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The Journal of Immunology, 2002, 169: 6154-6161.
Copyright © 2002 by The American Association of Immunologists

Mouse Vascular Endothelium Activates CD8+ T Lymphocytes in a B7-Dependent Fashion1

Daniel Kreisel*, Alexander S. Krupnick*, Keki R. Balsara*, Markus Riha*, Andrew E. Gelman*, Sicco H. Popma*, Wilson Y. Szeto*, Laurence A. Turka{dagger} and Bruce R. Rosengard2,*

Departments of * Surgery and {dagger} Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-{gamma}-activated C57BL/6 (H-2b) vascular endothelial cells as stimulators and CD8+ T lymphocytes isolated from CBA/J (H-2k) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-{gamma}. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8+ T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44low naive CD8+ T lymphocytes. Activated CD8+ T lymphocytes had the ability to produce IFN-{gamma} and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-xL. Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-xL. Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8+ T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8+ direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial cells may play an active role in mediating immune processes that contribute to acute and chronic rejection of vascularized allografts (1, 2). In fact, our laboratory has recently demonstrated that direct activation of CD8+ T lymphocytes by allograft nonhemopoietic cells such as vascular endothelium alone can lead to allograft rejection. Furthermore, we have shown that the presence of donor-derived hemopoietic APC does not influence the tempo of CD8+ direct allorecognition-mediated acute rejection (3). Despite numerous in vitro studies evaluating the interaction between human T lymphocytes and vascular endothelium, little mechanistic data regarding the costimulatory requirements are available in a murine system (4, 5, 6). Although human and murine endothelial cells have different characteristics, a better understanding of interactions between endothelial cells and T lymphocytes in the mouse is necessary for a proper interpretation of biological processes in this commonly used transplantation model.

To date, there have been few studies examining the Ag-presenting capacity of mouse vascular endothelium in vitro (7, 8, 9). This can be explained by the relative difficulty in isolating and culturing vascular endothelium from these animals (10). In addition, the validity of a mouse model has been challenged due to interspecies differences in the phenotypes of vascular endothelium both at a resting state and after treatment with proinflammatory cytokines. The presence of MHC class II molecules on the surface of HUVEC was thought to be unique to humans, and has been used as a particular argument to question the relevance of studies in small animal models (11). Moreover, the few studies that have examined interactions between mouse vascular endothelium and T lymphocytes have yielded conflicting results. It has been previously reported that mouse vascular endothelium is capable of direct activation of CD8+ T lymphocytes with greater ease than CD4+ T lymphocytes (7). However, another study found that IFN-{gamma}-activated endothelium failed to induce activation and proliferation of CD8+ T cells in a model of indirect presentation (9). Aside from using different modes of Ag presentation, the conflicting results of these studies might be explained by differing sources of vascular endothelium used in these experiments. For example, endothelium from the lung, which is in constant contact with environmental Ags, may not necessarily model vascular endothelium of organ allografts such as heart and kidney (12, 13).

The purpose of this study was to characterize the mechanism underlying direct activation of CD8+ T lymphocytes by mouse vascular endothelium. We have recently described a method to culture mouse vascular endothelium from the thoracic aorta (14). This endothelium shares numerous characteristics with human endothelium, including up-regulation of MHC class I molecules and induction of MHC class II molecules under proinflammatory conditions. Interestingly, cultured mouse vascular endothelium expresses CD80, but lacks both CD40 and CD86. Because CD80 is known to be critical to activation of T cells by engaging CD28, we hypothesized that vascular endothelium activates alloreactive CD8+ T lymphocytes in a B7-dependent fashion. The results of this study demonstrate that CTLA4-Ig inhibits proliferation of CD8+ T cells in response to both resting and activated vascular endothelial cells. These observations have been extended to an in vivo model in which we demonstrate that proliferation of CD8+ T lymphocytes in response to direct presentation by allogeneic nonhemopoietic cells, such as vascular endothelium, can be similarly inhibited by CTLA4-Ig. These findings could have important implications for the design of immunosuppressive strategies for preventing acute and chronic allograft rejection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male C57BL/6 (H-2Kb) (designated as B6) and {beta}2-microglobulin-deficient mice on a C57BL/6 background (designated as B6I-) were purchased from Taconic (Germantown, NY), and male CBA/J (H-2Kk) (designated as CBA) 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.

Vascular endothelium cell culture

Small pieces of thoracic aorta were isolated from B6 or CBA mice and plated endothelial side down onto 24-well plates coated with a three-dimensional type I rat tail collagen gel (14). After a 48-h period, the cultures were supplemented with RPMI medium containing 20% heat-inactivated FCS, 2-ME (1 µl/ml), gentamicin (1 µl/5 ml) (Life Technologies, Gaithersburg, MD), and endothelial cell growth supplement (50 µg/ml) (BD Biosciences, Bedford, MA). Endothelial cells were initially passaged onto tissue culture-treated plastic flasks using 0.3% collagenase H solution (Sigma-Aldrich, St. Louis, MO). Further passaging was conducted with 5 mM EDTA (Life Technologies). No trypsin was used during passaging to preserve surface protein expression. For functional assays, early passage endothelial cells were used either at a resting state or after activation with 500 U/ml murine rIFN-{gamma} (R&D Systems, Minneapolis, MN) for a period of 72 h.

Purification of CD8+ T lymphocytes and cell labeling

Responder cells for coculture assays consisted of CD8+ T lymphocytes isolated from unprimed CBA mice sacrificed by cervical dislocation. Spleens and pooled lymph node 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-CD8 mAbs and positively selected by passage though a magnetic column (midiMACS system; Miltenyi Biotec, San Diego, CA). Purity of CD8+ T lymphocytes was greater than 96%, as determined by flow cytometry. CD8+ T lymphocytes were labeled with the fluorescent dye CFSE (Molecular Probes, Eugene, OR) before their coculture with endothelial cells by incubation with CFSE in PBS at a final concentration of 2 µM for 3 min. To evaluate the response of naive CD8+ T lymphocytes, cells were sorted based on their surface expression of CD44. Briefly, CBA cells were labeled with CD44 conjugated to PE and CD8 conjugated to allophycocyanin, and sorted on FACSVantage sorter (BD Biosciences), with a purity exceeding 99%. CD44lowCD8+ T lymphocytes were then labeled with the fluorescent dye CFSE before their coculture with endothelial cells.

