The Journal of

Alloimmunity to Human Endothelial Cells Derived from Cord Blood Progenitors1

Yajaira Sua´rez, Benjamin R. Shepherd, Deepak A. Rao, and Jordan S. Pober2

There is considerable interest in exploiting circulating endothelial progenitor cells (EPCs) for therapeutic organ repair. Such cells may be differentiated into endothelial cells (ECs) in vitro and then expanded for use in tissue engineering. Vessel-derived ECs are variably immunogenic, depending upon tissue source, and it is unknown whether ECs derived from cord blood EPCs are able to initiate an allogeneic response. In this study, we compare the phenotype and alloantigenicity of human cord blood progenitor cell-derived ECs with HUVECs isolated from the same donors. Human cord blood progenitor cell-derived ECs are very similar to HUVECs in the expression of proteins relevant for alloimmunity, including MHC molecules, costimulators, adhesion molecules, , chemokines, and IDO, and in their ability to initiate allogeneic CD4؉ and CD8؉ memory responses in vitro and in vivo. These findings have significant implications for the use of cord blood EPCs in regenerative medicine or tissue engineering. The Journal of Immunology, 2007, 179: 7488–7496.

oth the regeneration of injured tissues and the construc- blood derived ECs (HCBECs). Because of their greater replicative tion, through bioengineering, of synthetic tissue substi- potential and their ability to form stable tubes in vivo, late out- tutes depend upon the formation of new blood vessels growth cells are a much more useful source of ECs for therapeutic B 3 (1–4). Endothelial cells (ECs) isolated from the luminal lining of purposes than early outgrowth EPCs. mature blood vessels can spontaneously organize into tubes that Immunological rejection of allogeneic stem cells following dif- may serve as the nucleus for new vessel development (3–6). How- ferentiation represents a major hurdle for both organ regeneration ever, vessel-derived ECs have only limited replication potential and tissue engineering. This is likely to be particularly true for and rapidly senesce when expanded in vitro (7). ECs may also be vascularized structures because some vessel-derived human ECs differentiated from endothelial progenitors cells (EPCs) that are are capable of initiating an allogeneic immune response, directly found in peripheral blood (8). Umbilical cord blood is a particu- presenting both non-self allelic forms of class I and class II MHC larly rich source of EPCs that can be used for tissue engineering (9, molecules to alloreactive memory CD8ϩ and CD4ϩ T cells, re- 10). The EPCs found in human cord blood may themselves be spectively (22, 23). Specifically, coculture of purified peripheral heterogeneous. Some EPCs, when cultured in basal medium sup- blood memory T cells subsets with allogeneic vessel-derived hu- plemented with FCS and vascular endothelial growth factor man ECs leads to expression of T cell activation markers, by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. (VEGF), rapidly produce colonies of EC-like cells. These early production, and proliferation (21, 22, 24, 25). Moreover, experi- outgrowth cells express some EC markers, like CD31 or von Wil- ments using immunodeficient mouse hosts have revealed that hu- lebrand factor, but also express markers of , like CD14 man ECs are capable of triggering graft rejection by adoptively and CD45. Such cells have limited replicative capacities and do transferred alloreactive effectors or memory T cells (24). The ma- not form stable tubes when implanted in immunodeficient mice jority of experiments involving vessel-derived ECs have used ei- (11–13,15). Other EPCs give rise to EC-like colonies only after a ther cells isolated from human umbilical veins (HUVEC) or hu- delay of 7 days or more (10, 14, 16–21). Such late outgrowth cells man dermal microvessels. It has been reported that ECs from other have high replicative capacity, express EC but not mark- vascular beds may differ from HUVECs in their capacities to ac-

http://classic.jimmunol.org ers, and form stable tubes in vivo (14). Hereafter we will refer to tivate allogeneic T cells (26). It is unknown whether ECs derived the differentiated progeny of late outgrowth EPCs as human cord from cord blood EPCs are able to activate allogeneic T cells, and if they can do so, how they compare with other types of ECs

