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Bcl-2 Transduction Protects Human Endothelial Cell Synthetic Microvessel Grafts from Allogeneic T Cells In Vivo¹

Lian Zheng^*, Thomas F. Gibson^*, Jeffrey S. Schechner^,, Jordan S. Pober^*,, and Alfred L. M. Bothwell^2,*,

^* Section of Immunobiology and Interdepartmental Program in Vascular Biology and Transplantation, Departments of Dermatology and Pathology, Yale University School of Medicine, New Haven, CT 06520

	Abstract

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T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis. Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings. In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues. Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.

	Introduction

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The vascular endothelium of an allograft is a primary target for immune-mediated rejection (1, 2). Either CD4 or CD8 T lymphocytes can be mediators of this response. The CD4 T cell response depends on IFN-, because this cytokine is necessary for induction of MHC class II on endothelial cells (EC)³ (3, 4, 5), which are required for the recognition by CD4 T cells (6). MHC class I molecules, which are recognized by CD8 T cells, are constitutively expressed by EC at sufficient levels to trigger T cell recognition even without further induction by cytokine (7). In contrast to most non-bone marrow-derived cells, EC express functional costimulatory molecules that can result in activation of resting memory T cells (7). Although activation of resting CD8 T cells to effector CTL can occur after recognition of MHC class I-expressing cells and appropriate costimulatory molecules, clonal expansion of effector cell populations requires an adequate source of the growth factor IL-2, which often must be provided by CD4 T cells (8).

Calcineurin inhibitor-based immunosuppressive therapies, using cyclosporine or tacrolimus, are designed to prevent IL-2 synthesis and thus prevent CTL expansion, because, once generated, CTL may be resistant to these agents (9). Although calcineurin inhibitor-based immunosuppression has achieved remarkable success, it is often limited by toxicity, and, even when optimal, CTL-mediated rejection episodes can still occur. A complementary strategy, not yet applied in the clinic, would be to increase the resistance of graft cells, especially graft EC, to the effects of CTL. We have investigated the potential cytoprotective effects of overexpression of human Bcl-2 on the functional cytolytic activity of alloreactive CD8 T cells in vitro (10). HUVECs retrovirally transduced with Bcl-2 showed nearly complete resistance to a variety of inducers of apoptosis, including alloreactive CTL. It is difficult to predict whether Bcl-2 transduction will protect human EC from T cell injury in vivo. We have found that implantation of human skin or artery into immunodeficient SCID/bg mice followed by adoptive transfer of allogeneic human PBMC provides a useful model for analyzing in vivo T cell reactions. We use SCID/bg mice as hosts, because these animals have compromised NK cell as well as T and B cell functions and thus accept human lymphoid cells as well as solid tissue grafts. Following inoculation i.p. with 1–3 x 10⁸ human PBMC, human CD4 and CD8 T cells appear in the circulation 1 wk after injection and persist for 2 mo. B cells also engraft and produce human Ab, but human NK cells, mononuclear phagocytes, and dendritic cells do not engraft. In previous studies with this model, a human alloresponse to well-healed orthotopic skin or coronary artery interposition grafts occurs spontaneously by 2 wk and involves significant infiltration of both CD4 and CD8 T cells (11). Removal of B cells from the inoculum does not alter the response to human skin, indicating that this is a wholly T cell-mediated reaction (12). By 3 wk, there is extensive immune-mediated injury with nearly complete destruction of human vessels. Histopathologic analyses suggest that CTL are the major effector populations, but other mechanisms could also contribute.

Despite their utility as models for studying T cell-mediated injury, skin or artery grafts have not lent themselves to studies of gene therapy-mediated cytoprotection. This is because human EC in these tissues have been difficult to transfect or transduce with high efficiency. In the present report, we extend the use of human-mouse chimeric models to study interactions between human lymphocytes and genetically modified EC in vivo by using a system we developed for forming a synthetic microvascular bed in SCID/bg mice (13). Normal HUVEC spontaneously form tubes when cast in collagen/fibronectin gels, which, when implanted into the abdominal wall of SCID/bg mice, spontaneously anastomose with the mouse microvessels at the gel edge and become perfused with mouse blood after a period of 2–3 wk. The overexpression of Bcl-2 protein results in recruitment of smooth muscle-like cells (SMC) of mouse origin. Bcl-2-transduced HUVEC also form a more extensive network of tubes than do control-transduced or untransduced HUVEC. In the present report, we have used this model to analyze the effect of Bcl-2 transduction on T cell-mediated injury in vivo. We find that Bcl-2 expression completely protects EC from T cell-mediated injury, although the persistence of human EC may actually enhance allogeneic T cell graft infiltration and expansion.

