Human as Intermediaries between Allogeneic Endothelial Cells and Allospecific T Cells: A Role for Direct Scavenger Receptor-Mediated Endothelial Membrane This information is current as Uptake in the Initiation of of September 30, 2021. He Xu, Kiran K. Dhanireddy and Allan D. Kirk J Immunol 2006; 176:750-761; ; doi: 10.4049/jimmunol.176.2.750 http://www.jimmunol.org/content/176/2/750 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Monocytes as Intermediaries between Allogeneic Endothelial Cells and Allospecific T Cells: A Role for Direct Scavenger Receptor-Mediated Endothelial Membrane Uptake in the Initiation of Alloimmunity1

He Xu,2 Kiran K. Dhanireddy, and Allan D. Kirk

Recipient monocytes, T cells, and donor endothelial cells (ECs) are recognized as critical components of allograft rejection. We have recently shown that human monocytes infiltrate vascularized allografts before clinical rejection and have thus hypothesized that monocytes, rather than costimulation-poor ECs, initiate an alloimmune response. However, the nature of the interactions between ECs, monocytes, and T cells has been incompletely defined. Specifically, it is not clear whether these cells interact in a Downloaded from hierarchical manner, nor is it apparent what constitutes an interaction. We therefore studied human ECs, monocytes, and T cells in various isolated in vitro combinations to define the salient features of their contact and to determine whether their interactions were sequential in nature. We find that T cells proliferate poorly to allogeneic ECs and autologous monocytes but well to autologous monocytes following allogeneic EC contact. We show that monocytes gain their stimulatory capacity by phagocytizing allogeneic but not autologous EC membranes in a process governed by scavenger receptors. This process facilitates the subsequent presentation of intact donor HLA molecules to T cells (semidirect presentation). Moreover, monocytes are receptive to help http://www.jimmunol.org/ only after exposure to ECs and require CD4؉ T cells to optimally express costimulatory molecules and foster Ag presentation. Our results indicate that monocytes engage allogeneic ECs through scavenger receptors and are then primed to facilitate T cell activation in a codependent manner. This reciprocal codependence allows for monocytes to serve as a regulated bridge between the allograft and T cells. The Journal of Immunology, 2006, 176: 750–761.

nitial acute allograft rejection is thought to be a T cell-me- it has been shown that human allograft rejection can occur in the diated process driven predominantly by direct alloantigen absence of donor-derived APCs (5). Thus, there are likely other recognition. However, the manner in which T cells recognize intermediaries between the graft and naive T cells.

I by guest on September 30, 2021 donor Ags and are activated in vivo remains incompletely defined, One cell with potential mobile APC capabilities is the host particularly in humans. Specifically, it has been shown in some . Unlike peripheral tissues such as the graft endothelium, animal models that allograft parenchymal and endothelial cells activated monocytes have the ability to express CD40 and B7 co- (ECs)3 are incapable of directly presenting alloantigen in vivo and stimulatory molecules (6–12), and these molecules have been are thus unable to initiate rejection even when T cells have access shown to be critical for optimal naive alloimmune responses (13– to the graft (1–3). Secondary lymphoid organs are thought to pro- 15). Recently, several human studies have implicated monocytes vide an environment where naive T cells receive the costimulatory as playing a more immediate role in allograft rejection than pre- signals required for activation, presumably by intermingling spe- viously suspected. For example, when human renal allograft re- cialized APCs with a critical number of T cells (1). Donor-derived cipients are profoundly T cell-depleted using mAb preparations, dendritic cells (DCs) are well known for carrying intact alloantigen rejection still occurs despite severe lymphopenia and is character- to secondary lymphoid tissues for direct presentation (4). Although ized by intense infiltration of the allograft by monocytes (16). In- this fact is clearly applicable to organs rich in DCs like skin grafts, deed, rejection does not occur until monocytes are available in the kidneys are less replete with these specialized APCs, and, even so, peripheral circulation, and graft dysfunction occurs upon the ar- rival at the graft of activated monocytes even without a prominent T cell infiltrate. Monocytes have also been shown to be among the Transplantation Branch, National Institute of Diabetes and Digestive and Kidney first cells infiltrating human renal allografts immediately upon Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892 reperfusion (17, 18). Thus, monocytes could be envisioned as in- Received for publication April 19, 2005. Accepted for publication October 25, 2005. teracting with graft cells such as the vascular endothelium and The costs of publication of this article were defrayed in part by the payment of page conveying alloantigen and costimulation to T cells. charges. This article must therefore be hereby marked advertisement in accordance Several questions exist when considering mobile APCs. For ex- with 18 U.S.C. Section 1734 solely to indicate this fact. ample, what specifically defines an “interaction” with allogeneic 1 This work was funded by the Division of Intramural Research of the National In- cells? How does the Ag get taken in by the cell and in what form stitute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. Partial salary support for K.K.D. was generously supplied by the Georgetown is it conveyed? Acute rejection has been typically thought to be University Department of Surgery. predominantly a process driven by direct alloantigen presentation 2 Address correspondence and reprint requests to Dr. He Xu, Room 5-5832, Building (4), and recipient-derived monocytes would be expected to be ca- 10 CRC, Center Drive, Bethesda, MD 20892. E-mail address: hex@intra. pable only of indirect alloantigen presentation. Also, given the niddk.nih.gov minimal injury to allografts using modern transplantation and pres- 3 Abbreviations used in this paper: EC, endothelial cell; DC, ; HLA-A1, anti-human HLA class I Ag A1; poly(G), polyguanylic acid; poly(C), polycytidylic ervation techniques (histologically, human live donor grafts have acid. no detectable necrosis), is Ag limited to the cells liberated by cell