Department of Immunobiology, Interdepartmental Program in Vascular Biology and isolated from mature vessels. Therapeutics, Yale University School of Medicine, New Haven, CT 06520 In the present study, we have compared HCBECs with HUVECs Received for publication June 26, 2007. Accepted for publication September in a variety of assays for immunological functions. In most in- 21, 2007. stances, we have been able to isolate HCBECs and HUVECs from Downloaded from The costs of publication of this article were defrayed in part by the payment of page the same umbilical cord to control for genetic variations. Our key charges. This article must therefore be hereby marked advertisement in accordance finding is that HCBECs are largely indistinguishable from with 18 U.S.C. Section 1734 solely to indicate this fact. HUVECs in their expression of molecules of relevance to alloge- 1 This work was supported in part by National Institute of Health Grants HL-51015, HL85416, and HL-70295 and Programme 3ϩ3 Fellowship from the Centro Nacional neic T cell activation and in their ability to stimulate an allore- de Investigaciones Cardiovasculares (CNIC), Spain (to Y.S.). sponse, a result that has important implications for the use of HC- 2 Address correspondence and reprint requests to Dr. Jordan S. Pober, Yale University BECs for regenerative medicine or tissue engineering School of Medicine, 10 Amistad Street, Room 401D, New Haven, CT 60509. E-mail address: [email protected] 3Abbreviations used in this paper: EC, endothelial cell; EPC, endothelial progenitor Materials and Methods cell; HUVEC, human umbilical veins; HCBEC, human cord blood progenitor cell- Cell culture derived EC; VEGF, vascular endothelial growth factor; Uea-1, Ulex europeus agglu- tinin 1; MFI, mean fluorescence intensity. All human cell populations were obtained using protocols approved by the Yale Human Investigation Committee. HUVECs were released from can- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 nulated and perfusion-cleared umbilical veins, by collagenase digestion,

www.jimmunol.org The Journal of Immunology 7489

and serially cultured on 0.1% gelatin-coated flasks in M199/20% FBS sup- mAbs reactive with human IDO and Hsp-90 (loading control) from Chemi- plemented with L-glutamine, penicillin/streptomycin (Invitrogen Life con International and BD Biosciences, respectively. Technologies), and endothelial cell growth supplement (Calbiochem) with heparin from porcine intestine as described (27). HCBECs were differen- Cytokine and chemokine assays tiated from cord blood mononuclear cells in vitro as “late outgrowth” cells, Supernatants of cultured ECs exposed to 10 ng/ml TNF-␣ (R&D Systems), as previously described (10), and then expanded in culture. Cultures were 50 ng/ml IFN-␥ (BioSource International), or mock-treated for the times serially propagated on gelatin-coated flasks in EGM-2/15% FBS and step- specified in the text were collected, and the samples were assessed for wise transitioned to grow in HUVEC culture medium. IP-10 or IL-8 using ELISA kits from BD Biosciences and BioSource In- Cell surface immunostaining ternational, respectively. Cell lysates of cultured ECs were prepared as indicated in Western blot analysis section and were used to analyze the ECs were analyzed for surface expression of specific markers by fluores- expression of IL-1␣ using an ELISA kit from PeproTech. cence flow cytometry. Where indicated, cultured EC were exposed to 10 T cell isolation ng/ml TNF-␣ (R&D Systems) or 50 ng/ml IFN-␥ (BioSource International) for the times specified in the text before harvest. Confluent monolayers PBMCs were isolated by density gradient centrifugation of were washed twice in HBSS (Invitrogen Life Technologies) and then in- products by using Separation Medium (Invitrogen Life Tech- cubated with trypsin-EDTA for 1 min. Enzyme activity was then quenched nologies) according to the directions of the manufacturer. Isolated PBMCs with 20% FBS/M199 and suspended cells were collected and washed in 10 were stored in 10% DMSO-90% FBS at Ϫ196°C, thawed, and washed ml of cold 1% BSA (Sigma-Aldrich)/PBS, centrifuged, and washed again before use (28). CD4ϩ or CD8ϩ T cells were isolated from PBMCs by in cold 1% BSA/PBS. Cells were then incubated with 2 ␮g/ml primary Ab positive selection using Dynabeads (Dynal Biotech). The selected popula- or control directly conjugated to FITC or PE for 45 min. Immu- tion obtained by this procedure was routinely Ͼ95% positive for the de- nostained cells were then washed twice with cold PBS and analyzed on a sired marker by flow cytometry (data not shown). Activated T cells and FACSort flow cytometer (BD Biosciences) using CellQuest analysis soft- monocytes were removed by negative selection using an anti-HLA-DR ware collecting 10,000 gated viable cells per sample. Specific Abs used in mAb at a concentration of 5 ␮g/ml (LB3.1; gift of J. Strominger, Harvard these analyses were reactive with human CD31, CD45, CD14, HLA-ABC, University, Cambridge, MA) for 20 min, washed twice, and depleted by HLA-DR, CD40, CD80, and CD86 from Beckman Coulter, with CD34 using magnetic beads conjugated to goat anti-mouse Ab (Dynal Biotech). from Miltenyi Biotec, with VEGFR2, glucocorticoid-induced TNF recep- Naive and memory subsets of CD4ϩ and CD8ϩ T cells were isolated from tor ligand, and E-selectin from R&D Systems, and with CD58 (LFA-3), the purified T cell subset populations by further negative selection using PD-L1 (CD274), PD-L2 (CD273), 41BB ligand (CD137L), Ox-40 ligand anti-CD45RO or anti-CD45RA mAbs, respectively, at a concentration of 2 (CD134L), VCAM-1, and ICAM-1 from BD Pharmingen. Anti-human ␮g/ml (BioSource International). The selected subset population obtained ICOS ligand mAb PE-labeled was a gift from H. W. Mages (Forschungs by this procedure was routinely Ͼ95% positive for the nondepleted CD45R Institut for Molekulare Pharmacologie, Berlin, Germany). Alternatively, isoform by flow cytometry (data not shown). cells were stained with FITC-conjugated Ulex europeus agglutinin (Uea-1) (Sigma-Aldrich), which reacts with the blood group ABH expressed on T cell-EC cocultures human ECs. ECs (ϳ1.5 ϫ 105 cells) were plated into gelatin-coated wells of 24-well Western blot analysis culture plates (Falcon; BD Biosciences) and treated with IFN-␥ (50 ng/ml) (BioSource International) where indicated. Purified T cell subsets were HUVECs or HCBECs were lysed in ice-cold buffer (containing 50 mM then added to each well (ϳ2 ϫ 106 per well). All cultures were maintained

