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Departments of
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Surgery,
Pathology and Laboratory Medicine, and
Medicine, University of Pennsylvania Health System, Philadelphia, PA 19104
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a requirement for concomitant expression of both MHC class II molecules and costimulatory molecules by APCs to activate alloreactive CD4+ T lymphocytes. Interestingly, cells that are not typically considered APCs can express MHC class II (4). Although Ag presentation by keratinocytes, pancreatic islets, and thyroid epithelial cells has been shown to induce anergy in CD4+ T cells, the ability of vascular endothelial cells to act as APCs for CD4+ T cells remains one of the most controversial issues in transplantation biology (5, 6, 7). Although some investigators have reported that CD4+ T cell clones fail to proliferate in response to endothelial cells unless trans-costimulation is provided, most studies have shown that purified CD4+ T cells proliferate and produce cytokines after coculture with activated allogeneic endothelial cells (8, 9, 10).
As studies addressing the allostimulatory capacity of endothelium have been predominantly performed in vitro, the critical question of whether allograft endothelial cells can activate CD4+ T cells in vivo remains unanswered. It has long been argued that the lack of endothelial expression of MHC class II makes the mouse an unsuitable animal model to study this important biological issue (11). However, we and others have recently shown that, similar to human vascular endothelial cells, treatment with IFN-
induces surface MHC class II expression on cultured mouse vascular endothelial cells (12, 13). In the present study we show that despite surface expression of MHC class II and B7.1, mouse vascular endothelium is a poor APC for allogeneic CD4+ T cells. Moreover, we show that direct recognition of MHC class II on graft endothelium does not play an important role in acute murine cardiac allograft rejection, and that the hemopoietic APCs are the critical triggers of the initial alloimmune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male wild-type C57BL/6 (H-2Kb; designated B6) mice and MHC class II-deficient mice (I-A
-deficient) on a C57BL/6 background (designated B6II–) were purchased from Taconic Farms (Germantown, NY). Male C3H/HeJ (H-2Kk; designated C3H) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They were housed under pathogen-free conditions and used at 4–7 wk of age. Experimental protocols were approved by the University of Pennsylvania institutional animal care and use committee and followed guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Purification of CD4+ T lymphocytes and cell labeling
Responder cells for in vitro and in vivo assays consisted of CD4+ T lymphocytes isolated from unprimed C3H mice killed by cervical dislocation. Spleens and pooled lymph nodes cells were isolated, and adherent cells were depleted from splenocytes by passage through nylon wool columns (Polysciences, Warrington, PA). Cells were then incubated with magnetic bead-coated anti-CD4 mAbs and isolated via positive selection by passage though a magnetic column (midiMACS system; Miltenyi Biotec, Auburn, CA). The purity of CD4+ T lymphocytes was >96%, as determined by flow cytometry. CD4+ T lymphocytes were labeled with the fluorescent dye CFSE (Molecular Probes, Eugene, OR) before coculture with endothelial cells by incubation with CFSE in PBS at a final concentration of 2 µM for 3 min. CFSE-labeled CD4+ T cells cultured alone in vitro did not show any evidence of activation, such as surface expression of CD25 or CD69.
In vitro MLRs
Endothelial cells were isolated from thoracic aorta of B6 and C3H mice as previously described and used as stimulators at early passage (12). Endothelial cells (2.5 x 104/well) were plated in 48-well plates and allowed to reach confluence (
1.5 x 105 cells/plate). These cells were incapacitated by treatment with 25 Gy of gamma irradiation from a 137Cs source immediately before the addition of T lymphocytes. For some experiments, endothelial cells were activated by treatment with recombinant murine IFN- (500 U/ml) 72 h before their use as stimulators. CFSE-labeled C3H CD4+ T lymphocytes, resuspended in 1 ml of RPMI 1640 medium containing 2-ME (1 µl/ml), gentamicin (1 µl/5 ml), and 10% FCS (all from Invitrogen Life Technologies, Gaithersburg, MD) were added to each well at a stimulator to responder ratio of 1:3. Analogous in vitro MLRs were performed with hemopoietic APCs as stimulators. Briefly, CFSE-labeled C3H CD4+ T lymphocytes were cocultured in round-bottom, 96-well plates with B6 splenocytes that had been irradiated with 25 Gy of gamma irradiation from a 137Cs source at a stimulator to responder ratio of 1:3. Proliferative responses were analyzed by flow cytometry after 7 days of coculture. These cells were identified by APC-labeled, CD4-specific (clone GK1.5; BD Pharmingen, San Diego, CA) Abs.