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 751 death or are live cells capable of delivering Ag for presentation? Leukocyte purification Furthermore, if monocytes are able to imbibe Ag, how do they Human monocytes and were obtained by from avoid promiscuous activation of cells that they encounter during normal volunteers enrolled in a National Institutes of Health Institutional transit? Review Board product procurement protocol following informed To investigate human monocytes and their potential role in con- consent. Human PBMCs were isolated by Ficoll (Sigma-Aldrich) density gradient centrifugation. Cells were washed three times with Ca2ϩ- and veying alloantigen to T cells, we have initiated studies investigat- ϩ Mg2 -free PBS. Human monocytes were either isolated from PBMCs by ing the nature of monocytic interactions with the allogeneic endo- negative selection methods or obtained as elutriated monocytes from the thelium and autologous T cells. We have specifically evaluated the Department of Transfusion, National Institutes of Health. Briefly, PBMCs effect of isolated interactions, because in vivo it is apparent that were resuspended in RPMI 1640 supplemented with 5% FCS, incubated monocytes encounter graft cells remote from substantial numbers with anti-CD2, anti-CD16, and anti-CD20 Abs at 4°C for 60 min, and washed three times with Ca2ϩ- and Mg2ϩ-free PBS. Cells were incubated of T cells (16). We find that monocytes directly engulf live allo- with goat anti-mouse IgG-coated Dynabeads (Dynal) at 4°C for 60 min. geneic but not autologous EC membranes using a scavenger re- After magnetic removal of the beads, cells were washed twice in RPMI ceptor-dependent process. This process facilitates the representa- 1640 and diluted with tissue culture medium (10% FCS in RPMI 1640 tion of intact MHC molecules to T cells and subsequent T cell medium) at 2 ϫ 106 cells/ml. Cell phenotype was verified by FACS with CD14ϩCD86ϩ cells at Ͼ90% (mean, 91 Ϯ 2.8%), CD3ϩCD4ϩ cells at activation. ϩ ϩ ϩ ϩ 2.2 Ϯ 1.2%, CD3 CD8 cells at 1.0 Ϯ 0.9%, CD3 CD20 cells at 0.5 Ϯ 0.6%, and CD3ϩCD16ϩ cells at 0.9 Ϯ 0.5%. Materials and Methods Human CD4ϩ T cells were purified from PBMCs by negative selection. Reagents and mAbs Briefly, PBMCs were incubated with anti-human CD8, CD11a, CD14,