Tris-HCl (pH 7.5), 10% glycerol, 125 mM NaCl, 1% Nonidet P-40, 5.3 in 5% CO2 at 37°C. The medium for coculture consisted of RPMI 1640 mM NaF, 1.5 mM Na2PO4), 1 mM orthovanadate, 175 mg/ml octylgluco- supplemented with 10% FBS serum, 2 mM L-glutamine, 100 U/ml peni- pyranoside, 1 mg/ml protease inhibitor mixture (Roche), and 0.25 mg/ml cillin, and 100 ␮g/ml streptomycin all from Invitrogen Life Technologies. 4-(2-aminoethyl)-benzenesulfonyl fluoride (Roche). Cell lysates were ro- Supernatants collected from cocultures of T cells and ECs after the by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. tated at 4°C for 30 min before the insoluble material was removed by indicated times of coculture were assayed using an ELISA kit for IFN-␥ centrifugation at 12,000 ϫ g for 10 min. After normalizing for equal pro- (BioSource International) or IL-2 (eBioscience). All ELISAs were per- tein concentration, cell lysates were heated in SDS sample buffer before formed as described by the manufacturers. separation by SDS-PAGE. Following overnight transfer of the proteins To measure proliferation by CFSE dilution, CD4ϩ or CD8ϩ T cells were onto nitrocellulose membranes, Western blots were performed using the stained with 250 nM CFSE (Molecular Probes) for 20 min before coculture

Table I. Flow cytometric analysis of proteins of immunological significance in HUVECs and HCBECs isolated from the same donora http://classic.jimmunol.org HUVEC HCBEC

Control IFN␥ (36 h) TNF (6 h) TNF (24 h) Control IFN␥ (36 h) TNF (6 h) TNF (24 h)

PECAM-1 (CD31) 353 358 377 329 369 362 382 348 CD45 ND ND ND ND ND ND ND ND CD14 ND ND ND ND ND ND ND ND

Downloaded from HLA-ABC 54 749 88 247 50 688 74 285 HLA-DR ND 27 ND ND ND 23 ND ND LFA-3 (CD58) 28 29 31 39 29 25 27 30 CD40 35 61 36 38 16 53 19 47 B7-1 (CD80) ND ND ND ND ND ND ND ND B7-2 (CD86) ND ND ND ND ND ND ND ND ICOS ligand 18 23 35 101 22 19 36 100 4-1BB ligand (CD137L) 20 19 20 21 16 19 20 18 Ox40 ligand (CD134L) 337 331 390 343 102 69 128 76 GITR ligand 7 9 11 8 10 13 10 4 PD-L1 (CD274) 11 37 12 15 17 50 22 22 PD-L2 (CD273) 13 54 18 23 19 53 28 49 VCAM-1 (CD106) ND 1 20 39 2 6 21 52 ICAM-1 (CD54) 80 526 971 1174 187 363 695 1478 E-selectin (CD62E) 1 2 48 11 ND 1 28 5

a Cells were exposed to 10 ng/ml TNF, 50 ng/ml IFN␥ , or mock treated for the indicated times. Cells were harvested and labeled as described in Materials and Methods. The results are expressed in terms of corrected median fluorescence intensity (MFI), in arbitrary units of fluorescence, after subtracting the MFI of isotype control from the MFI of the specifically stained cells. Each cell type was analyzed from at least two separate cultures with similar results. ND, Not detected. 7490 ALLOIMMUNITY TO ENDOTHELIUM FROM CORD BLOOD PROGENITORS