In vitro proliferation assays

Endothelial cells (2.5 x 104/well) were plated in 48-well plates. At this initial seeding density, they reached confluence 5 days after plating. For certain conditions, endothelial cells were activated by treatment with murine rIFN-{gamma} (500 U/ml) 72 h before their use as stimulators and were irradiated with 25 Gy from a 137Cs source immediately before the addition of T lymphocytes. After several washes to remove all traces of IFN-{gamma} from the endothelium, 5 x 105 CD8+ T lymphocytes, resuspended in 1 ml of RPMI medium containing 2-ME (1 µl/ml), gentamicin (50 pg/5 ml), and 10% FCS, were added to each well. Some cultures received CTLA4-Ig (15 µg/ml) (Bristol-Myers Squibb Pharmaceuticals, Princeton, NJ) or control hamster Ig (15 µg/ml) (The Jackson Laboratory). CFSE-labeled CBA CD8+ T lymphocytes were also cocultured with B6 splenocytes at a stimulator:responder ratio of 1:3 in 96-well round-bottom plates. B6 splenocytes were irradiated in a manner identical with the vascular endothelial cells. Proliferative responses were analyzed after 3, 5, and 7 days of coculture. CD8-specific Abs identified CBA CD8+ T lymphocytes. When splenocytes were used as stimulators, Abs specific for H-2Kk were also used.

Flow cytometry

All Abs and reagents for flow cytometric analysis of surface markers on endothelial cells and T lymphocytes were conjugated with PE or allophycocyanin and were purchased from BD PharMingen (San Diego, CA). Abs used for surface staining included anti-H-2Kb (clone AF6-88.5), anti-H-2Kk (clone 36-7-5), anti-CD40 (clone 3/23), anti-CD54 (ICAM-1) (clone 3E2), anti-CD80 (B7.1) (clone 16-10A1), anti-CD86 (B7.2) (clone GL1), anti-CD8a (clone 53-6.7), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD44 (clone IM7), and anti-CD95 ligand (Fas ligand) (clone MFL3). To determine the level of contamination with hemopoietic elements, the cultures were stained with the leukocyte common marker CD45 (leukocyte common Ag) (clone 30-F11). Appropriately labeled isotype control Abs of rat, hamster, and mouse origin were used. All samples were acquired on a FACSCalibur (BD Biosciences) and analyzed using the CellQuest software package (BD Biosciences).

Intracellular staining for Bcl-xL and cytokines

Expression of the antiapoptotic protein Bcl-xL and the ability to produce cytokines were assessed at the single cell level. PE-conjugated anti-Bcl-xL Ab (clone 7B2.5) and the appropriate isotype control were purchased from Southern Biotechnology Associates (Birmingham, AL). PE-conjugated Abs against IL-2 (clone JES6-5H4), IL-4 (clone 11B11), IFN-{gamma} (clone XMG1.2), and the appropriate isotype control were purchased from BD PharMingen. After the indicated time points of coculture of CD8+ T lymphocytes with endothelial cells, T cells were surface stained for 30 min in a small volume of cold PBS, followed by a 60-min period of fixation in 2.5% paraformaldehyde (Sigma-Aldrich) on ice. Cells were then permeabilized with 0.1% saponin (Sigma-Aldrich) in PBS containing 2% FCS for 10 min on ice. Intracellular staining was performed for 30 min, with the cells resuspended in a small volume of 0.1% saponin. After two washes in 0.01% saponin, the cells were resuspended in PBS containing 2% FCS and analyzed by flow cytometry. The staining protocol for intracellular cytokines was similar. However, before surface staining, cells were cultured with 20 ng/ml PMA (Sigma-Aldrich) and 1 µM ionomycin (Calbiochem, La Jolla, CA) for 4 h, and 2 µM monensin (Sigma-Aldrich) was added for the last 3 h of culture.

Intracellular perforin staining

To detect intracellular perforin, cells isolated from individual wells of endothelial cell/T lymphocyte cocultures were fixed with 2% paraformaldehyde for 20 min on ice. The cells were then washed and permeabilized with 0.1% saponin containing 2% FCS for 10 min on ice. Cells were subsequently incubated with goat serum for 10 min and then stained with anti-perforin Ab (clone P1-8) (Kamiya Biomedical, San Diego, CA) in 0.3% saponin for 30 min on ice. Following one wash in 0.1% saponin, cells were incubated with PE-labeled polyclonal goat anti-rat Ab (Southern Biotechnology Associates) in 0.3% saponin for 30 min on ice. After two washes in 0.01% saponin, cells were resuspended in PBS containing 2% FCS and stained with APC-labeled anti-CD8a Ab for 30 min on ice. Subsequently, the cells were washed twice in cold PBS containing 2% FCS.

Creating chimeric organs by bone marrow transplantation

Bone marrow chimeras were created as previously described (15). Briefly, for bone marrow transplantation, male wild-type B6 were reconstituted with marrow from B6I- mice. Bone marrow was harvested from the femora of donor mice, and T lymphocytes were depleted with anti-CD90 (Thy)-labeled magnetic microbeads (Miltenyi Biotec). Recipient mice received an inoculum of 1 x 107 T cell-depleted donor bone marrow via lateral tail vein injection 6 h after lethal irradiation. These bone marrow chimeras were used for in vivo MLRs at least 90 days after the bone marrow transplant. These bone marrow chimeras lacked expression of H-2Kb on their hemopoietic APC such as dendritic cells and macrophages (15). The following designation is used to describe chimeric organs: nonhemopoietic cells (hemopoietic APC).