CD16, CD20, and HLA-DR at 4°C for 60 min and then washed three times Downloaded from Recombinant human IFN-␥ and TNF-␣ were purchased from R&D Sys- with RPMI 1640 medium at 4°C. Cells were then incubated with goat tems. Human AB serum, gelatin, PKH-26-GL fluorescent cell linker com- anti-mouse IgG-coated Dynabeads at 4°C for 60 min. After magnetic re- pound kits, polyguanylic acid (poly(G)), and polycytidylic acid (poly(C)) moval of the beads, cells were washed twice with RPMI 1640 at 4°C and were obtained from Sigma-Aldrich. diluted with tissue culture medium at 2 ϫ 106 cells/ml for the T cell- The mAbs and their FITC or PE conjugates used for flow cytometry monocyte interaction studies. Cell phenotype was verified by FACS. included the mouse control Abs IgG1, IgG2a, and IgG1 (BD Bio- Human splenocytes were isolated from human cadaveric spleen and sciences), human-specific mouse Abs against CD4, CD8, CD14, CD16, aorta discarded after clinical organ procurement for transplantation. CD20, CD40, CD58, CD80, CD83, CD86, CD134L, CD154 (5c8), annexin Briefly, spleen was minced, and the resultant suspension was filtered by a http://www.jimmunol.org/ V, and HLA-DR (BD Biosciences), and CD40, CD106, CD62E, and von fine nylon filter to retain connective tissue capsule fragments. Splenocytes Willebrand factor (Serotec). The biotin-conjugated, mouse anti-human were washed three times with cold PBS after lysing RBCs. Cell viability HLA class I Ag A1 (HLA-A1) was purchased from United States Biolog- was determined by trypan blue (Invitrogen Life Technologies) exclusion ical. Biotin-conjugated mouse IgM (BD Biosciences) was used as an iso- and was Ͼ85%. Splenocytes were stored in freezing medium (10% DMSO type control Ab for the HLA-A1. PE-conjugated streptavidin was pur- in FCS) at Ϫ80°C for 24 h and then stored in liquid nitrogen until used. chased from Caltag Laboratories. FITC-conjugated, monoclonal, active Cells were thawed rapidly before the experiments, and cell viability was caspase-3 apoptosis kit 1 was purchased from BD Pharmingen. The agonist confirmed by trypan blue exclusion. CD28-specific monoclonal IgG2a (clone 9.3) and Fab were gifts from Dr. D. Harlan (Bethesda, MD). Human EC growth factor was purchased from Allogeneic mixed -EC reactions Sigma-Aldrich. Mouse human-specific Abs against CD2 (clone 35.1, IgG2a), CD8 All studies were performed using nontrypsinized, intact, confluent EC by guest on September 30, 2021 (clone 51.1, IgG2a), CD11a (clone OKM 1, IgG2b), CD14 (clone 63D3, monolayers to mimic the in vivo conditions of initial PBMC-EC interac- IgG1), CD16 (clone 3G8, IgG1), CD20 (clone 1F5, IgG2a), and HLA-DR tions. Human ECs were grown to confluence in 96-well, flat-bottom tissue (clone 2.06, IgG1) were prepared from American Type Culture Collection culture plates and examined microscopically to ensure viability before each hybridomas. experiment. Monolayers were treated with mitomycin C (Sigma-Aldrich) at 50 ␮g/ml for 38 min and washed three times with RPMI 1640 medium. EC culture and activation PBMCs (2 ϫ 105) or purified CD4ϩ T cells were added to each well supplemented with RPMI 1640 containing 10% human AB serum. Mixed Human ECs were isolated for coculture studies from the cadaveric aorta by lymphocyte-EC reactions were conducted for 6 days, with cells pulsed (50 digestion with 0.01% collagenase (Crescent Chemical). Primary cultures ␮Ci/ml [3H]thymidine) during the final day of culture, harvested, and and subcultures were conducted in EC culture medium (Invitrogen Life counted as described above. To verify the costimulation requirements of Technologies) supplemented with 10% FCS (HyClone Laboratories) and this system, mixed lymphocyte-EC reactions were in some cases supple- EC growth factor. The cells were verified to be endothelial by staining for mented by the addition of anti-CD28 (50 ␮g/ml) or Fab (50 ␮g/ml) for 6 von Willebrand factor by FACS analysis. For positive control activation, days. These and all other experiments were repeated at least three times ␣ ␥ ECs were treated with TNF- (250 U/ml, for 4 h) or IFN- (10,000 U/ml, with equivalent results. for 48–72 h), and activation was verified by expression and/or up-regula- tion of CD54, CD62E, CD106, HLA class I, and HLA-DR as determined PBMC-EC or monocyte-EC cocultures and flow cytometry by FACS using a FACScan (BD Biosciences). Both resting and - stimulated ECs were stained with anti-human CD40, CD80, and CD86 to To detect EC membrane uptake, coculture experiments were performed determine the expression of these costimulatory molecules. using intact EC monolayers labeled with PKH-26, a fluorescent compound For assessment of functional CD40 on ECs, human D1.1 cells (Amer- that incorporates aliphatic reporter molecules into the cell membrane by ican Type Culture Collection), CD4-negative derivatives of the Jurkat line selective partitioning (20). ECs were labeled with PKH-26 at room tem- capable of providing a contact-dependent helper function (19), were used perature for 5 min and then washed twice with RPMI 1640, seeded in as a stimulant. D1.1 cultures were maintained at 1 ϫ 106 cells/ml in RPMI gelatin-coated 12-well flat-bottom tissue culture plates, grown to conflu- 1640 medium containing 10% FCS, penicillin, and streptomycin. Intact EC ence, and examined microscopically before each experiment. ECs were monolayers were coincubated with D1.1 cells (5 ϫ 106) in the presence or also verified to be viable before and after interactions with monocytes by absence of the anti-CD154 Ab 5c8 (100 ␮g/ml) or anti-CD80 h1f1 (100 trypan blue exclusion and negative staining with FITC-conjugated mono- ␮g/ml; Genetics Institute/Wyeth) at 37°C for 2 h. After removal of the clonal anti-active caspase-3 and annexin V Ab to exclude apoptosis by D1.1 cells, EC monolayers were stained with mouse IgG1-FITC (isotype FACS analysis. Labeled ECs were Ͼ95% positive for PKH-26 by FACS. control) or anti-CD62E-FITC for 30 min, washed three times with FACS Unlabeled ECs were also seeded in 12-well tissue culture plates and used buffer, and incubated with PBS (Invitrogen Life Technologies) containing after EC monolayers formed. Human PBMCs or purified monocytes were 20 mM HEPES (BioWhittaker) (pH 7.4), 10 mM EDTA, and 0.5% BSA diluted with tissue culture medium and then added to 12-well, flat-bottom (Sigma-Aldrich) at 4°C for 20 min and then at 37°C for 20 min. De- culture plates with EC monolayers at 4 ϫ 106 cells/well. In some experi- tached ECs were analyzed by FACScan. Resting ECs and ECs activated ments, PBMC were added to a Transwell chamber (Corning Costar), and by recombinant human TNF-␣ were used as negative and positive con- the chamber was placed in 12-well, flat-bottom plates containing unlabeled trols, respectively. Because monocytes were cocultured with ECs in EC monolayers or PHK26-labeled EC monolayers. several experiments, both resting and activated EC were confirmed to To determine whether EC membrane uptake was allogeneic specific, be CD14-negative. frozen human splenocytes were isolated from a cadaveric donor spleen 752 MONOCYTE UPTAKE OF ENDOTHELIAL MEMBRANES AND ALLOIMMUNITY from which aortic ECs had been recovered and stored in standard freezing to 12-well tissue culture plates and coincubated with EC-conditioned medium in liquid nitrogen. Splenocytes were thawed in a 37°C water bath monocytes for 24 h (T cell/monocyte ratio was 5:1) at 2.5 ϫ 106 cells/ml and then transferred to culture medium followed by centrifugation at 4°C. in tissue culture medium. Coincubation of resting monocytes with purified Cellular debris and dead cells were removed by Ficoll centrifugation. Cells autologous CD4ϩ T cells was conducted as a negative control. Cells were were washed three times with culture medium, and viability was confirmed collected, washed twice with FACS buffer, and stained with mAbs against using trypan blue exclusion. Cells were then coincubated with allogeneic human CD14, CD40, CD80, CD86, and HLA-DR at 4°C for 30 min. Cells ECs and autologous ECs labeled with or without PKH-26 for 16 h, and were washed twice with FACS buffer and evaluated by FACScan analysis cells were collected followed by FACS analysis gating on monocytes by gating on the CD14ϩ monocyte population. forward and side scatter and CD14 expression. To determine whether cell To determine whether EC-conditioned monocytes could induce purified membrane uptake by monocytes was EC-specific, in an additional exper- CD4ϩ T cell proliferation, a 5-day EC-conditioned monocyte-T