For the [3H]thymidine incorporation assays, responder CD4+ T lymphocytes were positively selected (midiMACS system; Miltenyi Biotec) from lymph nodes and spleens of naive C3H mice as well as C3H recipients of B6(B6II–) hearts at the time of allograft rejection, as assessed by cessation of heartbeat. Allograft rejection was also confirmed histologically. APCs were prepared from splenocytes through RBC lysis with ACK buffer (BioWhittaker, Walkersville, MD) and T cell depletion with CD90.2 magnetic beads (midiMACS system; Miltenyi Biotec). After isolation, all APC preparations were incubated with 10 ng/ml IFN- (R&D Systems, Minneapolis, MN) for 24 h, washed twice, and irradiated with 1000 rad before addition to culture, except for APCs pulsed with B6 allopeptide, which was first prepared by coincubating C3H APCs with freeze-thawed (three cycles) B6 APCs in the presence of 10 ng/ml IFN- for 24 h before washing and irradiation. CD4+ T cells (2 x 105) and APC preparations were cocultured at three different ratios (1:1, 1:3, and 1:9) in round-bottom, 96-well plates (BD Biosciences, Franklin Lakes, NJ) in quadruplicate wells for 72 h in 200 µl of complete medium. Each sample was pulsed with 1 µCi of [3H]thymidine (DuPont NEN, Boston, MA) in 25 µl of culture medium and incubated at 37°C in 5% CO2. After a 6-h incubation period, the incorporation was stopped by freezing the reaction at –30°C until analysis. Frozen cultures were thawed, immediately harvested (Tomtec, Hamden, CT), and [3H]thymidine incorporation of the samples was measured using a Wallac 1205 beta plate liquid scintillation system (Wallac, Gaithersburg, MD). Radioactivity was expressed as cpm.
Creating chimeric organs by bone marrow transplantation
Our laboratory has previously described a method for creating chimeric organs using bone marrow transplantation (14). Male wild-type B6 mice and B6II– were used to create bone marrow chimeras in the following strain combinations: B6
B6II– and B6II–B6. Bone marrow was isolated from the femora of donor mice, and T cells were depleted with anti-CD90-labeled magnetic microbeads (Miltenyi Biotec). Recipient mice received 1 x 107 T cell-depleted donor bone marrow cells by lateral tail vein injection 6 h after receiving lethal irradiation (10 Gy). We use the following designation to describe bone marrow chimeric mice or organs harvested from bone marrow chimeras: nonhemopoietic cells (hemopoietic cells). Thus, a B6II–(B6) animal or heart has MHC class II-deficient nonhemopoietic cells and wild-type hemopoietic cells. Bone marrow chimeras were used at least 90 days after engraftment. Replacement of hemopoietic cells was confirmed in all heart donors as previously described (14).
In vivo MLRs
Alloresponses of C3H CD4+ T lymphocytes in the presence and the absence of direct presentation by B6 hemopoietic cells were tested in vivo. B6 wild-type B6II–, B6(B6II–) and B6II–(B6) bone marrow chimeras were treated with supralethal irradiation (18 Gy), followed by adoptive transfer of 25 x 106 CFSE-labeled C3H CD4+ T lymphocytes via tail vein injection. Some B6(B6II–) chimeras were treated with an i.p. injection of 100,000 U of recombinant murine IFN- 72 h before the adoptive transfer to induce MHC class II expression on vascular endothelial cells. These mice were killed 84 h after the adoptive transfer, and their spleens were analyzed flow cytometrically for the proliferative responses of the adoptively transferred CD4+ T cells. These were identified by staining with PE-labeled H-2Kk-specific (clone 36-7-5) and APC-labeled CD4-specific (clone GK1.5) Abs. Live cells were identified by the exclusion of the vital dye 7-aminoactinomycin.