In vivo proliferation assays

Responses of CBA CD8+ T lymphocytes to direct allopresentation by nonhemopoietic cells were tested in vivo. Wild-type B6 or B6(B6I-) mice were treated with supralethal irradiation (18 Gy) from a cesium source and then received an adoptive transfer of 25 x 106 CFSE-labeled CD8+ CBA T lymphocytes via tail vein injection within 4 h of irradiation. For certain experiments, B6(B6I-) bone marrow chimeras were treated with 100,000 U IFN-{gamma} via i.p. injection 72 h before irradiation. Certain mice received CTLA4-Ig (200 µg) (Bristol-Myers Squibb Pharmaceuticals) or control hamster Ig (200 µg) (The Jackson Laboratory) at the time of the adoptive transfer of the CFSE-labeled CD8+ T lymphocytes. Animals were sacrificed 72 h later. To assess the proliferative responses of the adoptively transferred CBA CD8+ T cells, their spleens and peripheral lymph nodes were analyzed by flow cytometry. These cells were identified by staining with Abs specific for H-2Kk as well as CD8.

RT-PCR analysis of aortic tissue

Spleens were removed from wild-type B6 mice, and thoracic aortas were removed from wild-type B6 mice and IFN-{gamma}-treated wild-type B6 mice. RNA was then isolated from these tissues. A total of 2 µg RNA was reverse transcribed at 55°C for 45 min for MHC class I and 58°C for 60 min for CD45 and amplified with EZ RTth Taq kit (PerkinElmer/Cetus, Norwalk, CT) and with 300 µg primers: MHC I sense (5'-ACATGGAGCTTGTGGAGACC-3'), MHC I antisense (5'-CAAGGACAACCAGAACAGCA-3'), CD45 sense (5'-GCACCAGCTGATCTCCAGATA-3'), CD45 antisense (5'-CAAACACCTACACCCAGT-3'), {beta}-actin sense (5'-ATCACCATTGGCAATGAGCGGTTCC-3'), and {beta}-actin antisense (5'-CTCGTCATACTCCTGCTTGCTGAT-3'). Thermocycling was performed on a Hybaid 900 (Hybaid, Middlesex, U.K.) using the following parameters: for MHC class I, 94.5°C for 120 s, followed by 36 cycles of 94.5°C for 30 s and 55°C for 150 s. For CD45, 95°C for 120 s, followed by 36 cycles of 95°C for 30 s and 58°C for 90 s.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Vascular endothelium: phenotypic changes after activation

Mouse vascular endothelium isolated from thoracic aorta expresses low levels of MHC class I (H-2Kb) and moderate levels of CD80 (B7-1) on its surface at a resting state. Although it also expresses CD54 (ICAM-1), there is no detectable CD40 or CD86 (B7-2). MHC class I levels are markedly up-regulated after a 72-h period of treatment with IFN-{gamma} (500 U/ml), while expression levels of CD80 do not change (14). We have also observed a mild increase in CD54 expression, while treatment with IFN-{gamma} did not induce expression of CD40 or CD86. Lack of contamination with hemopoietic cells was documented by flow cytometry as well as RT-PCR, which demonstrated no expression of CD45 (3).

Proliferation of CD8+ T cells after coculture with vascular endothelium

The phenotype of mouse vascular endothelium indicates that it has the means to activate unprimed alloreactive CD8+ T lymphocytes both at a resting state and after activation. Treatment with IFN-{gamma} enhances signal 1 (MHC class I) to a greater extent than signal 2 (CD80). We have recently demonstrated the ability of resting B6 vascular endothelium to induce proliferation of CD8+ T cells isolated from a TCR transgenic mouse, in which the transgene reacts with the allogeneic MHC class I H-2Kb (3). To test whether resting or activated endothelial cells can induce proliferation of wild-type CD8+ T cells, we labeled the responder CD8+ T cell population with the fluorescent dye CFSE before initiation of coculture. Due to equal segregation of this dye between daughter cells upon cell division, halving of cellular fluorescence indicates one round of proliferation (16, 17). Similar to splenocytes, both resting and activated vascular endothelium induce proliferation of allogeneic CD8+ T lymphocytes (Fig. 1GoA). Interestingly, the CFSE division profiles indicate differences in the kinetics of clonal expansion between the stimulator conditions. Alloreactive CD8+ T lymphocytes start proliferating after longer time periods of coculture with resting endothelium when compared with both activated endothelium and splenocytes. However, while CD8+ T lymphocytes start proliferating after shorter time periods in response to activated vascular endothelium and a higher proportion of CD8+ T lymphocytes are recruited into the proliferating pool at earlier time points, both resting and activated vascular endothelium recruit a similar proportion of CD8+ T lymphocytes into the proliferating pool after 7 days of coculture. No significant proliferation was observed when CBA CD8+ T lymphocytes were cocultured with syngeneic endothelium (Fig. 1GoB). T cells that have undergone at least one round of cell division after coculture with resting or activated endothelium show the hallmarks of activation, such as CD69 and CD25 expression (Fig. 1GoC). Importantly, CFSE-labeled CBA CD8+ T cells cultured alone in vitro do not show any evidence of activation, such as surface expression of CD25 or CD69. Of note, activated vascular endothelium was also able to induce proliferation of naive CD44low CD8+ T lymphocytes, while only minimal proliferation of this cell population was observed after 7 days of coculture with resting endothelium (Fig. 1GoD). Taken together, our results indicate that vascular endothelium has the capacity to activate and induce proliferation of alloreactive wild-type CD8+ T lymphocytes in vitro.