cell co- iment a coculture of human monocytes or PBMCs with unlabeled or PKH- culture was performed. Briefly, live cells were washed three times and 26-labeled allogeneic fibroblasts (a gift from Dr. K. Pechhold, Bethesda, diluted with RPMI 1640 medium containing 10% human AB serum. EC- MD) was performed for 16 h, followed by FACS analysis. conditioned monocytes (2 ϫ 104) were incubated with 1 ϫ 105 purified In additional experiments, PKH-26-labeled gelatin was coated on cul- CD4ϩ T cells in 96-well round-bottom culture plates with or without ture plates and incubated at 37°C for 2 h. Unlabeled, washed ECs were blocking mAbs directed against CD80, CD86, and CD154 at 37°C for 5 transferred into these wells. Cocultures were conducted at 37°C, and cells days. After incubation with 50 ␮Ci/ml [3H]thymidine on the final day, cells were collected at varying time points. Unstimulated PBMCs or monocytes were harvested onto glass fiber filters, and proliferation was measured by were used as controls. Uptake inhibition studies were performed using incorporation of [3H]thymidine using a liquid scintillation counter PBMCs or monocytes that were preincubated with poly(G) (a scavenger (PerkinElmer Life Sciences). All coculture experiments were repeated at receptor inhibitor) at 1.0, 0.5, 0.25, 0.125, or 0.0625 mg/ml for 60 min and least three times with equivalent results. then added to unlabeled or PKH-26-labeled EC monolayers, and cocultures were carried out for 16 h. Poly(C), a structural homolog of poly(G), was used as a specific negative control for poly(G) in inhibitory experiments. Downloaded from PBMCs or monocytes collected from cocultures were washed once with Results 4°C FACS buffer and stained with FITC-labeled Abs specific for CD4, Human ECs remain costimulation-poor despite activation by CD8, CD20, CD14, CD40, CD58, CD80, CD86, CD134L, and/or or exposure to T cells HLA-DR at 4°C for 30 min. After a final wash with cold FACS buffer, cells were analyzed by FACS with the electronic gate set on CD14ϩ or PKH26- Effective APCs express the costimulatory molecules CD80 and stained CD14ϩ cells. All coculture experiments were repeated at least three CD86. However, human ECs typically do not express these mol- times with equivalent results. Additionally, these cells were also stained for ecules even when optimally activated. To verify this assumption, http://www.jimmunol.org/ CD83 to determine whether the monocyte population changed over time to we evaluated both resting and activated ECs. Resting ECs ex- DC-like cells. pressed minimal CD40 and were devoid of CD80, CD86, and the Confocal microscopic evaluation of monocyte from monocyte- adhesion molecules CD62E, CD54, and CD106 (Fig. 1, a and b). EC coculture Following incubation with rIFN-␥, ECs expressed CD40, CD54, Monocytes collected from PKH-26-labeled EC-monocyte cocultures were CD62E, CD106, and HLA-DR but remained negative for surface washed three times with PBS and then incubated with anti-CD14-FITC at CD80, CD86 (Fig. 1b), and CD134L (not shown). TNF-␣ stimu- 4°C for 30 min. Cells were washed three times with PBS and then fixed lation improved CD40 expression but did not alter HLA-DR ex- with 1.6% paraformaldehyde on ice for 20 min. Cells were washed twice pression or induce CD80 or CD86 expression. Stimulation with with PBS and then diluted with FACS buffer, and cell slides were prepared ␣ ␥ by guest on September 30, 2021 by Cytospin (Shandon Southern Products) and examined by confocal mi- TNF- or rIFN- did not alter CD58 expression (not shown). ECs croscopy (MRC600; Bio-Rad). simultaneously stimulated with both rIFN-␥ and TNF-␣ also re- mained CD80-, CD86-, and CD134L-negative (data not shown). Tissue typing and verification of intact HLA class I transfer The CD40 was functional in that D1.1 T cells augmented an EC Human ECs and leukocytes were HLA-typed by the clinical HLA typing adhesion molecule expression that was inhibitable by an anti- laboratory at the Walter Reed Army Medical Center using a complement- CD154 Ab but not by an isotype control Ab (data not shown). dependent microcytotoxicity assay. To verify the expression of HLA-A1 However, even after activation by CD154-expressing T cells, ECs before the experiments, EC monolayers were examined microscopically before each experiment and stained with a biotin-conjugated mAb specific remained CD80-, CD86-, and CD134L-negative. Consistent with for the HLA-A1 on ice for 30 min. EC monolayers were washed twice with these findings, activated human ECs (those expressing HLA-DR, PBS buffer and then incubated with PE-conjugated streptavidin for 30 min, CD40, CD54, CD62E, and CD106) had no ability in vitro to ac- followed by three washes. ECs were detached by incubation in PBS con- tivate purified CD4ϩ T cells under the conditions of this study taining 20 mM HEPES (pH 7.4), 10 mM EDTA, and 0.5% BSA at 4°C for (Fig. 1c). These conditions were sufficient to induce robust resting 20 min and then at 37°C for 20 min. Detached ECs were analyzed by ϩ FACScan, and these ECs were positive for the HLA-A1. CD4 T cell proliferation when allogeneic monocytes served as To determine the uptake of EC-derived HLA-A1 Ag by human mono- stimulators (data not shown). Thus, the inability of ECs to induce cytes, human PBMCs were evaluated by FACS analysis to confirm the proliferation was not the result of overly stringent in vitro condi- absence of HLA-A1 Ag expression. An EC-PBMC coculture was carried tions. Interestingly, T cells did proliferate when monocytes were for 18 h. Cells were collected after incubation and washed twice with FACS buffer. Cells were then incubated with mouse mAb specific for present during the interaction (as did PBMCs) (Fig. 1c). Thus, HLA-A1 and CD14 as well as mouse isotype control Ig at 4°C for 30 min. human ECs were poor candidates for primary APCs and would be PE-conjugated streptavidin was added to cells after the final wash and unlikely to activate naive CD4ϩ T cells under physiological con- incubated at 4°C for 30 min. Cells were analyzed by FACS gating on ϩ ditions despite direct alloantigen expression. CD14 after two washes with FACS buffer. To confirm that the failure of ECs to serve as effective T cell Coculture of purified CD4ϩ T cells and EC-conditioned stimulators was related to their lack of B7 expression, ECs were ϩ monocytes coincubated with purified CD4 T cells in the presence of anti- CD28 Ab, an isotype control, or anti-CD28 Fab. Intact anti-CD28 To generate EC-conditioned monocytes, purified monocytes were coincu- bated with intact EC monolayers for 48 h in tissue culture medium at 37°C (but not isotype control Ab or Fab) induced T cell proliferation in and collected by repeated gentle washes without destroying the EC mono- the presence of allogeneic ECs (but not autologous cells), indicat- layers. Monolayers were confirmed to be intact by microscopic examina- ing that the lack of B7 by the ECs was a fundamental defect in tion. Cellular debris was removed by Ficoll centrifugation. Cells were then their ability to primarily stimulate isolated T cells (Fig. 1d). Thus, washed three times with PBS at 4°C, and the viability of cells was con- firmed by trypan blue exclusion. ECs require an external source of costimulation (for example, B7 Washed, EC-conditioned monocytes were diluted with tissue culture molecules) to activate isolated T cells, and ECs are not capable of medium. Purified autologous CD4ϩ T cells were rested for 48 h then added providing this source despite cytokine activation. The Journal of Immunology 753 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Human aortic ECs do not express CD80 or CD86 even after exogenous cytokine-mediated activation and fail to stimulate purified CD4ϩ T cells. This failure to activate CD4ϩ T cells is due to inadequate B7 costimulation. a, Flow cytometric data shows that human ECs derived from cadaveric aorta up-regulate the adhesion molecules CD62E, CD54, and CD106 (open histogram) when TNF-␣-stimulated. b, Cytokine-stimulated human ECs up-regulate CD40 (open histogram). IFN-␥ but not TNF-␣ stimulation results in HLA-DR up-regulation on human EC (open histogram). In contrast, cytokine-stimulated human ECs fail to up-regulate costimulatory molecule CD80 and CD86 (open histogram). c, Purified human CD4ϩ T cells proliferate poorly in response to human ECs but well when cultured as bulk PBMCs. d, ECs can be induced to stimulate purified CD4ϩ T cells if CD28 is cross-linked by the addition of anti-CD28 but not CD28-specific Fab.