Heterotopic heart transplantation
Chimeric and control hearts were transplanted heterotopically into the abdomen of recipient male C3H mice as previously described (14). Cardiac allograft function was evaluated by daily transabdominal palpation. The recipients were killed, and grafts were explanted when palpable contractions ceased. Routine histologic examination using H&E staining was performed on tissue sections from each heart.
CD8+ T lymphocyte depletion of recipient mice
For a subset of the experiments, C3H mice were used as heart recipients after in vivo depletion of CD8+ T lymphocytes. For this purpose, these mice received two doses of a purified CD8-depleting mAb (53-6.72; American Type Culture Collection, Manassas, VA; 800 µg i.p.) 48 and 24 h before transplantation. To maintain depletion of CD8+ T lymphocytes, recipient animals were given one dose of 53-6.72 (800 µg) on postoperative day 7 and weekly thereafter. Complete depletion of CD8+ T lymphocytes was demonstrated by flow cytometric analysis of splenocytes and pooled lymph node cells from treated animals.
Immunohistochemistry of mouse hearts
Immunohistochemistry was performed to assess the patterns of MHC II expression and inflammatory cell infiltrates in the rejected hearts and the IFN--treated hearts, which up-regulates MHC class II expression. Frozen sections from naive and rejected hearts were cut at 5 µm in a cryostat, air-dried, and stored at –80°C until use. After equilibration to room temperature, previously frozen slides were fixed in acetone and air-dried. Endogenous peroxidase activity was quenched with hydrogen peroxide. Slides were then incubated with the primary Ab, biotinylated anti-mouse I-Ab (-chain, B6 MHC class II, clone 25-9-17; BD Pharmingen), biotinylated anti-mouse CD31 (PECAM-1, clone MEC 13.3; BD Pharmingen), biotinylated anti-mouse CD4 (L3T4, clone RM4-5; BD Pharmingen), biotinylated anti-mouse CD8a (Ly-2, clone 53-6.7; BD Pharmingen), and biotinylated anti-mouse CD45R/B220 (clone RA3-6B2; BD Pharmingen). Staining intensity for all Ags was calibrated using appropriate positive and negative controls. Based on these controls, biotinylated anti-mouse I-Ab was incubated overnight at 4°C, whereas the remaining Abs were incubated for 1 h at room temperature. Positive and negative controls were included with every staining experiment. Streptavidin-HRP (Research Genetics, Huntsville, AL) was applied for 45 min at 37°C. Stable 3,3'-diaminobenzidine (Research Genetics) was applied for 10 min at room temperature, followed by counterstaining with aqueous hematoxylin.
Statistics
Actuarial survival curves were calculated by the Kaplan-Meier product-limit method using the computer-based statistics program STATISTICA (StatSoft, Tulsa, OK). The equality of the survival curves was tested by Cox-Mantel analysis. A value of p < 0.05 was considered statistically significant.
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Results |
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We have recently described a method to isolate mouse vascular endothelium, devoid of hemopoietic APCs. Resting mouse vascular endothelium lacks MHC class II expression. Although it expresses CD80 (B7.1) and CD54 (ICAM-1), there is no detectable CD40 or CD86 (B7.2). Similar to human endothelium, treatment with IFN- induces the expression of MHC class II on the surface of mouse vascular endothelial cells. Although expression levels of CD80 do not change, CD54 expression is mildly increased after treatment with IFN-. Furthermore, treatment with IFN- does not induce the expression of CD40 or CD86 (12). We examined whether vascular endothelium could activate allogeneic CD4+ T lymphocytes in vitro. Based on its lack of MHC class II expression and consequent inability to engage the TCR on CD4+ T lymphocytes, we did not expect resting B6 vascular endothelium to induce proliferation of C3H CD4+ T lymphocytes (Fig. 1A). However, based on the surface phenotype of endothelial cells after treatment with IFN-, with expression of both signals 1 and 2, we hypothesized that cytokine-activated mouse vascular endothelium would induce proliferation of allogeneic CD4+ T lymphocytes. We were surprised at the lack significant proliferation of CD4+ T lymphocytes after coculture with activated endothelium (Fig. 1B). Alternatively, significant proliferative responses were seen when C3H CD4+ T lymphocytes were stimulated with B6 splenocytes in vitro (Fig. 1C).