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  		FIGURE 1. A, Kinetics of CD8+ T cell division (CBA/J) in response to allogeneic (C57BL/6) resting and activated vascular endothelium after 3, 5, and 7 days of coculture. Histograms depict CFSE fluorescence of CD8+ T cells. Undivided parent generation CD8+ T cells are represented by the peak of highest CFSE fluorescence in each histogram, and daughter generations are represented by leftward peaks of diminishing CFSE fluorescence. Numbers indicate the percentage of CD8+ T cells that had divided at least once. B, CD8+ T cell (CBA/J) proliferation in response to syngeneic (CBA/J) resting and activated vascular endothelium as well as allogeneic (C57BL/6) splenocytes after 7 days of coculture. Histograms depict CFSE fluorescence of CD8+ T cells. Undivided parent generation CD8+ T cells are represented by the peak of highest CFSE fluorescence in each histogram, and daughter generations are represented by leftward peaks of diminishing CFSE fluorescence. Numbers indicate the percentage of CD8+ T cells that had divided at least once. C, CBA/J CD8+ T cells that have proliferated after 5 days of coculture with resting vascular endothelium (C57BL/6) express CD25 and CD69. Only a small number of T cells that had not progressed through the cell cycle express these activation markers. Staining with isotype-matched Abs is in the right and left lower quadrants for undivided and divided T lymphocytes, respectively. Dot plots are gated on live CD8+ T cells, identified by their forward and side scatter characteristics. D, Proliferation of CD44low CD8+ T cells (CBA/J) in response to allogeneic (C57BL/6) resting and activated vascular endothelium after 7 days of coculture. Histograms depict CFSE fluorescence of CD8+ T cells. Undivided parent generation CD8+ T cells are represented by the peak of highest CFSE fluorescence in each histogram, and daughter generations are represented by leftward peaks of diminishing CFSE fluorescence. Numbers indicate the percentage of CD8+ T cells that had divided at least once.

 
Cytokine profile and effector phenotype

Similar to CD4+ T cells, CD8+ T cells have the capacity to express several cytokine programs and differentiate into either Tc1 or Tc2 cells (18, 19). The type of APC has been discussed as a contributory factor in determining this differentiation of the responding T cells (20). In addition to cytokine secretion by APC, their relative expression of MHC molecules and costimulatory molecules have been discussed as contributing factors (21, 22). Consequently, we were interested in examining the ability of CD8+ T cells, activated through vascular endothelium by direct allorecognition, to produce cytokines. Cells were stimulated in vitro with PMA and ionomycin for 4 h before cytokine staining. This assay determines whether CD8+ T cells that have been activated by coculture with vascular endothelium have been primed to produce cytokines. A large percentage of cells that had undergone division were able to produce IFN-{gamma} after coculture with both resting and activated vascular endothelium (Fig. 2GoA). Additionally, we were able to detect the ability to produce IL-2 among cells that had divided (Fig. 2GoB). However, this percentage was smaller than those producing IFN-{gamma}, which is consistent with previous studies (23). We were unable to detect production of cytokines known to be associated with Tc2 differentiation, such as IL-4 (Fig. 2GoC).



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  		FIGURE 2. CFSE-labeled CBA/J CD8+ T lymphocytes were cultured for 4 h with 20 ng/ml PMA and 1 µM ionomycin, with 2 µM monensin present for the last 3 h after 5 days of coculture with resting vascular endothelium of C57BL/6 origin. Staining of T cells that had undergone division was positive for IFN-{gamma} (A) and IL-2 (B), while IL-4 (C) could not be detected. Staining with isotype-matched Abs is in the right and left lower quadrants for undivided and divided T lymphocytes, respectively. Dot plots are gated on live CD8+ T cells, identified by their forward and side scatter characteristics.

 
CD8+ T lymphocytes that produce cytokines associated with a Tc1 phenotype are known to have the potential to further differentiate into effector cells. To this end, we determined their expression of perforin and Fas ligand, both of which are known to contribute to the cytotoxic ability of CD8+ T lymphocytes (24, 25). We found that the majority of CD8+ T lymphocytes that had undergone cell division after coculture with resting or activated vascular endothelium expressed perforin (Fig. 3GoA). Similarly, Fas ligand was up-regulated on CD8+ T cells that had progressed through the cell cycle (Fig. 3GoB). Thus, we have established that CD8+ T cells acquire a cytotoxic armamentarium after activation through vascular endothelium.



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  		FIGURE 3. CFSE-labeled CBA/J CD8+ T lymphocytes that had undergone cell division have acquired intracellular perforin stores (A) and express Fas ligand (B) on their surface, here shown after 6 days of coculture with resting vascular endothelium of C57BL/6 origin. Staining with isotype-matched Abs is in the right and left lower quadrants for undivided and divided T lymphocytes, respectively. Dot plots are gated on live CD8+ T cells, identified by their forward and side scatter characteristics.

 
Costimulatory blockade

Given the presence of CD80 on cultured vascular endothelium, we postulated that its expression is important in the ability of endothelial cells to activate CD8+ direct allorecognition. We found that when CD28 costimulation is blocked in the presence of CTLA4-Ig, the proliferative response of alloreactive CD8+ T lymphocytes to resting and activated vascular endothelium is markedly blunted (Fig. 4GoA). The minimal proliferative response after 7 days of coculture in the presence of CTLA4-Ig is comparable with that after coculture with syngeneic endothelium, indicating that direct activation of alloreactive CD8+ by vascular endothelium is B7 dependent. Consistent with the lack of CD40 expression on resting as well as activated cultured vascular endothelium, addition of the CD40-blocking agent MR-1 did not inhibit proliferative responses of CD8+ T lymphocytes to allogeneic endothelial cells. Moreover, disrupting costimulation through ICAM-1 did not lead to diminished proliferative responses (data not shown).