Human monocytes require T cell help after exposure to source of B7 in the periphery. Unstimulated human monocytes, allogeneic ECs to optimally express costimulation molecules evaluated as part of freshly obtained or incubated PBMCs, ex- Human monocytes are typically thought to behave as mobile APCs pressed CD58, CD86, and HLA-DR, but not CD40, CD80, or and are known to be able to costimulate T cells via B7 molecule CD134L. Following coculture with ECs and T cells (as PBMCs), expression (21, 22). Thus, these cells could serve as the required monocytes increased their surface expression of CD40, CD80, and 754 MONOCYTE UPTAKE OF ENDOTHELIAL MEMBRANES AND ALLOIMMUNITY

HLA-DR (Fig. 2) but not of CD58 or CD134L (not shown). How- monocyte activation did not require T cell exposure to be simul- ever, this up-regulation was not an independent effect of exposure taneous with EC exposure. Monocytes stimulated with EC for 72 h to allogeneic ECs. Unlike the monocytes in PBMC-EC cocultures, did not express CD40 or CD80, and monocytes that were not ex- purified monocytes cocultured with ECs in isolation did not ex- posed to ECs did not up-regulate CD40 and CD80 when incubated press surface CD40 or CD80 (Fig. 2). HLA-DR expression was with T cells (Fig. 3). Thus, monocytes required help, in this case increased in isolated coculture, and there was no significant change in the form of exposure to autologous T cells, some time after a in CD86 expression for either coculture condition. Thus, although priming exposure to allogeneic ECs for optimal costimulation mol- monocytes could express B7 molecules, their exposure to ECs ecule expression. alone was insufficient to induce this expression. Rather, some other component of PBMCs was needed to facilitate monocyte APC function. Therefore, under peripheral lymphopenic conditions, Monocytes are primed for interactions with T cells by monocytes, like ECs, could be expected to be suboptimal APCs. internalizing allogeneic EC membranes via scavenger receptors To determine whether CD4ϩ T cells were permissive for EC- ϩ Given that monocytes were activated only after EC exposure and induced monocyte activation, purified autologous CD4 T cells ϩ were added back to the isolated monocyte-EC cocultures and then subsequent CD4 T cell exposure, we next examined the nature of evaluated after 3 days by FACS. CD4ϩ T cells reconstituted the the priming interaction between monocytes and ECs. EC mono- ability of monocytes in monocyte-EC cocultures to express CD40 layers were labeled with PKH-26, a membrane dye, and were then and CD80 (Fig. 2). To determine whether this effect required tem- incubated with PBMCs or purified monocytes for various lengths poral colocalization between T cells, EC-conditioned monocytes of time and analyzed by FACS to determine whether monocytes Downloaded from were collected from monocyte-EC cocultures after 48 h and sep- physically engulfed EC membranes. Monocytes derived from arated from the ECs. These EC-conditioned monocytes were neg- PBMC (with T cells present)-EC cocultures took up PKH-26 fol- ative for CD40 and CD80; however, following coincubation with lowing incubation with labeled ECs (Fig. 4a) as early as 60 min autologous CD4ϩ T cells in the absence of ECs for an additional following coculture and increased their PKH-26 content after 2, 4, 24 h, these monocytes expressed CD40 and CD80 (Fig. 3). Thus, and 24 h of coincubation (Fig. 4a). PKH-26-positive monocytes http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. The up-regulation of CD40 and CD80 on human monocytes is dependent on the presence of CD4ϩ T cells. Shown are flow cytometric analyses of human monocyte populations gating on CD14ϩ as purified resting monocytes in resting PBMCs, undergoing allogeneic ECs stimulated in the presence or absence of purified human CD4ϩ T cells, or as PBMCs. Isotype control results are shown in the gray histograms, whereas the experimental results are shown in the open histograms. Resting purified monocytes and monocytes in resting PBMCs express CD86 and HLA-DR but not CD40 or CD80 expression following a 3-day culture. Purified monocytes, stimulated by allogeneic ECs for 3 days, similarly failed to express CD40 or CD80. In contrast, allogeneic EC-stimulated PBMCs markedly up-regulated the costimulatory molecules CD40 and CD80. Purified monocytes were able to express CD40 and CD80 following allogeneic EC stimulation in the presence of CD4ϩ T cells. The Journal of Immunology 755

FIGURE 3. Monocyte expression of CD40 and CD80 following EC exposure does not require simultaneous exposure to ECs and T cells. Shown is a flow cy- tometric analysis of human monocyte populations from purified resting mono- cytes, EC-conditioned monocytes, and EC-conditioned monocytes following their coculture with CD4ϩ autologous T cells with gating on CD14ϩ monocytes. Forty-eight- and 72-h EC-stimulated Downloaded from monocytes as well as unstimulated monocytes were negative for surface ex- pression of CD40 and CD80. EC-condi- tioned monocytes, when removed from the EC and subsequently incubated with CD4ϩ autologous T cells, up-regulated http://www.jimmunol.org/ CD40 and CD80 expression (row 4). Co- incubation of resting monocytes with resting autologous T cells did not result in up-regulation of CD40 and CD80. by guest on September 30, 2021

expressed surface CD40 and CD80, but PKH-26-negative mono- the splenocytes of a deceased human donor in which we had es- cytes remained surface CD40- and CD80-negative (Fig. 4b), indi- tablished an aortic EC population. When monocytes were cocul- cating that EC priming for subsequent T cell help was associated tured with autologous PKH-26-labeled ECs for 16 h, they re- with membrane uptake. The engulfment of EC membranes was mained PKH-26-negative (Fig. 5). In contrast, when incubated monocyte-specific, because neither CD4ϩ nor CD8ϩ T cells be- with two allogeneic sources of ECs, the monocytes became posi- came positive for PKH-26 (data not shown). In addition, purified tive for PKH-26, suggesting that these monocytes were able to CD4ϩ T cells from CD4ϩ T cell-EC cocultures also remained discriminate between self and foreign sources of endothelium. In negative for PKH-26 (data not shown). To determine whether the an additional study, coincubation of monocytes or PBMCs with engulfment of EC membranes was dependent on the presence of T PKH-26-labeled allogeneic fibroblasts resulted in positive PKH-26 cells (as activation was shown to be) or some other PBMC-derived staining on monocytes, indicating that this effect was not EC-spe- factor, purified monocytes were cocultured with PKH-26-labeled cific (data not shown). ECs. Interestingly, PKH-26 transfer to purified monocytes was de- In addition to active monocyte of EC membranes, tected both by FACS (data not shown) and confocal microscopy several trivial explanations could explain the observed PKH-26 (Fig. 4c). However, as shown above, despite membrane uptake the uptake results. We therefore investigated a variety of passive monocytes did not express costimulation molecules in the absence mechanisms to determine whether membrane uptake was an active of T cells. Thus, monocytes, but not T cells, engulfed EC mem- process. To determine whether labeled ECs released PKH-26 into branes during EC-monocyte contact independently of the presence the culture medium that could then be taken up via passive scav- of T cells or T cell-derived factors, but EC membrane uptake was enging, human PBMCs were incubated with PKH-26-labeled ECs not sufficient for monocyte activation and costimulation of mole- in a Transwell system that prohibited cell-to-cell contact. Mono- cule expression. Membrane uptake was a distinct event from ac- cytes separated from ECs did not become PKH-positive, indicating tivation, although activation was predicated on EC exposure and a requirement for direct cell-to-cell contact (data not shown). To uptake. determine whether the uptake required contact with live cells or To determine whether the uptake of EC membranes was allospe- merely contact with any PKH-26-labeled entity, PBMCs were cific (perhaps driven by unique carbohydrate differences but not cocultured with unlabeled ECs that were seeded on PKH-26-la- necessarily HLA specificities), we studied monocytes derived from beled, gelatin-coated culture plates. PBMCs collected from these 756 MONOCYTE UPTAKE OF ENDOTHELIAL MEMBRANES AND ALLOIMMUNITY