FIGURE 1. Time course of surface expression of E-selectin, VCAM-1, and ICAM-1 after cytokine treatment on HCBECs and HUVECs. HUVECs (f) and HCBECs (Ⅺ) isolated from the same donor were exposed to 10 ng/ml TNF (continuous line) or 50 ng/ml IFN-␥ (dotted line) for the indicated times. Cells were harvested and labeled as described in Materials and Methods. The results are expressed in terms of corrected MFI, in arbitrary units of fluorescence, after subtracting the MFI of isotype control from the MFI of the specifically stained cells.

with EC. Following 1 wk of coculture, T cells were collected and stained ume of EGM-2MV was added to the well. Twelve to 18 h after gel poly- with anti-CD4 or CD8 PE-labeled mAbs (Beckman Coulter), respectively, merization, the gels were removed, bisected, and implanted in the s.c. and analyzed using two-color FACS. Duplicate samples were labeled with position on the abdominal wall. Three weeks after implantation, one third anti-CD25 PE-labeled mAb, also from Beckman Coulter. of the animals were euthanized and the grafts harvested for analysis of the human microvasculature. A second third of animals were given 3 ϫ 108 In vivo analysis of EC alloantigenicity allogeneic human PBMCs by intraperitoneal inoculation. The remaining All animal protocols were approved by the Yale Institutional Animal Care animals were injected with PBS and enrolled as nonreconstituted controls. and Use Committee. Human microvessels were generated and implanted in Before the termination of the experiment, the number of circulating human the s.c. position on the abdominal wall of C.B-17/Scid-beige mice as pre- T cells was evaluated. Heparinized retro-orbital venous blood samples viously described (5). Briefly, HCBECs or HUVECs were suspended in a were obtained and the erythrocytes were lysed using ammonium chloride. rat tail type I collagen-human plasma fibronectin gel and ϳ650 ␮lofthe Leukocytes were incubated with PE-conjugated rat anti-mouse CD45 (BD cell suspension was gently poured into a single well of a 24-well tissue Pharmingen) mAb and FITC-conjugated mouse anti-human CD3 mAb culture plate. The protein gel was polymerized at 37°C and an equal vol- (Immunotech/Beckman Coulter) in PBS/1% BSA/5% NGS plus 2 ␮g/ml by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 2. Chemokine secretion (IL-8 and IP-10), inflammatory cytokine expression (IL-1␣), and the immunoinhibitory enzyme IDO expression in HUVECs and HCBECs isolated from the same donor. Cells were exposed to 10 ng/ml TNF (u), 50 ng/ml IFN-␥ (F), or mock-treated (E) for 24 h. Supernatants were collected and the samples were assessed for IL-8 (A) or IP-10 (B) using ELISA as described in Materials and Methods. Cells were treated as indicated and cell lysates of cultured ECs were prepared as indicated in Materials and Methods. Lysates analyzed for expression of IL-1␣ (C) using ELISA and IDO (D) by Western blottign. Hsp-90 protein levels were used as loading control. For panels A–C, data represent mean Ϯ SEM of quadruplicate determinations from one of two independent experiments with similar results. For panel D, blots are from one of two independent experiments with similar results. The Journal of Immunology 7491

FIGURE 3. Activation of CD4ϩ T cells by allogeneic HUVECs and HCBECs isolated from the same donor (donors I and II). HUVECs and HCBECs were treated with IFN-␥ to up-regulate MHC class I/II or mock-treated and subsequently cocultured with CD4ϩ T cells. A and B, Medium were collected for ELISA from HUVECs and HCBECs cultured with CD4ϩ T cells for 24 h. Significant production of both IFN-␥ (A and B, left panels) and IL-2 (A and B, right panels) was observed when compared with resting ECs (not pretreated with IFN-␥)orCD4ϩ T cells cultured in the absence of ECs. Data are mean Ϯ SEM of quadruplicate determinations in one of two independent experiments with similar results. C, After 1 wk of cocultures, CFSE-labeled T cells were stained with anti-CD4 and anti-CD25 mAbs and subjected to FACS analysis. CD4ϩ T cells demonstrate significant proliferation in HUVECs or HCBECs cocultures of Donor I and Donor II, as judged by CFSE dilution, and CD25 expression was enhanced in proliferating cells (upper and lower panels, respectively). Data are from one of two independent experiments with similar results.