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  		FIGURE 4. A, Proliferative responses of CBA/J CD8+ T lymphocytes cultured with resting allogeneic vascular endothelium (C57BL/6) in the presence of control Ig or CTLA4-Ig were examined at 3, 5, and 7 days. The proliferative response is markedly diminished in the absence of CD28 costimulation, indicating that this is a B7-dependent process. Numbers indicate the percentage of CD8+ T cells that had divided at least once. B, CBA/J CD8+ T lymphocytes that had undergone cell division up-regulate the antiapoptotic protein Bcl-xL. The number of T cells proliferating and expressing Bcl-xL is decreased in the presence of CTLA4-Ig (15 µg/ml), here shown after 4 days of coculture with resting vascular endothelium of C57BL/6 origin. Staining with isotype-matched Abs is in the right and left lower quadrants for undivided and divided T lymphocytes, respectively.

 
Expression of antiapoptotic protein Bcl-xL

Expression of the antiapoptotic protein Bcl-xL in activated T lymphocytes alters their apoptotic threshold and protects them from apoptotic stimuli, such as that of growth factor withdrawal or treatment with glucocorticoids (26). Up-regulation of Bcl-xL has been described in T cells following activation through B7-dependent mechanisms (27). Having shown that the interaction between CD80 on the surface of vascular endothelium and CD28 on the T cells is critical for the division and activation of alloreactive T cells, we set out to examine whether vascular endothelium has the capacity to up-regulate Bcl-xL in CD8+ T lymphocytes. We found that the vast majority of T cells that have progressed through the cell cycle at least once expressed high levels of Bcl-xL after coculture with both resting and activated endothelium. Treatment of the cultures with CTLA4-Ig reduced the absolute number of T lymphocytes that entered the cell cycle and consequently reduced the number of T lymphocytes expressing Bcl-xL. Of note, the few T cells that divide in cultures treated with CTLA4-Ig express Bcl-xL, raising the possibility that costimulation through CD28 may not be the sole mechanism inducing the expression of this antiapoptotic protein (28, 29). Nevertheless, the observation that cell division and the number of T lymphocytes expressing Bcl-xL are both reduced by CTLA4-Ig indicates that mouse vascular endothelium has the capacity not only to induce proliferation and differentiation, but also potentially regulate survival in a B7-dependent fashion (Fig. 4GoB).

Proliferation of CD8+ T cells after in vivo activation by nonhemopoietic cells

To assess whether nonhemopoietic cells such as vascular endothelium had the ability to induce proliferation of alloreactive CD8+ T lymphocytes in vivo, we used an assay in which CFSE-labeled CBA CD8+ T lymphocytes are adoptively transferred into whole body irradiated B6 mice. Vigorous proliferation was seen when CBA CD8+ T lymphocytes were adoptively transferred into wild-type B6 hosts (Fig. 5GoA). In this experimental group, both hemopoietic and nonhemopoietic cells of B6 origin can contribute to the activation of the CBA CD8+ T lymphocytes. To determine whether nonhemopoietic B6 cells can induce proliferation of alloreactive CD8+ T lymphocytes in vivo, we created B6 bone marrow chimeras in which the hemopoietic APC are derived from {beta}2-microglobulin-deficient mice and therefore lack surface expression of MHC class I (B6I-). Our laboratory has previously shown that the level of hemopoietic APC replacement in all tissues of these bone marrow chimeras amounts to over 99.99%, as determined by semiquantitative RT-PCR, with no contribution to allorecognition as assessed by MLR (15). Hemopoietic cells of such B6(B6I-) bone marrow chimeras are, therefore, unable to stimulate alloreactive CD8+ T lymphocytes. Only minimal proliferation was seen after adoptive transfer of CBA CD8+ T lymphocytes into B6(B6I) chimeras (Fig. 5GoB). Because this assay is limited by the death of the majority of irradiated hosts by ~96 h after irradiation and we have observed differences in kinetics between stimulation by resting and cytokine-activated vascular endothelium in vitro, we treated B6(B6I-) bone marrow chimeras with systemic IFN-{gamma} 72 h before adoptive transfer of CBA CD8+ T lymphocytes. Consistent with previous reports, this regimen leads to up-regulated expression of MHC class I on vascular endothelial cells, as confirmed by RT-PCR (30) (Fig. 6GoA). Furthermore, these cells were negative for CD45 by RT-PCR analysis, indicating that the up-regulated MHC class I expression was due to increased transcription in vascular endothelium and not to the presence of professional APCs (Fig. 6GoB). Approximately one-third of CBA CD8+ T lymphocytes present 84 h after adoptive transfer had undergone at least one round of cell division, indicating that nonhemopoietic cells are able to induce the proliferation of alloreactive CD8+ T lymphocytes (Fig. 5GoC). Having shown that the proliferative response of CD8+ T lymphocytes to vascular endothelium is dependent on B7 in vitro, we next questioned whether the proliferative response to cytokine-activated nonhemopoietic cells in vivo could also be inhibited by CTLA4-Ig. Systemic administration of CTLA4-Ig inhibited the proliferation of CBA CD8+ T lymphocytes, which had been adoptively transferred into IFN-{gamma}-treated B6(B6I-) bone marrow chimeras. This suggests that nonhemopoietic cells can activate alloreactive CD8+ T lymphocytes in a B7-dependent fashion in vivo (Fig. 5GoD).



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  		FIGURE 5. In vivo proliferation of CFSE-labeled CBA/J CD8+ 84 h after transfer into supralethally irradiated C57BL/6 wild-type mice (A), B6(B6I-) bone marrow chimeras (B), B6(B6I-) bone marrow chimeras that had received 100,000 U IFN-{gamma} i.p. 72 h before the adoptive transfer treated with either control-Ig (C) or CTLA4-Ig (D) (200 µg i.p. on days 0 and 2). Plots represent 20,000 live CD8+ T cells.