FIGURE 4. Direct allogeneic cell uptake by monocytes. Shown is flow

cytometric analysis of human mono- Downloaded from cyte populations from PBMC-EC co- cultures. a, Monocyte populations stain positive for PKH-26 as early as 2 h following their interaction with PKH-26-labeled ECs and increase their uptake of PKH-26 following 4- and 24-h coincubation with labeled http://www.jimmunol.org/ ECs. b, PKH-26-positive monocytes have enhanced expression of CD40 and CD80 following a 3-day coincu- bation (open histograms) as compared with PKH-26-negative monocytes. c, Shown are the results of confocal mi- croscopic evaluation of monocytes af- ter interaction with PKH-26-labeled EC monolayers showing dual staining by guest on September 30, 2021 for CD14-FITC and PKH-26 (ϫ60).

cocultures similarly did not become PKH-26-positive (data not membrane uptake. Thus, membrane engulfment was not dependent shown). Thus, monocytes engulfed membranes specifically from upon EC activation, injury, or apoptosis. live, nonself cells after direct cell-to-cell contact and not passively One mechanism known to facilitate live cell engulfment by as a result of adherence to cellular debris. Furthermore, membrane monocytes involves the function of scavenger receptors, a broad uptake did not result in EC disruption, lysis, apoptosis, or death. family of integral membrane proteins (SR-A, SR-B, SR-D, and EC monolayers remained intact and viable after 24 h of coincu- SR-E class) that function as innate pattern recognition receptors bation with PBMCs or purified monocytes. Thus, uptake was not and mediate the cellular binding and internalization of many neg- simply a clearing of apoptotic ECs. Additionally, although mono- atively charged macromolecules (23). A scavenger receptor block- cyte adhesion to EC monolayers increased when EC monolayers ade with scavenger receptor ligands such as poly(G) has been were preconditioned with activating cytokines or hypoxia/hypo- shown to prevent phagocytosis of apoptotic cells and glycosylated thermia, these maneuvers did not lead to demonstrably increased pathogens by (24). To evaluate the role of scavenger The Journal of Immunology 757

FIGURE 5. Monocytes do not engulf autologous EC membranes. Shown are flow cytometric analyses of human monocyte populations from spleno- cyte-EC cocultures. Monocytes derived from splenocytes became positive for PKH-26 following their interaction with two separate PKH-26-labeled allogeneic EC populations (EC-1 and EC-2, open histograms) but did not stain positive when incubated with stained autologous ECs. The gray histograms in each panel show background from cocultures of splenocytes with unlabeled ECs.

receptors in this process, human PBMCs were pretreated with tion of HLA-A1-positive ECs with HLA-A1-negative PBMC re- Downloaded from poly(G) or poly(C), a structural homolog of poly(G), and were sulted in transfer of the surface expression of HLA-A1 on CD14ϩ then coincubated with PKH-26-labeled ECs. Poly(G) but not monocytes (Fig. 7). Thus, EC uptake not only facilitated monocyte poly(C) inhibited the uptake of EC membranes by monocytes in a priming and monocyte-T cell interactions, it also provided a mech- dose-dependent manner (Fig. 6). Thus, the monocyte uptake of EC anism for alloantigen presentation and transport. membranes was a contact-dependent process mediated by scaven- ger receptors. EC-conditioned monocytes costimulate, activate, and induce ϩ http://www.jimmunol.org/ One potential consequence of membrane uptake that would have proliferation in CD4 T cells a bearing on alloimmunity is alloantigen internalization and rep- Because it was evident that monocytes were primed for interaction resentation. It has been demonstrated that monocytes pulsed with with T cells by exposure to the allogeneic endothelium, the func- allogeneic cell membranes can process immunodominant peptides tional relevance of monocyte-EC conditioning was then assessed. for indirect presentation, and it has been equally established that Purified resting CD4ϩ T cells proliferated poorly in response to EC monocytes can directly present MHC Ag to allogeneic T cells monolayers and when incubated with autologous monocytes (Figs. (reviewed in Ref. 25). Recently, the prospect of intact MHC mol- 1C and 8). However, when EC-conditioned monocytes were gen- ecule transfer has been proposed as a mechanism for direct MHC erated from purified monocyte-EC cocultures, these monocytes presentation by an autologous cell (26), so-called “semidirect pre- stimulated robust T cell proliferation that could not be augmented by guest on September 30, 2021 sentation” (27). We therefore looked to see whether monocyte EC by additional anti-CD28 stimulation. Although, EC-conditioned engulfment facilitated intact HLA molecule transfer and surface monocytes did not express surface CD40 and CD80 before coin- expression using an Ab that recognized intact HLA as opposed to cubation with T cells, the monocyte-induced T cell proliferation the HLA peptide. We evaluated HLA-A1-positive ECs coincu- was inhibited by mAbs specific for CD80, CD86, or CD154 (Fig. bated with PBMC that were HLA-A1-negative as determined by 8), and a combined blockade with these Abs completely blocked T molecular HLA typing as well as by FACS. The cells were inter- cell proliferation. Thus, monocyte EC uptake facilitated a prolif- rogated with FACS by gating on monocytes both by forward and erative T cell response that was costimulation-sensitive, yet co- side scatter and on CD14 expression to rule out the possibility of stimulation molecule expression was predicated on exposure to T false-positive PKH-26 staining of contaminating allogeneic ECs cells. Monocytes, even though primed with foreign cell mem- (neither resting nor activated ECs express CD14). The coincuba- branes, reserved their costimulation molecule expression until they were in direct contact with T cells, at which time they up-regulated CD40 and CD80 and facilitated reciprocal T cell activation. As such, peripheral monocytes engaged with an allogeneic endothe- lium would be expected to reserve their costimulatory capabilities until they are in a T cell-rich environment, most typically a sec- ondary lymphoid organ. Accordingly, monocytes in transit likely remain suboptimal APCs until they are in an appropriate environ- ment, with appropriateness defined by excess T cell presence.