	Materials and Methods

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Animals

C.B-17 SCID/bg mice (Taconic, Germantown, NY) were used at 5–6 wk of age. The animals were housed in microisolator cages and fed sterilized food and water. All animal experimental manipulations were performed under a protocol approved by the Yale Animal Care and Use Committee. Serum IgG levels were determined by ELISA using reagents from Cappel (Durham, NC). SCID/bg mice were considered leaky at IgG levels of >1 µg/ml and excluded from experimental use.

Cells and retroviral transduction

HUVEC were isolated by collagenase treatment of human umbilical veins under a protocol approved by the Yale Human Investigation Committee as previously described and cultured on 0.2% gelatin-coated plastic in Medium 199 with 20% FCS, 50 µg/ml EC growth supplement (Collaborative Research/BD Biosciences, Bedford, MA), 100 µg/ml porcine intestinal heparin (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All of the HUVEC used in these experiments were at passage 1 through 6. Such cultures are homogeneous for EC markers (vWF, CD31, inducible E-selectin) and are free of contaminating CD45⁺ leukocytes.

Retroviral vectors expressing the caspase-resistant form of Bcl-2 have been previously described (14). HUVEC were infected with retroviral vectors in the presence of polybrene (hexadimethrine bromide; 8 µg/ml; Sigma-Aldrich) daily for up to 3 days. One day after the last infection, the cells were cultured in medium with 600 µg/ml G418, and after 10-day incubation, 95% of the cells expressed transgene.

Formation of HUVEC vascular construct in collagen/ fibronectin gels

HUVEC were isolated and cultured as described. Microvessel grafts in collagen/fibronectin gels were prepared using a modified protocol from that previously described (13). EC (3 x 10⁶ cells) were suspended in a 1.2 ml of a solution of rat tail type 1 collagen (1.5 mg/ml), human plasma fibronectin (100 µg/ml) (both from BD Biosciences), 25 mM HEPES, 1.5 mg/ml NaHCO₃, 10% FCS, and 10x Medium 199 (Sigma-Aldrich) for HUVEC at 4°C. The pH was adjusted to 7.5 by 0.1 M NaOH. The HUVEC collagen-fibronectin suspension was pipetted into rat tail type 1 collagen-coated C-6 Transwells (BD Biosciences) and warmed to 37°C for 15 min to allow polymerization of the collagen. M199 or DMEM medium with 20% FCS was added to the Transwells to cover the solidified gels. For implantation into SCID/bg mice, gels were harvested and trisected 24 h after gel formation. Each resulting 1 x 1 x 0.2-cm gel segment was implanted into a bluntly dissected s.c. pouch in the anterior abdominal wall of a 5- to 6-wk-old mouse. The wound was closed with a skin staple.

PBMC isolation and reconstitution

Normal PBMC were obtained from healthy donors by leukapheresis under a protocol approved by the Yale Human Investigation Committee. PBMC were isolated by density gradient centrifugation using lymphocyte separation medium (Organon Teknika, Durham, NC) as previously described. The cells were stored in 10% DMSO in liquid nitrogen for up to 12 mo, thawed, and washed before use. Microvessel-grafted SCID/bg mice were reconstituted with 3 x 10⁸ human PBMC by i.p. inoculation. The number of circulating human T cells was evaluated by indirect immunofluorescence flow cytometry. In brief, heparinized retro-orbital venous blood samples were obtained at different time points after reconstitution. The erythrocytes were lysed using ammonium chloride, and leukocytes were incubated with PE-conjugated rat anti-mouse CD45 (BD Pharmingen, San Diego, CA) mAb and FITC-conjugated mouse anti-human CD3 mAb (Immunotech/Beckman Coulter, Marseille, France), FITC-conjugated mouse anti-human CD4 mAb (Immunotech/Beckman Coulter), or FITC-conjugated mouse anti-human CD8 mAb (Immunotech/Beckman Coulter), and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences).