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Having shown that vascular endothelial cells do not induce proliferation of alloreactive CD4+ T lymphocytes, we tested the Ag-presenting capacity of nonhemopoietic cells in vivo. To examine this question, we created bone marrow chimeras to control for MHC class II expression on nonhemopoietic and hemopoietic cells. As recently reported by our laboratory, bone marrow transplantation leads to functionally complete replacement of hemopoietic APCs (14). We have also established that the level of replacement in all tissues amounts to >99.99%, as determined by semiquantitative RT-PCR, with no contribution to allorecognition, as assessed by MLR. Specifically, we created B6(B6II–) bone marrow chimeras, in which hemopoietic cells are unable to activate allogeneic CD4+ T lymphocytes due to their lack of MHC class II expression. We also created B6II–(B6) chimeras, in which nonhemopoietic cells cannot activate allogeneic CD4+ T lymphocytes. Vigorous proliferation was seen after adoptive transfer of CFSE-labeled, purified C3H CD4+ T lymphocytes into supralethally irradiated, wild-type B6 mice (Fig. 2A). Theoretically, CD4+ T lymphocytes could have been activated by both nonhemopoietic and hemopoietic APCs. No proliferation was observed when C3H CD4+ T lymphocytes were transferred into B6II– mice (Fig. 2B). We did not observe proliferation when C3H CD4+ T lymphocytes were injected into B6(B6II–) bone marrow chimeras, suggesting that nonhemopoietic cells do not contribute to the activation of allogeneic CD4+ T lymphocytes in this model (Fig. 2C). Furthermore, no proliferation was seen even after pretreatment of the B6(B6II–) bone marrow chimeras with systemic IFN- 72 h before the adoptive transfer of allogeneic T lymphocytes (Fig. 2D), a regimen that induces MHC class II expression on vascular endothelial cells in vivo (Fig. 2F) (15). Alternatively, vigorous proliferation was seen when C3H CD4+ T lymphocytes were transferred into B6II–(B6) bone marrow chimeras, where only hemopoietic cells can contribute to the activation of alloreactive CD4+ T lymphocytes (Fig. 2E). Taken together, these findings indicate that nonhemopoietic cells are poor APCs for CD4+ direct allorecognition in vivo.
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Our in vitro and in vivo findings suggested that vascular endothelial cells of allografts do not contribute to CD4+ direct allorecognition, a pathway known to be critical for the vigor of acute allograft rejection. To test this question, we first transplanted cardiac allografts derived from B6II– mice into C3H mice to eliminate CD4 direct allorecognition. Although B6 hearts bearing B6 hemopoietic cells were acutely rejected with a median survival time of 7 days, the survival of MHC class II-deficient hearts was significantly prolonged, with a mean survival time of 14 days (Fig. 3A). Because this rejection is mediated via the CD4 indirect and/or the CD8 direct pathway, this suggests that CD4 direct allorecognition is a critical determinant of the tempo of acute rejection. We next examined whether vascular endothelium contributes to the direct activation of CD4+ T lymphocytes. To this end, we transplanted cardiac allografts derived from B6II–(B6) and B6(B6II–) bone marrow chimeras. When B6II–(B6) cardiac allografts were transplanted, only hemopoietic APCs had the capacity to activate CD4 direct allorecognition. B6II–(B6) hearts were acutely rejected with a median survival time of 7 days, identical with that seen in B6 hearts bearing B6 hemopoietic cells (Fig. 3B). Alternatively, when B6(B6II–) hearts were transplanted, only nonhemopoietic cells of graft origin can theoretically activate CD4+ direct allorecognition. Interestingly, B6(B6II–) grafts were rejected with a mean survival time of 14 days, identical to that seen for B6II– hearts (Fig. 3B). Cardiac allografts from all experimental groups showed histological evidence of acute rejection. In rejecting hearts from all experimental groups the cellular infiltrate consisted predominantly of CD8+ T lymphocytes, with a lesser contribution by CD4+ cells (data not shown).
