Endothelial cells present antigens in vivo

To determine the state of β-gal specific lymphocytes in transgenic mice expressing β-gal in their vascular EC, VWF-lacZ transgenic and wild type mice were immunized with a β-gal expression vector DNA using a gene gun. DNA immunization can induce both humoral and cellular responses [42‐44] and B lymphocytes require specific T cell help to form a high titer antiserum against β-gal [45]. Thus, antiserum titers demonstrated the activity of antigen specific B cells and also provided valuable surrogate markers for assessing the state of antigen specific T cell populations. The antiserum titer and antibody composition were determined by an Ig isotype specific ELISA for antibodies to β-gal.

Immunization raised high titer, β-gal specific antisera in both transgenic VWF-lacZ and congenic, wild type FVB mice (Figure 1A, filled symbols). Ig isotypes characteristic of both Th1-like responses (IgG2a) and Th2-like responses (IgG1) were detected at equivalent levels in transgenic and wild type mice (Figure 1A). Serum collected from these mice before immunization (preimmune) or from mice immunized with a control expression vector (CMV-GFP) did not contain β-gal specific antibodies (open symbols, Figure 1A), demonstrating that the antisera were formed in response to the immunization. Both VWF-lacZ and FVB mice generated primary antisera that were much stronger than sera from non-immune mice and the secondary responses were even stronger, demonstrating maturation of the immune response in both transgenic and wild type mice (Figure 1B). Similarly, immunized TIE2-lacZ transgenic mice also generated strong antisera against β-gal (Figure 1C). Thus, expression of β-gal in the EC of these transgenic mice did not appear to alter their immune responses against β-gal.

B lymphocytes secreting high affinity antibodies can be the most susceptible to tolerance induction [35]. To compare the affinities of the β-gal specific antisera from transgenic and wild type mice, we incubated the sera with soluble β-gal before adding them to the coated wells. Once bound to antigen, high affinity antibodies are less likely to dissociate and bind the plated antigen, providing a good measure of affinity [46]. Equivalent levels of soluble β-gal inhibited sera from wild type and transgenic mice, suggesting that they have equivalent affinities for β-gal (Figure 1D). Plate binding was halved (IC50) between 10–100 pM free β-gal, demonstrating the generation of high affinity antisera in both strains. Surface plasmon resonance assays also suggested equivalent antibody affinities in wild type and transgenic mouse sera (Biacore, data not shown).

We also compared immune responses to immunization with β-gal protein. Transgenic and wild-type mice were immunized with β-gal protein emulsified in complete Freund's adjuvant (CFA). Both strains of transgenic mice and the wild type mice responded with high titer antisera containing both IgG1 and IgG2a isotype antibodies (Figure 2).

To analyze directly the T cell responses in transgenic mice, we measured the responses of lymph node T cells from immunized mice in vitro. Proliferation was measured by tritiated ([³H]) thymidine incorporation. The expansion of CD4⁺ (helper) and CD8⁺ (cytotoxic) T cell populations was measured by immunostaining and FACS analysis. Activated T cells were detected by their expression of CD25 (IL-2 growth factor receptor) and effector functions were assessed by measuring IFN-γ secretion.

In cultures that received β-gal protein, both CD4⁺ and CD8⁺ T cells from wild type and transgenic mice were activated (CD25⁺) on day 4 in vitro (Figure 3A). Cultures that received β-gal protein antigen contained many more cells than those that did not. We compared the kinetics of activation in the three mouse strains. The data compiled from several independent experiments revealed that activation of CD4⁺ and CD8⁺ T cells from transgenic and FVB mice peaked between days 4 and 6 (Figure 3B). To determine the antigen presentation pathways, monoclonal antibodies that bind FVB (H-2^q) MHC class I or class II molecules were added at the beginning of the culture (34-5-3S or 15-1-5P hybridoma supernatants, ATCC). Each antibody partially blocked T cell proliferation, suggesting that β-gal peptides are presented by both MHC class I and class II molecules (data not shown).

Tolerance may be most evident in the T cell populations with highest avidity for the antigen [47]. T cell avidity can be estimated by reducing the antigen dose, which limits antigen more rapidly for lower avidity T cells. T cells from transgenic and FVB mice showed equivalent dose-response relationships (Figure 3C), suggesting that T cells with similar avidities respond against β-gal in wild type and transgenic mice.