Human CD3⁺ T cell isolation and reconstitution

CD3⁺ T cells were isolated from PBMC by negative selection. In brief, PBMC were first depleted of monocytes by adherence to culture flasks for 1 h at 37°C. Nonadherent cells were incubated with a mixture of mAb containing anti-MHC class II Ab (BD Pharmingen), anti-CD14 Ab (BD Pharmingen), anti-CD16 Ab (BD Pharmingen), anti-CD16b Ab (BD Pharmingen), and anti-CD56 Ab (BD Pharmingen) for 25 min at 4°C. The cells were then washed to remove excess Ab. Further enrichment was done by magnetic immunodepletion using BioMag goat anti-mouse IgG-bound bead (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The purity of the CD3 T cells was >90% as determined by direct immunofluorescence staining with anti-CD3 Ab (BD Pharmingen). Microvessel-grafted SCID/bg mice were reconstituted with 3 x 10⁷ purified CD3⁺ T cells by i.p. inoculation.

Immunohistochemistry and quantitative analysis of sections

HUVEC gels were harvested from SCID mice at various times after PBMC cell injection, fixed in 10% buffered formalin, and embedded in paraffin. Sections (5-µm thick) were cut for immunostaining. In some experiments, specimens were instead snap frozen in Tissue-Tek OCT (Sakura Finetek, Zoeterwoude, The Netherlands) and 6-µm cryosections were prepared. Ab staining was conducted using the Vectastain ABC (avidin/biotin complex) kit (Vector, Burlingame, CA). Specifically, single Ab staining was performed with primary Ab, such as anti-human collagen IV (Sigma-Aldrich), anti-MHC class I (BD Pharmingen), anti-MHC class II (BD Pharmingen), anti-human ICAM-1 (BD Pharmingen), anti-human VCAM-1 (BD Pharmingen), anti-human CD45RO mAb (DakoCytomation, Carpinteria, CA), anti-human CD4 (Serotec, Raleigh, NC), anti-human CD8 mAb (Serotec, Raleigh, NC), or anti-human perforin (BD Pharmingen), for 1 h and washing with PBS, followed by incubation with biotinylated secondary Ab (Vector) for 1 h and washing with PBS. Slides were then incubated for 1 h with the ABC reagent and incubated with diaminobenzidine (DAB) peroxide substrate until the desired color developed, followed by a light hematoxylin stain (Sigma-Aldrich). For double staining with CD45RO and collagen IV Ab, the sections were first treated with anti-human CD45RO mAb, followed by incubation with biotinylated secondary Ab and visualized by DAB-NiCl2 (dark blue color). Then, the sections were incubated with anti-human collagen IV Ab, followed by incubation with biotinylated secondary Ab and visualized by DAB (brown color).

For quantitation of PBMC-mediated injury, the numbers of human type IV collagen-outlined human microvessels or human CD45RO T cells within the cross-section of the entire gel were either counted manually under the microscope, or a picture of the cross-section of the entire gel was taken, and the entire gel area was measured using Scion Image 4.02 for Windows 95 software (downloaded from www.scioncorp.com/frames/fr_download_now.htm). In that case, the density of human microvessels (numbers of vessels per square millimeter) or human CD45RO T cells was calculated by dividing the numbers of microvessels or human CD45RO T cells within the entire gel by the cross-sectional area of the gel.

For quantitation of CD3⁺ T cell-mediated injury, the numbers of human type IV collagen-outlined human microvessels within the cross-section of the entire gel were counted manually under the microscope. The survival of the microvessels on CD3 T cell-injected samples was calculated by dividing the number of microvessels on CD3 T cell-injected samples by the number of microvessels on saline-injected samples. The p value was determined to be <0.001, indicating that there was significant difference of microvessel survival between the CD3 T cell- and saline-injected samples.

