessary for the activation of antigen-experienced buttolerant tumor-specific CD8 T cells will require fur-

ther experimental efforts and may further optimize theeffectiveness of immunotherapy.

Acknowledgment: We thank A. Dolf, P. Wurst, andE. Endl of the flow cytometry core facility for techni-cal assistance.

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