We next tested the effector functions of β-gal specific T cells. IFN-γ is an important antiviral cytokine produced by T cells. Recently, IFN-γ was shown to also induce blood vessel remodeling in the absence of leukocytes, suggesting that IFN-γ may link pathological changes in blood vessels to immune responses occurring within the vessel wall [48]. To test IFN-γ secretion, lymph node cells were restimulated immediately ex vivo with β-gal. Equivalent amounts of IFN-γ induced by β-gal were detected in the supernatants of cultured lymph node cells from transgenic and wild type mice (Figure 3D). This was true at all times after the initiation of culture, with the peak accumulation occurring between days 2 and 3. However, substantial variation was observed in the responses of the TIE2-lacZ mice. Whereas lymph node cells from FVB and transgenic VWF-lacZ mice responded to β-gal by secreting equivalent amounts of IFN-γ in 5 out of 5 experiments, lymph node cells from TIE2-lacZ mice produced equivalent or higher amounts of IFN-γ twice, lower amounts twice, and were unresponsive once (not shown). Although the reason for variation in the TIE2-lacZ responses is uncertain, it is clear that effector T cell populations from the transgenic mice are capable of responding normally following immunization with β-gal.

It was possible that conventional immunization protocols did not efficiently induce mature T cells capable of responding aggressively against intracellular proteins. Immune responses against microbial products can produce autoimmunity when they cross-react with self-proteins [49]. We attempted to initiate self-reactivity by infecting transgenic mice with a recombinant vaccinia virus encoding β-gal (β-gal-VV).

Transgenic mice remained apparently healthy for over 9 months after immunization with DNA or protein. Antiserum specific for β-gal would not be expected to bind EC in transgenic mice because the enzyme is expressed in the cytoplasm and not on the cell surface. However, specific T cells could perhaps respond against β-gal peptides presented on EC. To detect any immune effects on the vasculature, organs were harvested, fixed and sectioned, and blood vessels were stained and analyzed by morphometry. The heart (left ventricle), liver, lung, and spleen, the infrarenal aorta, ascending aorta, coronary artery, carotid artery, and iliac artery were examined in cross section. There were no histological abnormalities. No changes were observed in the areas of the lumen, intima, media or vessel (defined as within the external elastic lamina) of any of the large vessels examined (data not shown).

It was possible that mice reduced their expression of the lacZ transgene upon mounting an immune response. For example, transgenic mice expressing hepatitis B antigens were shown to reduce their expression of the viral transgenes upon infection with virus [50].

To compare overall lacZ expression in immunized and unimmunized mice, whole tissues were stained with the chromogenic β-gal substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). Cardiac EC expressed equivalent, easily detectable amounts of β-gal in immunized and unimmunized TIE2-lacZ and VWF-lacZ mice but not in control FVB mice (Figure 4A). Dermal EC also continued to express β-gal at comparable levels in immunized and unimmunized TIE2-lacZ mice (Figure 4B). Therefore, EC continued to express β-gal despite the strong humoral and cellular immune responses specific for β-gal.

In three series of transplantations, only 9 out of 35 transplanted hearts or hosts did not survive the procedure, failing within 1 week for technical reasons. Nearly all transgenic donor hearts that survived 1 week continued to beat for over 60 days after transplantation into FVB mice (11 out of 12 TIE2-lacZ; 10 out of 12 VWF-lacZ). Two control FVB transplants showed similar graft survival. β-gal specific antisera were not induced in FVB hosts of transgenic hearts (data not shown). Transgenic grafts survived even when the recipients were sensitized before transplantation by TIE2-lacZ skin graft or by immunization with β-gal protein in adjuvant (data not shown).

It was possible that the donor EC were replaced or stopped making β-gal. To test β-gal expression in the donor EC, hearts from long-term graft survivors were harvested and stained for β-gal expression. The vascular EC within the donor hearts continued to express β-gal (Figure 5) and the graft vessels were histologically normal (data not shown). Therefore, the transplanted hearts survived despite the continued expression of β-gal by their EC.

Skin grafting is a more rigorous test of immunological tolerance, capable of detecting very small differences between host and donor tissues. We performed full thickness skin transplants between TIE2-lacZ mice and wild type FVB mice. Full thickness grafts included the blood vessels underlying the muscle layer.

Skin grafts between transgenic TIE2-lacZ and wild type FVB mice appeared to survive equivalently on both hosts (5 separate experiments, totaling over 20 host mice of each strain). When skin grafts were reversed before placement and stapling, the direction of the re-grown fur also indicated that both hosts accepted the grafts. However, β-gal specific antibodies were detected in the serum of FVB mice engrafted with TIE2-lacZ skin (Figure 6A, representative of 5 separate experiments). The antibodies were largely of the IgG2a isotype, which predominate in cellular, Th1-mediated responses. No β-gal specific antibodies were detected in TIE2-lacZ mice engrafted with FVB skin or TIE2-lacZ mice engrafted with TIE2-lacZ skin.