	Results

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Previously, we have described an in vivo model system for creating synthetic microvessels using HUVEC in SCID/bg mice (13). In the original description of these microvessels, there was a substantial recruitment of mouse SMC to the synthetic microvessels when the HUVEC were transduced with Bcl-2 compared with reporter gene (EGFP)-transduced HUVEC. The experiments described in this study use a minor modification, which is the inclusion of FBS when the gels are cast in the presence of collagen/fibronectin. This enhances the reproducibility and density of the microvessels that form from untransduced HUVEC. Moreover, it also allows HUVEC not transduced with Bcl-2 to recruit mouse SMC. Although Bcl-2-transduced HUVEC have even greater numbers of SMC surrounding the microvessels, the inclusion of serum enhances the utility of this model, because Bcl-2 transduction is not required to form good microvessels.

Because we had found a high degree of cytoprotection in vitro from human CTL by overexpression of Bcl-2 in HUVEC, we wished to assess whether this strategy would work in vivo. We first implanted groups of mice with gels containing either unmodified HUVEC or Bcl-2-transduced HUVEC as described above. The optimal conditions for reconstitution of these mice with PBMC had been established previously (15, 16). Intraperitoneal injection of 3 x 10⁸ PBMC per mouse results in effective reconstitution in all recipients. Based on a series of pilot experiments, we allowed the microvascular bed to heal for 2 wk in vivo and then injected i.p. either saline (control) or PBMC. Grafts were harvested and stained with anti-CD45RO to visualize T cells at various times after PBMC injection. At 10 days following introduction of the allogeneic PBMC, there is no alteration of the structure of HUVEC-derived microvessels compared with control animals, and no T cells are detected within the gel graft (Table I). Untransduced microvessel grafts analyzed at 2 wk show minimal evidence of microvessel destruction as well as the presence of a few T cells in samples that received PBMC (one mouse of three showed a 50% reduction in the number of microvessels). When mice were examined 3 wk after introduction of PBMC, we note consistent destruction of untransduced microvessels in the grafts (60–100%) and infiltration by a significant number of T cells within grafts containing either untransduced or HUVEC-Bcl-2-transduced microvessels in animals receiving PBMC but not in no-PBMC control animals (Figs. 1 and 2; Table I). In other words, despite the fact that microvascular bed grafts formed from Bcl-2-transduced HUVEC showed infiltration of T cells in all samples that received PBMC, there was little evidence of microvessel loss.

View larger version (65K): [in this window] [in a new window]   FIGURE 1. Identification of synthetic human microvessels. HUVEC or HUVEC transduced with Bcl-2 were used to form synthetic microvessels and implanted into SCID mice. Either saline (control) or 3 x 108 PBMC/mouse were injected i.p. into grafted mice 2 wk after implantation. Human microvessels were recovered 3 wk after PBMC injection and prepared as paraffin sections. Human collagen IV Ab staining was used to detect human microvessels. The experiment shown is representative of six similar experiments.

View larger version (88K): [in this window] [in a new window]   FIGURE 2. Identification of human CD45RO T cells in human microvessels 3 wk after PBMC injection. Mice were injected with PBMC 2 wk after implantation. Human microvessels were recovered 3 wk after PBMC injection and embedded in paraffin. A (saline control) and B (+PBMC) contain HUVEC microvessels stained with anti-CD45RO; C (saline control) and D (+PBMC) contain HUVEC-Bcl-2 microvessels stained with CD45RO; and E (saline control) and F (+PBMC) contain HUVEC-Bcl-2 microvessels stained with anti-CD45RO (dark blue), anti-collagen IV (brown), and a light blue counterstain. Human CD45RO Ab staining was used to detect human CD3+ T cells. The experiment shown is representative of six similar experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Enumeration of human T cells in peripheral blood of grafted SCID/bg mice. The number of circulating human T cells was evaluated by indirect immunofluorescence flow cytometry. The leukocytes were incubated with PE-conjugated rat anti-mouse CD45 mAb, FITC-conjugated mouse anti-human CD3 mAb, FITC-conjugated mouse anti-human CD4 mAb, and FITC-conjugated mouse anti-human CD8 mAb, and analyzed using a FACScan flow cytometer. The experiment shown is representative of three similar experiments.

View larger version (70K): [in this window] [in a new window]   FIGURE 4. Expression of HLA class I on synthetic human microvessels. Experiments were performed as described in Fig. 1 and analyzed for expression of HLA class I using frozen sections. The experiment shown is representative of four similar experiments.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Expression of HLA class II on synthetic human microvessels. Experiments were performed as described in Fig. 1 and analyzed for expression of HLA class II using frozen sections. The experiment shown is representative of four similar experiments.