Fc-Block (BD Biosciences) and analyzed by FACS. Three weeks following pattern of lineage marker expression was indistinguishable from

by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. PBMC inoculation, all remaining mice were euthanized and the grafts har- that of HUVECs and defines HCBECs as true endothelium. How- vested for analysis of human EC-lined microvessels and the degree of ever, compared with HUVECs, HCBECs grew faster (data not allogeneic PBMC infiltration. Recovered gels and surrounding soft tissue were fixed in 10% buffered formalin and embedded in paraffin. Sections shown) and appeared capable of a greater number of population (5-␮m thick) were cut for immunostaining and for H&E staining. In some doublings without evidence of senescence, consistent with the experiments, the harvested grafts were bisected and half of the specimen growth characteristics previously reported for late outgrowth EPC- was fixed in 10% buffered formalin and half snap frozen in Tissue-Tek OCT (Sakura Finetek), the latter used to prepare 6-␮m cryosections. Sec- derived ECs (14, 16, 21). Because of their robust growth potential, tions were also stained with tetramethylrhodamine isothiocyanate-Uea-1- HCBECs are of special interest to tissue engineering. labeled lectin for detection of human EC-lined vessels within engrafted Having established that HCBECs appear to be true ECs, we protein gels. proceeded to analyze the expression of surface proteins of immu- http://classic.jimmunol.org Statistical analysis nological significance under basal conditions and after cytokine (TNF or IFN-␥) activation. There is considerable genetic variabil- All data are expressed as means Ϯ SEM. Statistical differences were mea- sured by StudentЈs t test. A value of p Յ 0.05 was considered as statisti- ity among humans in EC expression of molecules of immunolog- cally significant. ical relevance (29). Therefore, six independent isolates were com- pared and the results were generally consistent with some minor Results quantitative variations. This extent of variability among donors Downloaded from Phenotypic analyses was comparable to the variability among HUVECs from different We first characterized multiple individual isolates of both HC- donors (data not shown). To compare HCBECs with HUVECs, we BECs and HUVECs by extensive phenotyping. HCBECs were dif- isolated three pairs from the same donor, avoiding donor-depen- ferentiated from EPCs in vitro as “late outgrowth” colonies and dent variability. One representative data set from one of these ex- then serially expanded in culture (10). As we previously reported periments is shown in Table I. The expression levels in HCBECs (10), these HCBECs formed a confluent monolayer of nonover- of the strongest stimulators of alloimmune response, class I and lapping polygonal cells containing cytoplasmic granules that class II MHC molecules (HLA-ABC and HLA-DR, respectively), stained positive for von Willebrand factor and formed intercellular were very similar to those in HUVECs, showing only low levels of junctions expressing vascular endothelial-cadherin. By flow cy- class I MHC molecules in resting cells and expressing more class tometry, HCBECs uniformly bound Uea-1 and expressed CD34, I and de novo induction of class II MHC molecules after IFN-␥ VEGFR2 and CD31, all markers of differentiated ECs. The HC- treatment (30). Additionally, TNF induced-increases of class I BECs used in these experiments were uniformly negative for my- MHC molecules (31) were very similar in both EC types. HCBECs eloid markers CD133, CD45, CD18, and CD14 (data not shown), expressed the same set of costimulatory molecules as HUVECs, which may be coexpressed by “early outgrowth ECs” (14). This namely CD58, CD40, ICOS ligand, 4-1BB ligand, Ox40 ligand, 7492 ALLOIMMUNITY TO ENDOTHELIUM FROM CORD BLOOD PROGENITORS

FIGURE 4. Activation of CD8ϩ T cells by allo- geneic HUVECs and HCBECs isolated from the same donor. HUVECs and HCBECs were cocul- tured with CD8ϩ T cells. A and B, Medium were collected for ELISA from HUVECs and HCBECs cultured with CD8ϩ T cells for 48 h. CD8ϩ T cells cultured with both HUVECs and HCBECs showed significant production of both IFN-␥ (A) and IL-2 (B) when compared with CD8ϩ T cells cultured in the absence of ECs. Data are mean Ϯ SEM of qua- druplicate determinations from one of three indepen- dent experiments with similar results. C, After 1 wk of cocultures, CFSE-labeled T cells were stained with anti-CD8 or anti-CD25 mAbs and subjected to FACS analysis. CD8ϩ T cells demonstrate signifi- cant proliferation in HUVECs and HCBECs cocul- tures, as judged by CFSE dilution (4.3% and 3.5%, respectively), and the CD25 expression was en- hanced in proliferating cells (upper and lower pan- els, respectively). Data are from one of three inde- pendent experiments with similar results.