Discussion Monocytes are recognized as participants in human allograft re- jection, and their role has been made more apparent by clinical lymphocyte depletion protocols (16–17, 28, 29). It is unclear whether monocytes assume more responsibility for rejection in a lymphopenic environment or whether lymphopenia simply makes FIGURE 6. EC membrane uptake by monocytes is blocked in a dose- dependent fashion by scavenger receptor blockade with poly(G). Mono- them more readily observable. Nevertheless, monocytes are clearly cytes were incubated for 16 h with PKH-26-labeled allogeneic ECs in the present histologically before and during all functionally significant presence of the indicated concentration of poly(G) or poly(C), a structural cellular rejections. The data reported herein specifically address homolog of poly(G) that does not inhibit scavenger receptor function, and the initial events occurring when monocytes infiltrate a vascular- then assessed by flow cytometry gating on CD14ϩ monocytes. ized allograft and demonstrate a mechanism by which they can 758 MONOCYTE UPTAKE OF ENDOTHELIAL MEMBRANES AND ALLOIMMUNITY

FIGURE 7. HLA-A1-negative monocytes become surface A1-positive after incubation with A1-positive ECs. Shown are flow cytometric analyses of ECs that express HLA-A1 (left panel, open histograms). Monocytes not coincubated with EC monolayers lack HLA-A1 expression on their surface (middle panel). Monocytes incubated with HLA-A1-positive ECs express HLA-A1 on their surfaces (right panel, open histograms). Downloaded from initiate an alloimmune response. These data further show the spe- after Ag presentation, this would limit monocyte priming to the cific temporal and stoichiometric relationships required for a co- resting spots of T cells (such as the nodes) and limit peripheral ordinated response between ECs, T cells, and monocytes and re- activation. Clearly, mechanisms are required to limit peripheral T veal a novel mechanism (direct cell engulfment) for response cell activation during chance encounters of cells in transit from initiation. These studies begin to address the choreography be- sites of inflammation to lymph nodes, and additional regulation is

tween cells involved in rejection and suggest that there is a clear needed to avoid nonactivated monocytes from inappropriately http://www.jimmunol.org/ hierarchy required for delivering activating signals and Ags among stimulating T cells by chance encounters in nodal tissue. This or- the participants in cellular alloimmunity. dered response satisfies both of these requirements. It is known that APCs, specifically murine DCs, require priming This study is also consistent with others in showing that human by CD4ϩ T cells to gain the ability to activate a cytotoxic response ECs, unlike mouse ECs, are incapable of expressing CD80 and (30–33). We have now shown that monocytes require an interac- CD86 (38, 39), making them suboptimal costimulatory cells and tion with foreign cells to become receptive to priming and that this incapable alone of fostering a naive immune response or of pro- prepriming can be achieved through scavenger receptor-mediated moting naive T cell Th1 differentiation (40). These findings are cell uptake outside of a secondary lymphoid organ. This observa- also consistent with studies by Adams et al. (41) and, more re- tion is novel and is the first report of scavenger receptor involve- cently, Vudattu et al. (42) in showing that the presence of mono- by guest on September 30, 2021 ment in human alloimmunity. The scavenger receptor system is cytes markedly influences the proliferative capacity of naive T increasingly recognized as facilitating the engulfment of foreign cells. The conditions associated with the experiment are important cells and lipoprotein bodies based on carbohydrate recognition and in interpreting our findings and have been chosen to specifically is now seen as an important part of innate (23, 24, 34). relate to initial immune interactions in lymphopenic hosts. ECs Thus, we suspect that the ability to distinguish autologous from have been shown by many investigators to induce CD4ϩ T cell allogeneic cells is related to carbohydrate moiety recognition, proliferation under certain in vitro conditions, specifically when which is not a function of MHC. However, we cannot exclude the the cells are preincubated with IFN-␥ 2 (43–46). However, this role of the small number (Ͻ1%) of T cells facilitating this dis- condition is not consistent with the physiological absence of IFN-␥ crimination. Elucidating this role will require additional study. Be- at the time of initial monocyte contact with allogeneic EC post- cause allogeneic fibroblast membranes are also subject to uptake, transplant reperfusion (17) and, thus, may not reflect the most this may represent a more general incompatibility than previously likely conditions present during transplantation. Although CD8ϩ T recognized, one that facilitates the presentation of intact HLA Ags cells have been shown in vitro to provide IFN-␥ and to stimulate and provides a means for direct allorecognition in the absence of the proliferative capacity of ECs (40), we have shown in humans donor-derived APCs. However, the specific characterization of via serial protocol biopsies that CD8ϩ T cells are rare in the early scavenger receptor usage and the signaling involved will require infiltrates of renal allografts in depleted hosts (16). Additionally, considerable additional study. most prior studies have used ECs that have been trypsinized and Interestingly, the type A scavenger receptor has replated with responding T cells, often in dense aggregates (e.g., been recently identified as a differentially overexpressed transcript U-bottom plates). We have used intact EC monolayers for our during rat allograft rejection (35), and scavenger receptors have studies to more precisely mimic the early conditions seen clini- also been increasingly implicated in advanced atherosclerotic dis- cally, particularly in patients following depletional induction. ease (36). This finding is consistent with the advanced vasculopa- Thus, although conditions can be created in vitro to foster EC- thy typically seen in transplanted organs (37) and deserves more mediated T cell proliferation, we do not feel that these conditions intense scrutiny. These data also provide one explanation for why reflect those present posttransplant or that ECs independently in- alloimmune responses are delayed considerably by profound pe- duce naive T cell activation in vivo. ripheral monocyte depletion but eventually proceed upon mono- It is also important to draw a distinction between naive T cell cyte resurgence (16). responses and those of committed memory T cells. Purified These data suggest that monocytes in vivo become partially CD8ϩCD45ROϩ T cells can proliferate in response to allogeneic primed and capable of direct alloantigen recognition in the periph- ECs directly, and studies have shown that CD2, LFA-3, and CD59 ery but do not become capable of mature reciprocal responses until play a major role in this response. Similarly, many of the non-B7 they gain access to T cells. Because T cells are mobilized only costimulatory molecules present on the human ECs in this study The Journal of Immunology 759