To test whether β-gal expression by cardiac EC had any effect on the host immune response, VWF-lacZ mice were grafted with TIE2-lacZ skin. In 3 separate experiments, all but one of the control FVB mice (7 out of 8) and none of the VWF-lacZ mice (8 out of 8) developed antibodies, suggesting the VWF-lacZ mice accepted the β-gal⁺ EC in the donor TIE2-lacZ skin grafts (Figure 6B). In addition, the β-gal expression by EC within the skin grafts was analyzed by X-gal staining. All TIE2-lacZ skin grafts transplanted onto VWF-lacZ mice contained many β-gal⁺ EC whereas virtually no β-gal⁺ EC were observed in the grafts transplanted onto FVB mice (Figure 6C). The organization of the β-gal⁺ vessels within the TIE2-lacZ graft was disrupted, suggesting that the dermal vasculature had undergone remodeling but not replacement during engraftment (Figure 6C, compare TIE2-lacZ donor vs. VWF-lacZ host). The continued presence of β-gal⁺ EC in grafts on VWF-lacZ mice demonstrates that EC normally remain in the graft and suggests that EC were cleared from the grafts on FVB mice, probably by the host immune response.

VWF-lacZ transgenic mice were provided for this study by Dr. William Aird (Beth Israel Deaconess Medical Center, Boston) [40]. TIE2-lacZ transgenic mice were generated by Sato and colleagues [41] and purchased from the Jackson Laboratories (Bar Harbor, ME). Both transgenic strains were generated and maintained on the FVB background. The lines were maintained by backcrossing hemizygous transgenic mice to FVB/NJ mice (Jackson Laboratories). Transgenic offspring were identified from ear punches at approximately 6 weeks of age. The punches were digested (100 μl, 10 μg proteinase K, 10 mM KCl, 10 mM Tris pH 8, 2.5 mM MgCl₂, 0.45% NP-40, 0.45% Tween-20) for 3 h at 55°C then heated for 10 min. at 95°C. PCR was performed using 1 μl of the digest with primers for lacZ (Bgal.51: GCGTTGGCAATTTAACCG and Bgal.31: TGGTAATGGTAGCGACCG, each at 0.5 μM) and a control sequence tagged site (STS) from the X chromosome (M37335, each primer at 0.1 μM). The cycling conditions were: 1 min at 94°C, then 30 cycles of 30 s at 94°C, 30 s at 53°C, and 30 s at 72°C, then 10 min. at 72°C. The PCR products were separated on an agarose gel and detected by staining with ethidium bromide (788 bp β-gal, 200 bp M37335).

For generating antisera, mice were immunized by intraperitoneal (i.p.) injection of 50 μg β-gal protein emulsified in complete Freund's adjuvant (CFA, 50 μl final volume) and boosted 4 weeks later with 50 μg β-gal, i.p., in phosphate buffered saline. β-gal (CAS 9031-11-2) protein was purchased from Sigma (recombinant, G3153) and Calbiochem (345788). These preparations were used interchangeably and no differences were observed. Alternatively, mice were immunized with a gene gun. Briefly, an expression vector encoding β-gal (100 μg, CMV-β-gal, Clontech, CA) was precipitated with CaCl₂ onto gold beads (21 mg, 2.6 μm diameter, Aldrich or 1 μm BioRad), which were dried onto plastic tubing (1/8" × 3/32" Tefzel, Bio-Rad). The tubing was cut (25 × 1 cm) and loaded into a high pressure gun (Agracetus, Middleton, WI). With a burst of gas (300 psi), the beads were delivered into the skin on the backs of mice whose fur had been clipped. Mice were boosted with a second gene gun immunization after 6 weeks.

Serum samples were taken before and after immunizations, as noted in the figures. Blood was allowed to clot at room temperature then it was placed at 4°C for 2–12 h. The clots were spun down at 10,000 × g and the sera were collected and stored frozen at -20°C.

Wells of a 96-well plate (Microtest III, Becton-Dickinson) were coated with β-gal protein (2 μg/ml PBS, 50 μl/well, overnight), blocked (50 μl 1% BSA/PBS, 2 h), and rinsed with wash buffer (0.05% Tween 20, 138 mM NaCl, 2.7 mM KCl, 50 mM Tris, pH 8.0). Sera were diluted in dilution buffer (1% BSA/PBS) either 1:200 initially and 1:3 serially or 1:1,000 initially and 1:5 serially then added (50 μl). After incubation (2 h), the wells were washed and secondary antibodies were added (50 μl, 1:2,000 diluted alkaline phosphatase conjugated goat anti-mouse IgG1 or IgG2a, ICN Biochemicals). The specificity of these secondary antibodies was confirmed in a sandwich ELISA on plates coated with a capture antibody (goat anti-mouse Fc, Jackson Immunoresearch) and using the mAbs WT31 (IgG1) and M9144 (IgG2a) as positive and negative controls. After incubation (1 h), wells were washed and substrate was added (50 μl p-nitrophenyl phosphate, Sigma). After color developed (<1 h), the absorption at 405 nm wavelength was measured on a plate reader (Bio-Rad). Antiserum titers were determined from a curve fit to the data.