 


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  		FIGURE 6. MHC class I expression on aortic tissue. A, RT-PCR analysis demonstrates up-regulation of MHC class I transcription in animals treated with IFN-{gamma}. MHC class I appears above, and {beta}-actin appears below. B, RT-PCR analysis demonstrates the absence of CD45 in vascular endothelium obtained from both untreated and IFN-{gamma}-treated mice. C57BL/6 splenocytes served as positive control. CD45 appears above, and {beta}-actin appears below.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although hemopoietic professional APC of donor origin are critical stimuli for direct allopresentation in the early postengraftment period, their number decreases substantially over time (31). Vascular endothelium, however, remains present at the graft-host interface for the duration of the graft and could, therefore, provide a lasting stimulus for direct allorecognition. We have recently demonstrated that nonhemopoietic cells, such as vascular endothelium, can serve as potent stimulators for CD8+ direct allorecognition (3). A better understanding of endothelial-driven direct Ag presentation could lead to novel therapeutic strategies for the prevention and treatment of both acute and chronic rejection. Furthermore, studies analyzing the underlying mechanism of activation of lymphocyte subsets by mouse vascular endothelium may prove significant in correctly interpreting observations made in commonly used murine transplantation models that examine allorecognition pathways.

Similar to observations previously made for endothelial cells derived from human umbilical veins, our results show that both resting and activated vascular endothelial cells are capable of activating and inducing proliferation of alloreactive CD8+ T lymphocytes (4). Moreover, CD8+ T lymphocytes start proliferating sooner, and the response appears more vigorous at earlier time points when they are cocultured with activated endothelium within our system. This observation is in apparent contradiction to a recent study that reported lack of CD8+ lymphocyte proliferation stimulated by activated microvascular endothelial cells isolated from murine lung tissue (9). Similar to the phenotype of the endothelial cells used in the present study, the authors report that MHC class I molecules are up-regulated after treatment with IFN-{gamma}, while CD80 expression is unchanged after activation. Marelli-Berg et al. (9) attribute the reduced immunogenicity of activated endothelium to a failure to up-regulate CD80 after treatment with IFN-{gamma}, which leads to an imbalance between TCR engagement and CD28 engagement after activation. In contrast, our results suggest that higher levels of signal 1 in activated endothelium probably lead to an enhancement in TCR occupancy, with more T cells being recruited into the proliferating pool at earlier time points. However, it should be pointed out that the apparent discrepancies between these studies could be attributed to the different sources of endothelium used. Microvascular endothelium isolated from the pulmonary system is constantly exposed to environmental Ags. Because tolerance for inhaled Ags may be due to altered function of APC at these sites, conclusions drawn from endothelium isolated from lung tissue may not reflect the immunologic function of macrovascular endothelium from an internal, protected source, as described in this work. As we have recently shown that nonhemopoietic cells such as vascular endothelium can trigger acute rejection of cardiac allografts via CD8+ direct allorecognition, it would be interesting to explore whether vascular endothelial cells can trigger rejection of vascularized allografts exposed to environmental Ags, such as lungs (3). Another important difference between our experimental system and Lechler’s study (9) is that we have focused on CD8+ direct allorecognition, important to the field of solid organ transplantation, while the other group has focused on self MHC-restricted recognition of a nominal Ag. The ability of vascular endothelium to induce proliferation of naive CD8+ T lymphocytes has also been the subject of controversy. Although it was initially reported in a human system that resting endothelium is incapable of inducing proliferation of naive CD8+ T cells, a subsequent finding in a murine model suggests otherwise (4, 9). Similarly, we have recently reported that resting endothelium is capable of activating CD44low CD8+ T cells in a TCR transgenic system (3). In this study, while we did not observe proliferation of CD44low CD8+ wild-type CBA T cells after coculture with resting endothelium, activated endothelium did induce proliferation of CD44low CD8+ T cells. Taken together, these observations may be a reflection of different experimental systems.

Similar to human vascular endothelium, expression of MHC class I molecules is strongly up-regulated on our mouse endothelial cells after treatment with proinflammatory cytokines (32). An important distinction between HUVEC and our endothelial cells is the inability to induce expression with CD40 after treatment with IFN-{gamma} (33). Furthermore, it is well documented that interspecies differences exist with regard to the endothelial expression of B7. Cultured human endothelial cells do not express B7, and endothelium-driven activation of alloreactive T lymphocytes is contingent upon the costimulatory molecules LFA-3 and CD2 (34, 35). However, it is important to note that CD86 and CD80 are expressed on porcine and mouse vascular endothelium, respectively (14, 35). Despite these differences in surface expression of costimulatory molecules, our findings in the mouse system extend observations made in large animal and human models that vascular endothelium has the ability to directly activate alloreactive T cells.

Several studies in murine models have documented the ability to suppress alloimmune responses with blockade of costimulatory signals, particularly the B7-CD28 and CD40-CD40 ligand interactions (36, 37). Marked suppression of the proliferative response of CD8+ T cells to cultured vascular endothelium in vitro and to nonhemopoietic cells in vivo after treatment with CTLA4-Ig demonstrates that this direct activation of CD8+ T lymphocytes occurs in a B7-dependent fashion. In addition to its effect on IL-2 transcription, B7 molecules have been shown to up-regulate the expression of the antiapoptotic protein Bcl-xL (27). Increasing the number of T lymphocytes expressing Bcl-xL as a consequence of interaction with endothelial cells raises the interesting possibility that the survival of alloreactive T lymphocytes may be regulated at the level of the allograft. Our results suggest that the administration of CTLA4-Ig in a mouse allotransplantation model could have the additional benefit of blocking the survival signal that alloreactive T lymphocytes receive at the level of the allograft.