View larger version (96K): [in this window] [in a new window]   FIGURE 6. Expression of ICAM-1 and VCAM-1 on synthetic human microvessels. Experiments were performed as described in Fig. 1, but only HUVEC-Bcl-2 implants were analyzed for expression using frozen sections. The experiment shown is representative of three similar experiments.

View larger version (108K): [in this window] [in a new window]   FIGURE 7. Expression of CD8 and perforin on synthetic human microvessels. Experiments were performed as described in Fig. 1, but only HUVEC-Bcl-2 implants were analyzed for expression using frozen sections. The experiment shown for CD8 expression is representative of six similar experiments and three for the analysis of perforin expression.

View larger version (13K): [in this window] [in a new window]   FIGURE 8. Purified CD3+ T cells develop cytotoxic function in vivo. T cells were purified from fresh PBMC, and 3 x 107 CD3+ T cells were injected i.p. 2 wk following gel implantation and harvested 6 wk later.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanism of the development of T cell cytolytic function appears consistent with certain expectations. The T cells expand in vivo, and activation of human T cells, especially CD8 cells, results in secretion of cytokines. Expression of HLA class II is virtually undetectable in the absence of PBMC but dramatically increased after injection, probably as a consequence of IFN-. Adhesion molecules ICAM-1 and VCAM-1 are induced on the HUVEC-Bcl-2 microvessels that are resistant to cytolysis as a consequence of Bcl-2 expression.

In murine systems, CD4 T cells play an important role in facilitating secondary expansion and development of a CD8 memory response (18). The role of CD4 T cells in a human CD8 response is less clear. The provision of growth factors, especially IL-2, may be the only requirement. In vitro-purified human CD8 T cells do generate an effector T cell response to HUVEC (19, 20). This in vivo model should be very valuable in these types of studies of basic requirements for development of an immune response. Recently, there has been considerable evidence that regulatory T cells can have a potent effect in promoting graft survival. This system is also well suited to examine at a mechanistic level the function of human regulatory cells in an in vivo model.

Mechanistic studies and preclinical evaluation of therapeutics of human lymphocyte interactions with blood vessels and tissues in vivo are severely limited for ethical reasons, and proof of principle of gene therapy has almost exclusively depended upon animal models or in vitro systems. The results presented clearly show that our model is well suited to assess the functional properties of a broad range of gene products and it is possible that other protective genes that show limited effects in in vitro assays may show significant functional phenotypes in the in vivo model. The genetic modifications that could be made to the EC are essentially unlimited. Factors that enhance recognition can be overexpressed or inhibited in HUVEC. For example, assessment of genes that affect homing or activation of lymphocytes (e.g., adhesion molecules, cytokines, chemokines, or costimulators) could also be effectively studied.

In conclusion, we have developed a novel model system involving transplantation of cultured human EC into immunodeficient mice, resulting in formation of a synthetic microvascular bed. We have shown in this study that this system can be combined with adoptive transfer of human T cells to evaluate the efficacy of gene therapy as a strategy for reducing allograft injury. Strikingly, the protective effect of Bcl-2 is even more pronounced in vivo, where the possibilities of injury are more complex, than it was in vitro.

	Acknowledgments

 
We thank Agnes Keh for technical assistance with the immunohistochemistry, and Louise Camera-Benson and Gwendolyn Davis in the Boyer Center for Molecular Medicine Tissue Culture facility for providing HUVEC. Retroviral vectors and packaging cell lines were obtained from Dr. Garry Nolan at Stanford University (Stanford, CA).

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants HL051448 and HL65744 (to A.L.M.B.) and HL62188 (to J.S.P.).

² Address correspondence and reprint requests to Dr. Alfred L. M. Bothwell, Section of Immunobiology, P.O. Box 208011, 300 Cedar Street, Yale University School of Medicine, New Haven, CT 06520-8011. E-mail address: alfred.bothwell{at}yale.edu

³ Abbreviations used in this paper: EC, endothelial cell; SMC, smooth muscle-like cell; DAB, diaminobenzidine.

Received for publication March 29, 2004. Accepted for publication June 25, 2004.

	References

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