favor a primacy of B7 costimulation during initial alloimmune interactions. Monocytes are drawn to an allograft to some extent in propor- tion to the degree of reperfusion injury (17). The critical require- ment of this cell type for T cell activation described herein helps explain the marked augmentation of alloimmunity that occurs commensurate with aggressive reperfusion injury and monocyte graft infiltration after T cell depletion (16). It is interesting to note that one of the most striking effects of methylprednisolone treat- ment (a glucocorticosteroid widely used at the time of allograft reperfusion) is monocyte clearance from the circulation (K. K. Dhanireddy and A. Kirk, manuscript in preparation) that is inde- pendent of the use of depleting Abs. Although steroids have many effects that could mediate their antirejection effects, their influence ϩ FIGURE 8. EC-conditioned monocytes induced autologous CD4 T on monocyte mobilization may be one that has been overlooked. cell proliferation that was inhibited by costimulation blockade. Shown are Although our in vitro results suggest that membrane engulfment is the results of proliferation studies evaluating CD4ϩ T cell proliferation independent of EC activation, injury, or apoptosis, we cannot dis- from T cells or monocytes alone or from T cells cocultured with mono- cytes, EC-conditioned monocytes, or EC-conditioned monocytes in the count the possibility that increased adhesion would facilitate mem- presence of anti-CD80 and/or CD86 and/or CD154 (or isotype control brane uptake. Berliner and coworkers (52) have investigated in- Downloaded from Abs). CD4ϩ T cell proliferation was induced by EC-conditioned mono- teractions between monocytes and allogeneic ECs and have shown cytes and inhibited by costimulation-specific mAbs. that allospecific T cells do facilitate monocyte adhesion. Indeed, we favor a model in which EC activation brings monocytes into proximity and allows this membrane uptake to occur. (CD58, OX40L, and the ICOS ligand) have been shown to support Our observation of patients treated with potent lymphocyte-de- the proliferation and cytokine secretion of memory and/or poly- pleting agents combined with these in vitro studies lead us to sug- http://www.jimmunol.org/ clonally stimulated T cells independent of the effects of B7 co- gest a model for alloimmune activation in a naive lymphocyte- stimulation (47–51). However, there is no effect of these molecules depleted host (Fig. 9). We propose that alloimmunity is initiated by on naive T cells, and they cannot by themselves induce a de novo monocyte attraction to a reperfused vascularized allograft (17, 18) response. Because the addition of CD28 signaling markedly and the resultant scavenger receptor-mediated EC membrane en- changes the proliferative capacity of naive cells in this study, we gulfment. Monocytes then traffic without costimulation molecule by guest on September 30, 2021

FIGURE 9. A model for the role of monocytes in allograft rejection. Following reperfusion and the resultant increase in monocyte chemotactic/adhesion factors, monocytes are attracted to the allograft and initiate EC membrane engulfment driven by scavenger receptor recognition. Monocytes then traffic without costimulation molecule expression to the secondary lymphoid tissues. During transit, the monocytes are unable to support sporadic T cell activation. However, upon entering areas replete with T cells, they can present intact HLA Ags via a semidirect method and receive reciprocal priming signals from CD4ϩ lymphocytes. Once primed, monocytes express CD80 and CD40 and are thus able to mature a response. This reciprocal codepen- dence of T cells and monocytes helps insure that immune activation occurs only when there are alloantigen-expressing cells (in this case, ECs), monocyte activation, and a critical number of T cells. 760 MONOCYTE UPTAKE OF ENDOTHELIAL MEMBRANES AND ALLOIMMUNITY expression to areas replete with T cells, where they can present 12. Wang, J., H. Beekhuizen, and R. van Furth. 1994. Surface molecules involved in ␥ ␥ intact HLA Ags via a semidirect method (27) and receive recip- the adherence of recombinant interferon- (rIFN- )-stimulated human monocytes ϩ to vascular endothelial cells. Clin. Exp. Immunol. 95: 263–269. rocal priming signals from CD4 lymphocytes. This process ap- 13. Judge, T. A., Z. Wu, X. G. Zheng, A. H. Sharpe, M. H. Sayegh, and L. A. Turka. pears to be at least partially mediated by the IFN-␥ receptor on 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the monocytes (H. Xu and A. Kirk, manuscript in preparation). Once induction of long-term allograft survival. J. Immunol. 162: 1947–1951. 14. Kearney, E. R., T. L. Walunas, R. W. Karr, P. A. Morton, D. Y. Loh, primed, monocytes express CD80 and CD40 and have the neces- J. A. Bluestone, and M. K. Jenkins. 1995. -dependent clonal expansion of sary surface molecules to mature a cytotoxic T cell response (31, a trace population of antigen-specific CD4ϩ T cells in vivo is dependent on CD28 32). This reciprocal codependence of T cells and monocytes helps costimulation and inhibited by CTLA-4. J. Immunol. 155: 1032–1036. 15. Kirk, A. D., D. M. Harlan, N. N. Armstrong, T. A. Davis, Y. Dong, G. S. Gray, insure that immune activation occurs only when there are alloan- X. Hong, D. Thomas, J. H. Fechner, Jr., and S. J. Knechtle. 1997. CTLA4-Ig and tigen-expressing cells (in this case, ECs), monocyte activation, and anti-CD40 ligand prevent renal allograft rejection in primates. Proc. Natl. Acad. a critical number of T cells. This mechanism is not thought to be Sci. USA 94: 8789–8794. 16. Kirk, A. D., D. A. Hale, R. B. Mannon, D. E. Kleiner, S. C. Hoffmann, exclusive, because tissue-based DCs clearly have the capacity to R. L. Kampen, L. K. Cendales, D. K. Tadaki, D. M. Harlan, and S. J. Swanson. mediate Ag transfer to lymph nodes. However, this mechanism can 2003. Results from a human renal allograft tolerance trial evaluating the human- ized CD52-specific monoclonal alemtuzumab (CAMPATH-1H). Trans- substantially augment the capacity of the local environment to de- plantation 76: 120–129. liver and present Ags in response to local tissue injury and factors 17. Hoffmann, S. C., R. L. Kampen, S. Amur, M. A. Sharaf, D. E. Kleiner, K. Hunter, increasing chemotaxis. In circumstances like allotransplantation, S. S. John, D. A. Hale, R. B. Mannon, P. J. Blair, and A. D. Kirk. 2002. Molecular and immunohistochemical characterization of the onset and resolution of human this augmented capacity may take on a more prominent role. renal allograft ischemia-reperfusion injury. Transplantation 74: 916–923. We conclude that human monocytes are primed by a direct in- 18. McLean, A. G., D. Hughes, K. I. Welsh, D. W. Gray, J. Roake, S. V. Fuggle, teraction with allogeneic ECs through a process involving the in- P. J. Morris, and M. J. Dallman. 1997. Patterns of graft infiltration and cytokine Downloaded from gene expression during the first 10 days of kidney transplantation. Transplanta- corporation of allogeneic EC membranes and the representation of tion 63: 374–380. HLA. This process is mediated via monocyte scavenger receptors 19. Lederman, S., J. Michael, M. J. Yellin, A. Krichevsky, J. Belko, J. J. Lee, and and influences the mobile APCs to become receptive to subsequent L. Chess. 1992. Identification of a novel surface protein on activated CD4 T cells ϩ ϩ that induce contact-dependent differentiation (help). J. Exp. Med. 175: interactions with CD4 T cells. Interactions with CD4 T cells 1091–1101. induce the up-regulation of costimulatory molecules, “licensing” 20. Horan, P. K., and S. E. Slezak. 1989. Stable cell membrane labeling. Nature 340:

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