Differentiation of in vitro stimulated CD8+ T lymphocytes into effector cells involves the development of intracellular stores of proteins required for the granule exocytosis cytotoxic pathway as well as TCR-induced transcription and surface expression of Fas ligand (38, 39). We have demonstrated that proliferating CD8+ acquire intracellular perforin and express Fas ligand after coculture with mouse vascular endothelium. These findings parallel observations in human in vitro studies using HUVEC as stimulators (40). Interestingly, human CD8+ T cells that have been cocultured with endothelial cells acquire endothelial cell-specific cytotoxic activity, while failing to lyse B lymphoblastoid targets from the same donor (6). The authors speculate that this could be due to cell-specific interactions that occur during the differentiation of CD8+ T cells into effector cells. The question, whether mouse CD8+ T cells, which acquire cytotoxic characteristics after coculture with vascular endothelial cells, display target cell selectivity like their human counterparts, will be explored in future studies. Examining this issue will be particularly interesting in light of the differences in costimulatory interactions between mice and humans that account for the activation of alloreactive CD8+ T lymphocytes. The lack of B7 expression on human endothelial cells in particular may have important consequences for their differentiation into effector cells. A recent study found that either B7 or IL-2 is sufficient for the generation of CD8+ CTL in a mouse system (41). Unlike the case for the human studies, in which exogenous IL-2 is added to the cultures during CTL differentiation, no cytokines are supplemented within our murine cultures (6, 40, 42).

Many immunosuppressive strategies in murine allotransplantation models have focused on perioperative depletion of CD4+ T lymphocytes in the host or perioperative administration of agents that block costimulation (37, 43). The main goal of these approaches is to reduce the strength of CD4+ direct allorecognition triggered by donor-derived hemopoietic APC. Despite reports of prolongation of allograft survival, the success of these protocols often depends on the strain combination used, and many allografts that survive long-term show evidence of chronic rejection (37, 44). In this study, we have shown that direct activation of CD8+ T lymphocytes by mouse vascular endothelial cells is B7 dependent. Because allogeneic nonhemopoietic cells such as vascular endothelium remain in the graft indefinitely, long-term administration of costimulatory blockade, including CTLA4-Ig, might be necessary to blunt direct allorecognition and induce immunological tolerance.


   Footnotes
 
1 The work was supported in part by grants from the National Institutes of Health R01 (AI47257-01A1). Back

2 Address correspondence and reprint requests to Dr. Bruce R. Rosengard, Division of Cardiothoracic Surgery, 6 Silverstein, Department of Surgery, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, PA 19104. E-mail address: bruce.rosengard{at}uphs.upenn.edu Back