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To confirm that cardiac allografts undergoing acute rejection expressed MHC class II on nonhemopoietic cells, rejected cardiac allografts were stained with Abs to the MHC class II Ag I-Ab and to CD31, an adhesion molecule specific for vascular endothelium. Untransplanted B6 and B6II– hearts were also stained with these Abs. Untransplanted B6 hearts showed positive staining for MHC class II Ag on rare interstitial cells with dendritic cell morphology and had no staining on vascular endothelium (Fig. 5). Untransplanted B6II– hearts showed no staining for MHC class II Ag (data not shown). The MHC class II staining of rejected grafts was consistent with the predicted pattern of expression (Fig. 6). Of note, rejected B6 grafts bearing B6 hemopoietic cells and B6 grafts bearing B6II– hemopoietic cells expressed MHC class II on their vascular endothelium. These studies demonstrate that although mouse endothelium lacks constitutive expression of MHC class II, it is expressed in the inflammatory setting of acute allograft rejection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4 T cells can be activated via two distinct pathways of allorecognition, direct and indirect. In the direct pathway, CD4 T cells are activated by allogeneic graft-resident APCs. Alternatively, CD4 activation by host APCs that have processed alloantigen is referred to as the indirect pathway. Of note, a recent study has shown that donor MHC class II expression is a requirement for CD4-mediated allograft rejection, emphasizing the critical role of CD4+ direct allorecognition in the initiation of allograft rejection (21). It is generally believed that graft-resident hemopoietic cells are responsible for the activation of CD4 direct allorecognition. It is based on these concepts that strategies were designed to deplete donor-type APCs in an attempt to weaken the alloimmune response (14, 22). Interestingly, although some prolongation in survival has been documented with these approaches, these allografts are nevertheless eventually rejected. Although it is generally believed that rejection that occurs in the absence of allogeneic APCs proceeds by the indirect pathway, our recent observations with CD8+ T cells raise the possibility that nonhemopoietic cells of the allograft can activate CD4+ T cells directly (23).
There has been controversy in the literature about the ability of vascular endothelium to activate allogeneic CD4+ T cells. It has been demonstrated that treatment of cultured human vascular endothelium with IFN- induces the surface expression of MHC class II (24). Although some investigators have observed proliferation of and cytokine production by CD4+ T cells after coculture with cytokine-activated human endothelial cells, others have reported the inability of cytokine-activated endothelium to initiate CD4+ alloresponses (8, 25, 26, 27). Similar to human endothelium, MHC class II is induced after treatment with IFN- on cultured mouse vascular endothelium (12, 13). In addition, it expresses B7.1, a costimulatory molecule that engages CD28 on T cells and is important for the transcription of IL-2 and up-regulation of the anti-apoptotic protein Bcl-xL (28). We have previously shown that mouse vascular endothelium is able to induce proliferation of alloreactive CD8+ T lymphocytes in B7-dependent fashion (29). Surprisingly, despite the expression of both signal 1 and signal 2, cytokine-activated mouse endothelium is a poor stimulator for allogeneic CD4+ T cells in vitro. These observations are consistent with previous reports suggesting that although mouse vascular endothelium can activate CD8+ T cells, it is unable to activate CD4+ T cells via direct allorecognition (30). Moreover, in models of self-restricted responses to nominal Ags, cytokine-activated mouse vascular endothelium was able to induce DNA synthesis only in previously activated CD4+ T cells (31). Recent studies have illustrated the existence of differential activation requirements for CD4+ and CD8+ T lymphocytes, such as densities of MHC molecules and expression patterns of costimulatory molecules (32). Our observations raise the possibility that although the mouse vascular endothelial cells are capable of directly activating allogeneic CD8+ T cells, the MHC class II density and expression of costimulatory molecules on endothelial cells may not engage a sufficiently large number of TCRs on CD4+ T cells to attain the threshold for their activation (33). These data are further corroborated by the observations of our in vivo mixed lymphocyte reactions, although these experiments do not address the theoretic possibility that vascular endothelial cells could activate alloreactive CD4+ T lymphocytes in tissue locations other than secondary lymphoid organs.