Mice were immunized by subcutaneous injection in the base of the tail with 25 μg of β-gal emulsified in CFA in a total volume of 50 μl. The draining inguinal lymph nodes were harvested on day 7. Lymph node cells were cultured either alone or with irradiated filler cells (0.2 ml, 96-well plate (Microtest, Falcon) in complete medium (RPMI 1640, 10% FBS, 100 Units/ml penicillin, 100 μg/ml streptomycin, 2.5 mM L-glutamine, 1 mM Sodium pyruvate, 100 μM non-essential amino acids, 50 μM 2-mercaptoethanol, 5 mM HEPES (GIBCO, Grand Island, NY)) with antigen additions at 10 μg/ml or as noted in the Results. For flow cytometric analysis, cells from triplicate wells were harvested, pooled, and incubated with biotin-conjugated rat anti-mouse CD25 (clone PC 615.3, Caltag, Burlingame, CA), washed and then incubated with fluorescein-conjugated streptavidin (Amersham), Quantum Red-conjugated rat anti-mouse CD4 (clone H129.19, Sigma) and R-Phycoerythrin-conjugated rat-anti-mouse CD8a (clone 53-6.7, Sigma or Pharmingen) then analyzed by flow cytometry (FACS Scan, Becton-Dickinson, CA). For proliferation studies, triplicate wells were pulsed with ³H-thymidine 16 hours prior to harvesting. For measurement of secreted IFN-γ, supernatants from triplicate cultures were harvested, centrifuged to remove residual cells, and frozen until sandwich immunoassay (R & D Systems, Minneapolis, MN). Replicates supernatants were assayed individually and the results were averaged. For all T cell cultures, purified protein derivative (PPD, Mycobacterium tuberculosis H37Rv, Mycos Research LLC, Fort Collins, CO) was used as a positive control and ovalbumin (OVA) was used as a negative control. Responses to OVA were not significantly different from the responses of unstimulated cultures (data not shown).

A portion of skin (~4 cm²) and the heart were harvested from freshly sacrificed mice. Skin sections and the heart base were placed into neutral buffered formalin (10%, Sigma) for 5 min. then washed 4 times in PBS. Staining solution was added (0.25 ml, 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 1 mM MgCl₂, 10 mM K₃Fe(CN)₆, 10 mM K₄Fe(CN)₆ in PBS) and the reaction proceeded at room temperature overnight. Digital photographs were taken (DC290, Kodak, Rochester NY) through a microscope (SMZ1000, Nikon).

Blood vessel morphometry was performed on animals that were perfusion fixed immediately after sacrifice. Vessels were dissected, embedded in OCT, and flash frozen. Sections were cut, stained with hematoxalin and eosin or Elastic van Giesen, and scored under a microscope (Optiphot2, Nikon).

Recombinant vaccinia virus containing the lacZ gene (β-gal-VV) was generously provided for these studies by Dr. Nicholas Restifo (NCI, Bethesda, MD). Virus was grown and titered using HeLa cells. Virus stock was prepared by scrape harvesting in PBS, freeze thawing three times, and passing the suspension through a 70 μm sieve. Mice were infected with 10⁷ PFU (0.25–0.35 ml, i.p.).

Donor and recipient mice were anaesthetised (0.2 ml xylazene/ketamine per 20 g body weight i.p.). Ophthalmic balm was applied to protect their eyes. Effective anaesthesia was tested before proceeding by pinching. More anaesthetic (~0.05 ml) was administered if the mouse flinched. Fur was clipped off the upper back and a full thickness, ~1 cm square patch of skin was cut from the upper backs. The host skin patch was removed and replaced with the donor skin patch, which was held in place by surgical staples. Mice were kept warm and under observation until they recovered, then single housed until they were healed (~2 w).

Heterotopic vascularized cardiac transplantation was performed as previously described [68]. The donor heart was arrested by infusion of 0.5 ml of University of Wisconsin (UW) solution (DuPont, Wilmington, Delaware, USA) into the inferior vena cava and the aortic root was flushed with 0.1 ml of UW solution to expel intracoronary blood thoroughly. The heart was removed from the chest and placed into UW solution on ice. After the recipient abdominal aorta and inferior vena cava were isolated, the donor aorta and pulmonary artery were joined end-to-side to the recipient aorta and vena cava, respectively, using 10-0 nylon suture. After the completion of the anastomoses, the abdomen was closed with a single running suture to all layers. The recipient mouse was then warmed for a few hours during recovery from anesthesia and had free access to water and food. The function of the transplanted hearts was assessed daily by abdominal palpation