and glucocorticoid-induced TNF receptor ligand, but not CD80 or treatment, no detectable expression was found in either cell type CD86 (Table I). The levels of expression were generally compa- (data not shown). rable between HCBECs and HUVECs. The single notable differ- ence being that basal expression of CD40 was consistently lower Functional analyses in vitro

by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. on HCBECs than on HUVECs, although comparable expression Cultured HUVECs can present both class I and class II MHC mol- levels were seen after cytokine activation (32). The expression of ecules in a manner that results in the activation of allogeneic mem- the negative signaling molecules PD-L1 and PD-L2 on HCBECs ory T cells (25, 35). We analyzed the responses of multiple T cell were also very similar to HUVECs. We next compared the ex- isolates from adult human peripheral blood to multiple different pression levels of the TNF-induced leukocyte adhesion molecules cultures of HCBECs and HUVECs and found generally similar E-selectin, VCAM-1, and ICAM-1. No significant differences responses to both cell types (data not shown). Once again, to avoid were observed between HCBECs and HUVECs. The pattern and differences attributable to different donor origin we focused our level of expression of these adhesion molecules following either experiments on the response of T cells to pairs of HCBECs and TNF or IFN-␥ treatment was analyzed in a time course experiment HUVECs isolated from the same EC donors, and the results from http://classic.jimmunol.org and found to be similar between both cell types (Fig. 1). Cumu- two independent donors are shown in Fig. 3. In these experiments latively, these data suggest that HCBECs express the same pattern with CD4ϩ T cells, HUVECs and HCBECs were first treated with of surface molecules relevant for interactions with the immune IFN-␥ for three days (to induce HLA-DR expression) or mock- system as HUVECs. treated and then cocultured with allogeneic, CFSE-labeled CD4ϩ Many soluble factors secreted by ECs may also contribute to T cells. Untreated (HLA-DRϪ) HUVECs and HCBECs from both allogeneic responses. Therefore, we analyzed the EC secretion of donor I and II were less able to induce CD4ϩ T cells to secrete

Downloaded from some of these proteins including cytokine-induced proinflamma- either IFN-␥ or IL-2 (Fig. 3, A and B). In contrast, both types of tory chemokines (IL-8 and IP-10) and the inflammatory cytokine ECs that had been pretreated with IFN-␥ induced significant pro- (IL-1␣) (in our hands HUVECS synthesize very little IL-␤ and do duction of these two cytokines in cocultures with allogeneic CD4ϩ not synthesize TNF (33) and unpublished data). As shown in Fig. T cells (Fig. 3, A and B). The overall degree of secretion of both 2, A and B, the secretion of IL-8 and IP-10 in response to TNF and cytokines by CD4ϩ T cells was higher in response to donor I than IFN-␥, respectively, was very similar in both cell types and among in donor II. Although cytokine secretion by T cells stimulated with different donors. The levels of IL-1␣ after TNF treatment were EC from donor I was slightly less in HCBEC cocultures than in also very similar in both cell types but varied more highly among HUVEC cocultures (Fig. 3A, left and right panels), this difference donors (Fig. 2C). We also examined the expression of the immu- was not observed in cocultures donor II (Fig. 3B, left and right noinhibitory enzyme IDO, which may play a role in the regulation panels). Consistent with IL-2 secretion, CD4ϩ T cells demonstrate of adaptive immune responses (26, 34). The level of expression of both proliferation and activation in response to IFN-␥-pretreated IDO was undetectable under basal conditions but was induced to HUVECs or HCBECs from both donor I and II, as judged by comparable levels in both cell types following IFN-␥ treatment CFSE dilution and CD25 expression in proliferating cells, respec- (Fig. 2D). Additionally, when the surface expression of CD95L tively (Fig. 3C, upper and lower panels). Again, the degree of the (FasL) was analyzed under basal conditions and after cytokine T cell response was higher in cocultures with ECs from donor I The Journal of Immunology 7493