A recent study has shown that mice that lack a spleen and peripheral lymph nodes are unable to reject cardiac allografts (34). Hemopoietic APCs are known to leave the graft shortly after revascularization and home to the recipient’s lymphoid organs, where the initial alloimmune response occurs via direct allorecognition (35). When all pathways of allorecognition are functional, CD4+ direct allorecognition is generally considered to be the most potent and thought to be principally responsible for the tempo of acute rejection (20, 36). Consequently, we have observed prolongation in allograft survival when CD4+ direct allorecognition triggered by graft-resident hemopoietic APCs has been eliminated (14, 36). Based on our in vitro findings we hypothesized that vascular endothelium would not play an important role in CD4+ direct allorecognition in vivo. In this study we are the first to show that CD4+ direct allorecognition triggered by nonhemopoietic cells, such as vascular endothelium, does not influence the tempo of acute rejection when all other allorecognition pathways are functional. When CD4+ direct allorecognition triggered by graft-resident hemopoietic APCs is eliminated, MHC class II expression by nonhemopoietic cells cannot reconstitute the vigor of acute rejection. In fact, we have demonstrated that CD4+ T lymphocytes of recipients of cardiac allografts lacking MHC class II expression on hemopoietic cells are activated predominantly through indirect allorecognition. Such grafts are rejected with the same tempo as hearts that lack MHC class II expression on both hemopoietic and nonhemopoietic cells. In the final analysis, CD4+ direct allorecognition may depend on migration of APCs to the host’s secondary lymphoid organs (34, 37). Moreover, there exists no evidence in the literature that vascular endothelial cells have the capacity to migrate to the host’s secondary lymphoid organs. Nonetheless, our findings suggest that the inability of endothelial cells to activate CD4+ T cells is primarily related to their poor Ag-presenting capacity.
Although our study shows that despite expression of MHC class II, vascular endothelial cells are poor APCs for allogeneic CD4+ T cells in vitro and do not contribute to CD4+ direct allorecognition in vivo, we cannot exclude that MHC class II expression by endothelial cells is important for other immune processes. It has been previously shown that cognate recognition of MHC class II molecules on vascular endothelium by T lymphocytes facilitates their transendothelial migration in vitro (38). These observations suggest that expression of MHC class II molecules by the vascular endothelium may play an important role in recruiting T cells into the allograft. Although it was not the goal of this study to examine recruitment of CD4+ T cells at serial time points, we have observed similar graft infiltration with CD4+ T cells in rejected hearts regardless of endothelial MHC class II expression. Therefore, we believe that the impact of MHC class II expression on recruitment of CD4+ T cells does not influence the tempo of acute rejection. A possible role of CD4+ T cell recruitment by MHC class II expression on vascular endothelium in the development of chronic rejection needs to be explored.
Donor-type hemopoietic professional APCs are important in initiating alloimmune responses in the immediate postoperative period. However, these cells decrease in number over time and are eventually cleared from the host. Although our previous studies have shown that nonhemopoietic cells are potent APCs for CD8+ T lymphocytes, this is not the case for CD4+ T cells. In summary, the results of our experiments demonstrate that endothelial MHC class II Ag expression does not influence the time course of acute rejection. This suggests that, unlike the case for CD8+ direct allorecognition, the in vivo Ag-presenting capacity of endothelial cells for CD4+ direct allorecognition is quite limited. Rather, it is the Ag-presenting capacity of the graft-resident hemopoietic APCs that is the primary stimulus for CD4+ direct allorecognition.
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Footnotes |
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1 This work was supported in part by National Institutes of Health Grant RO1AI47257-01A1. 
2 D.K. and A.M.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Bruce R. Rosengard at the current address: British Heart Foundation Professor and Chair of Cardiothoracic Surgery, Papworth Hospital, University of Cambridge, Papworth Everard, Cambridge, U.K. CB3 8RE. E-mail address: bruce.rosengard{at}papworth.nhs.uk
Received for publication May 14, 2004. Accepted for publication June 28, 2004.
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cells. Nature 336:476.[Medline]
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