Received for publication May 2, 2002. Accepted for publication September 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Denton, M. D., C. S. Geehan, S. I. Alexander, M. H. Sayegh, D. M. Briscoe. 1999. Endothelial cells modify the costimulatory capacity of transmigrating leukocytes and promote CD28-mediated CD4+ T cell alloactivation. J. Exp. Med. 190:555.[Abstract/Free Full Text]
  2. Briscoe, D. M., S. I. Alexander, A. H. Lichtman. 1998. Interactions between T lymphocytes and endothelial cells in allograft rejection. Curr. Opin. Immunol. 10:525.[Medline]
  3. Kreisel, D., A. S. Krupnick, A. E. Gelman, F. H. Engels, S. H. Popma, A. M. Krasinskas, K. R. Balsara, W. Y. Szeto, L. A. Turka, B. R. Rosengard. 2002. Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition. Nat. Med. 8:233.[Medline]
  4. Epperson, D. E., J. S. Pober. 1994. Antigen-presenting function of human endothelial cells: direct activation of resting CD8 T cells. J. Immunol. 153:5402.[Abstract]
  5. Ma, W., J. S. Pober. 1998. Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells. J. Immunol. 161:2158.[Abstract/Free Full Text]
  6. Biedermann, B. C., J. S. Pober. 1998. Human endothelial cells induce and regulate cytolytic T cell differentiation. J. Immunol. 161:4679.[Abstract/Free Full Text]
  7. Lodge, P. A., C. E. Haisch. 1993. T cell subset responses to allogeneic endothelium: proliferation of CD8+ but not CD4+ lymphocytes. Transplantation 56:656.[Medline]
  8. Perez, V. L., L. Henault, A. H. Lichtman. 1998. Endothelial antigen presentation: stimulation of previously activated but not naive TCR-transgenic mouse T cells. Cell. Immunol. 189:31.[Medline]
  9. Marelli-Berg, F. M., D. Scott, I. Bartok, E. Peek, J. Dyson, R. I. Lechler. 2000. Activated murine endothelial cells have reduced immunogenicity for CD8+ T cells: a mechanism of immunoregulation. J. Immunol. 165:4182.[Abstract/Free Full Text]
  10. Moyer, C. F., E. Huggins, S. Sarantopoulos, J. C. Lewis, D. Sajuthi, C. A. Biron, C. L. Reinisch. 1992. Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells. Exp. Cell Res. 199:63.[Medline]
  11. Choo, J. K., J. D. Seebach, V. Nickeleit, A. Shimizu, H. Lei, D. H. Sachs, J. C. Madsen. 1997. Species differences in the expression of major histocompatibility complex class II antigens on coronary artery endothelium: implications for cell-mediated xenoreactivity. Transplantation 64:1315.[Medline]
  12. Tian, J., M. A. Atkinson, M. Clare-Salzler, A. Herschenfeld, T. Forsthuber, P. V. Lehmann, D. L. Kaufman. 1996. Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J. Exp. Med. 183:1561.[Abstract/Free Full Text]
  13. McMenamin, C., P. G. Holt. 1993. The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class I-restricted CD8+ T cell-mediated but MHC class II-restricted CD4+ T cell-dependent immune deviation resulting in selective suppression of immunoglobulin E production. J. Exp. Med. 178:889.[Abstract/Free Full Text]
  14. Kreisel, D., A. S. Krupnick, W. Y. Szeto, S. H. Popma, D. Sankaran, A. M. Krasinskas, K. M. Amin, B. R. Rosengard. 2001. A simple method for culturing mouse vascular endothelium. J. Immunol. Methods 254:31.[Medline]
  15. Krasinskas, A. M., S. D. Eiref, A. D. McLean, D. Kreisel, A. E. Gelman, S. H. Popma, J. S. Moore, B. R. Rosengard. 2000. Replacement of graft-resident donor-type antigen presenting cells alters the tempo and pathogenesis of murine cardiac allograft rejection. Transplantation 70:514.[Medline]
  16. Lyons, A. B., C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131.[Medline]
  17. Wells, A. D., H. Gudmundsdottir, L. A. Turka. 1997. Following the fate of individual T cells throughout activation and clonal expansion: signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100:3173.[Medline]
  18. Sad, S., R. Marcotte, T. R. Mosmann. 1995. Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 2:271.[Medline]
  19. Croft, M., L. Carter, S. L. Swain, R. W. Dutton. 1994. Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J. Exp. Med. 180:1715.[Abstract/Free Full Text]
  20. Metlay, J. P., E. Pure, R. M. Steinman. 1989. Distinct features of dendritic cells and anti-Ig activated B cells as stimulators of the primary mixed leukocyte reaction. J. Exp. Med. 169:239.[Abstract/Free Full Text]
  21. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, H. L. Weiner, N. Nabavi, L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[Medline]
  22. Grewal, I. S., H. G. Foellmer, K. D. Grewal, J. Xu, F. Hardardottir, J. L. Baron, C. A. Janeway, Jr, R. A. Flavell. 1996. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273:1864.[Abstract/Free Full Text]
  23. Fong, T. A., T. R. Mosmann. 1990. Alloreactive murine CD8+ T cell clones secrete the Th1 pattern of cytokines. J. Immunol. 144:1744.[Abstract]
  24. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.[Abstract/Free Full Text]
  25. Lowin, B., M. Hahne, C. Mattmann, J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370:650.[Medline]
  26. Grillot, D. A., R. Merino, G. Nunez. 1995. Bcl-xL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice. J. Exp. Med. 182:1973.[Abstract/Free Full Text]
  27. Boise, L. H., A. J. Minn, P. J. Noel, C. H. June, M. A. Accavitti, T. Lindsten, C. B. Thompson. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL. Immunity 3:87.[Medline]
  28. Maus, M. V., A. K. Thomas, D. G. Leonard, D. Allman, K. Addya, K. Schlienger, J. L. Riley, C. H. June. 2002. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20:143.[Medline]
  29. Rogers, P. R., J. Song, I. Gramaglia, N. Killeen, M. Croft. 2001. OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 15:445.[Medline]
  30. Skoskiewicz, M. J., R. B. Colvin, E. E. Schneeberger, P. S. Russell. 1985. Widespread and selective induction of major histocompatibility complex-determined antigens in vivo by {gamma} interferon. J. Exp. Med. 162:1645.[Abstract/Free Full Text]
  31. Lechler, R. I., J. R. Batchelor. 1982. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J. Exp. Med. 155:31.[Abstract/Free Full Text]
  32. Page, C., M. Rose, M. Yacoub, R. Pigott. 1992. Antigenic heterogeneity of vascular endothelium. Am. J. Pathol. 141:673.[Abstract]
  33. Hollenbaugh, D., N. Mischel-Petty, C. P. Edwards, J. C. Simon, R. W. Denfeld, P. A. Kiener, A. Aruffo. 1995. Expression of functional CD40 by vascular endothelial cells. J. Exp. Med. 182:33.[Abstract/Free Full Text]
  34. Hughes, C. C., C. O. Savage, J. S. Pober. 1990. Endothelial cells augment T cell interleukin 2 production by a contact-dependent mechanism involving CD2/LFA-3 interaction. J. Exp. Med. 171:1453.[Abstract/Free Full Text]
  35. Davis, T. A., N. Craighead, A. J. Williams, A. Scadron, C. H. June, K. P. Lee. 1996. Primary porcine endothelial cells express membrane-bound B7-2 (CD86) and a soluble factor that co-stimulate cyclosporin A-resistant and CD28-dependent human T cell proliferation. Int. Immunol. 8:1099.[Abstract/Free Full Text]
  36. Jones, N. D., A. Van Maurik, M. Hara, B. M. Spriewald, O. Witzke, P. J. Morris, K. J. Wood. 2000. CD40-CD40 ligand-independent activation of CD8+ T cells can trigger allograft rejection. J. Immunol. 165:1111.[Abstract/Free Full Text]
  37. Sayegh, M. H., X. G. Zheng, C. Magee, W. W. Hancock, L. A. Turka. 1997. Donor antigen is necessary for the prevention of chronic rejection in CTLA4Ig-treated murine cardiac allograft recipients. Transplantation 64:1646.[Medline]
  38. Carter, L. L., R. W. Dutton. 1995. Relative perforin- and Fas-mediated lysis in T1 and T2 CD8 effector populations. J. Immunol. 155:1028.[Abstract]
  39. Sad, S., D. Kagi, T. R. Mosmann. 1996. Perforin and Fas killing by CD8+ T cells limits their cytokine synthesis and proliferation. J. Exp. Med. 184:1543.[Abstract/Free Full Text]
  40. Dengler, T. J., J. S. Pober. 2000. Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype. J. Immunol. 164:5146.[Abstract/Free Full Text]
  41. McAdam, A. J., B. E. Gewurz, E. A. Farkash, A. H. Sharpe. 2000. Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL. J. Immunol. 165:3088.[Abstract/Free Full Text]
  42. Biedermann, B. C., J. S. Pober. 1999. Human vascular endothelial cells favor clonal expansion of unusual alloreactive CTL. J. Immunol. 162:7022.[Abstract/Free Full Text]
  43. Chen, Z., S. Cobbold, S. Metcalfe, H. Waldmann. 1992. Tolerance in the mouse to major histocompatibility complex-mismatched heart allografts, and to rat heart xenografts, using monoclonal antibodies to CD4 and CD8. Eur. J. Immunol. 22:805.[Medline]
  44. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434.[Medline]



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