and HCBEC cocultures from this donor were less able to activate also share this characteristic, allogeneic CD4ϩ and CD8ϩ T cells CD4ϩ T cells than HUVEC cocultures from this same donor. This were first separated into memory (CD45ROϩ) and naive difference was less clear in ECs from donor II. These results show (CD45RAϩ) T cells and then separately cocultured with HCBECs that HCBECs are roughly comparable to HUVECs in their ability or HUVECs (Fig. 5). As shown in Fig. 4, A and B, CD4ϩ T cells to activate allogeneic CD4ϩ T cells. (memory and naive) did not respond to HUVECs or HCBECs that We next compared the capacity of HCBECs to activate alloge- were not pretreated with IFN-␥, but IFN-␥ pretreated HUVEC and neic CD8ϩ T cells. As shown in Fig. 3, resting HCBECs were able HCBECs were both able to induce memory (CD45ROϩ) but not to induce CD8ϩ T cells to secrete IFN-␥ and IL-2 (Fig. 4, A and naive (CD45RAϩ) CD4ϩ T cells to secrete IFN-␥ and IL-2. By B) and to proliferate (Fig. 4C). As expected, similar percentages of CFSE dilution and FACS analysis, purified memory CD4ϩ T cells, proliferating cells were found when activated cells were analyzed but not naive CD4ϩ T cells, proliferated in cocultures with IFN-␥ (CD25high and CFSElow) (Fig. 4C, lower panels). The CD8ϩ T cell pretreated HUVECs and HCBECs (Fig. 5C). Similarly, only mem- response to HCBECs was similar to the response to HUVECs in ory CD8ϩ T cells were induced to secrete IFN-␥ and IL-2 (Fig. 5, the same experiments. D and E) and to proliferate significantly (Fig. 5F) in response to HUVECs preferentially activated allogeneic memory T cells allogeneic HUVECs or HCBECs, but in this case IFN-␥ pretreat- compared with naive T cells (22, 23). To assess whether HCBECs ment was not required. The overall magnitude of T cell responses by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 5. HUVECs and HCBECs activate CD4ϩ and CD8ϩ T cells memory but not naive T cells. A–C, HUVECs and HCBECs were treated with IFN-␥ as previously indicated and subsequently cocultured with CD4ϩ T memory (CD45ROϩ) or naive (CD45RAϩ) T cells. A and B, Media were collected for ELISA from HUVECs and HCBECs cultured with either memory or naive CD4ϩ T cells for 24 h. Memory CD4ϩ T cells cultured with both HUVECs and HCBECs showed significant production of both IFN-␥ (A) and IL-2 (B) when compared with naive CD4ϩ T or when compared with resting ECs (not pretreated with IFN-␥) or memory and naive CD4ϩ T cells cultured in the absence of ECs. Data are mean Ϯ SEM of quadruplicate determinations from one of two independent experiments with similar results. C, After 1 wk of cocultures, purified CFSE-labeled CD4ϩ memory and naive T cells were stained with anti-CD4 mAb and subjected to FACS analysis. Purified memory CD4ϩ T cells showed significantly greater proliferation than their purified naive counterparts in HUVECs and HCBECs cocultures. Data are from one of two independent experiments with similar results. D–F, HUVECs and HCBECs were cocultured with CD8ϩ memory (CD45ROϩ) or naive (CD45RAϩ) T cells. D and E, Media were collected for ELISA from HUVECs and HCBECs cultured either memory or naive CD8ϩ T cells for 48 h. Memory CD8ϩ T cells cultured with both HUVECs and HCBECs showed significant production of both IFN-␥ (A) and IL-2 (B) when compared with naive CD8ϩ T cells or memory and naive CD8ϩ T cells cultured in the absence of ECs. Data are mean Ϯ SEM of quadruplicates determinations from one of two independent experiments with similar results. E, After 1 wk of cocultures, CFSE-labeled T cells were stained with anti-CD8 mAb and subjected to FACS analysis. Purified memory CD8ϩ T cells showed greater proliferation than their purified naive counterparts in HUVECs and HCBECs cocultures. Data are from one of two independent experiments with similar results. 7494 ALLOIMMUNITY TO ENDOTHELIUM FROM CORD BLOOD PROGENITORS

to HCBECs were somewhat less than those elicited by allogeneic cification (Fig. 6A, right panels). Immunostaining identified only HUVECs, but differences did not reach statistical significance in rare human CD45ϩ cells within the gel, although focal accumu- multiple comparisons. To determine whether the variably lesser lations were noted at the edge of the gel in some specimens (data degree of stimulation induced by HCBECs from donor I correlated not shown). Staining of human ECs with Uea-1 lectin confirmed with the slightly higher level of expression of the IFN-␥-induced previous results with HUVECs that the perfused tubes within the IDO (Fig. 2D), we either inhibited IDO activity with 1-methyl- gels of control animals were lined with human ECs and that human (D)-tryptophan (200 ␮M) or supplemented the cocultures with ECs disappeared in animals receiving allogeneic human PBMCs tryptophan (200 ␮M). Neither of these strategies increased the (5, 36, 37). Quantitation of Uea-1 staining revealed that the num- ability of HCBECs (or HUVECs) to further stimulate T cell pro- ber of human EC-lined tubes increased between 3 and 6 wk in liferation (data not shown). saline inoculated animals, but were decreased in animals receiving PBMCs (Fig. 6B). Significantly, no differences were observed in Functional analyses in vivo this in vivo model of human graft rejection between gels contain- In a final series of experiments, we evaluated the interactions be- ing HCBECs or gels containing HUVECs. tween allogeneic and human EC-lined microvessels in vivo using a previously described model. Specifically, human ECs were suspended in proteins gels formed from rat tail type I Discussion collagen and human plasma fibronectin (5, 36, 37) and then im- Previous reports have shown that human EPCs isolated from pe- planted into the abdominal wall of C.B-17/SCID-beige mouse. ripheral blood or umbilical cord blood can be differentiated to ma- Consistent with previous reports, implanted HUVECs and ture ECs and expanded in vitro, providing a robust source of ECs HCBECs will form tubes that inosculate with the host vasculature for cell transplantation and for promoting vascularization of tissue and are perfused with mouse blood (Fig. 6A) (5, 14). Perfused engineered organs and tissues (38–44). In theory, the expansion of tubes are established by 3 wk and persist for as long as 6 wk (Fig. vascular cells from blood progenitors could allow patients to serve 6A). To detect T cell-mediated alloreactivity in vivo, 3 wk after as their own (autologous) stem cell donors, minimizing the poten- implantation of collagen-fibronectin gels containing HUVECs or tial for immunological rejection. However, most indications for the HCBECs, some of the animals were inoculated i.p. with 3 ϫ 108 use of allogeneic sources of ECs will of necessity be derived from PBMC allogeneic to the HUVECs or HCBECs lining the devel- allogeneic stem cell sources, e.g., in the treatment of acute isch- oped human microvessels. The remaining animals were injected emic events or in the prefabrication of bioengineered tissues for with saline as control groups. All grafts were harvested 3 wk later. clinical use. Moreover, cord blood EPCs appear more robust than Grafts containing either HCBECs or HUVECs showed a plexus of EPCs from peripheral blood of adults and most adult recipients simple EC-lined tubes containing mouse blood at 3 wk postim- will not have access to autologous cord blood. Differentiation and plantation and the appearance at 6 wk was largely unchanged in expansion of ECs from allogeneic EPCs will give rise to alloan- animals inoculated with saline (Fig. 6A, left and center panels). tigenic differences between donor and recipient. Before this study, Inoculation with PBMCs led similar changes in grafts formed from the significance of such differences for graft survival was un- HCBECs or HUVECs, namely loss of tubes, a sparse inflammatory known. It has been demonstrated that differentiated ECs isolated

by guest on October 1, 2021. Copyright 2007 Pageant Media Ltd. infiltrate comprised of mononuclear cells, and focal areas of cal- from mature blood vessels, such as HUVECs, are immunogenic,

FIGURE 6. HUVECs and HCBECs are subjected to T cell-mediated rejec- tion in vivo. A, H&E staining of colla- gen-fibronectin gels containing either

http://classic.jimmunol.org HCBECs or HUVECs. Stable, per- fused, microvessels were formed at 3 wk by either HCBECs or HUVECs in protein gels following s.c. implantation in immunodeficient mice (left panels) and were found to persist for at least 6 wk (middle panels). Three weeks after

Downloaded from implantation, the remaining animals were reconstituted with PBMCs or given saline. Three weeks after recon- stitution, vessel destruction was noted in gels containing either HCBECs or HUVECs in animals receiving PBMCs (right panels), but not saline injected controls (central panels). B, Quantifi- cation of human vessel density, calcu- lated as Uea-1 positive staining per gel area in arbitrary units. For each animal, 2–4 randomly selected fields (ϫ20) from two sections were quantified; n ϭ p Ͻ 0.05 ,ء .3 The Journal of Immunology 7495

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