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The Journal of Immunology, 2004, 173: 6660-6666.
Copyright © 2004 by The American Association of Immunologists

Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. II. Deletion of Effector Cells by Fas-Fas Ligand Apoptosis1

Shlomit Reich-Zeliger, Judith Gan, Esther Bachar-Lustig and Yair Reisner2

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8+ T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8+1B2+ effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8+ T cells were from 2C mice, which lack Fas expression ((2CX lpr)F2), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8+1B2+ effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the {alpha}3 domain of H2 class 1 on the effector cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The visualization by FACS of veto activity enabled by the 2C TCR transgenic (Tg)3 mouse model can be useful for mechanistic studies directed at understanding basic questions related to cell surface molecules and intracellular pathways triggered upon initiation of contact between effector T cells and veto cells. Previous studies making use of blocking Abs in the indirect functional Cr release cytotoxicity assay indicated potential involvement of CD8 on veto cells and the {alpha}3 domain of H-2-class 1 on effector cells (1, 2, 3, 4, 5). More recently, a role for Fas-FasL apoptosis was also indicated by this approach (4, 6, 7, 8). In the present study, by breeding 2C TCR Tg mice with a mutated strain of mice deficient in the expression of Fas, we were able to show direct evidence of the importance of this apoptosis pathway. An additional intriguing question related to the control of apoptosis of effector cells by veto cells is how Fas-FasL signaling can overcome intracellular apoptosis inhibitors (5, 9, 10, 11, 12, 13). Thus, we investigated the possibility that the expression of such inhibitory molecules during the MLR is reduced before the appearance of annexin V and the induction of apoptosis of 2C effector T cells.


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

Female 6- to 12-wk-old mice were used. BALB/c (H-2d), FVB (H-2q), SJL (H-2s), and C57BL/6 (H-2b) mice were obtained from the Weizmann Institute Animal Center (Rehovot, Israel). DBA/2 (H-2d) and C3H/HeJ (H-2k) mice were obtained from the Roscoe B. Jackson Memorial Laboratory (Bar Harbor, ME). A breeding pair of Tg H-2b mice expressing TCR from the CTL clone 2C with specificity for H-2Ld was provided by J. Nikolic-Zugic (Sloan-Kettering Institute, New York, NY). The progeny of these Tg mice were bred at the Weizmann Institute Animal Breeding Center. All mice were kept in small cages (five animals in each cage) and fed sterile food and acid water.

2C-lpr Tg mice were produced by breeding 2C Tg mice with C3H.MRL-Tnfrsf6lpr mice. The F1 2C/lpr mice were bred again with C3H.MRL-Tnfrsf6lpr

Preparation of host-nonreactive, donor anti-third-party veto CTLs

Responder splenocytes of BALB/c or DBA/2 mice were harvested, and the cell suspensions were treated with Tris-buffered ammonium chloride to remove RBC. The isolated cells (2 x 106/ml) were stimulated with irradiated (20 Gy) FVB, C3H/HeJ, or SJL (third-party stimulators) splenocytes (2 x 106/ml). Responders (2 x 106/ml) and stimulators (2 x 106/ml) were cultured for 6 days in RPMI 1640 complete tissue culture medium at 37°C in a 5% CO2/air incubator. On day 6 after seeding, cells were fractionated on Ficoll, and the lymphoid fraction was subjected to positive selection of CD8+ cells using magnetically labeled anti-CD8 Abs and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells (2 x 106/ml) were restimulated with the same irradiated (20 Gy) third-party stimulators (FVB, C3H/HeJ, or SJL; 2 x 106/ml), and human rIL-2 (40 U/ml; Eurocetus, Milan, Italy) was added, beginning that day, every second day to the MLR culture (days 6, 8, and 10). On day 10, the MLR cultures were harvested, fractionated on Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden), and analyzed by FACS for CD8 level and veto activity.

MLR cultures and cytotoxicity assay

Spleen cells of C3H/HeJ mice (responders) were harvested, and single-cell suspensions were prepared as described above. The cells (2 x 106/ml) were then stimulated with irradiated (20 Gy) BALB/c splenocytes (2 x 106/ml) or 2 x 106/ml irradiated (20 Gy) SJL splenocytes. Four to six replicates per group were cultured in 96-well, U-bottom plates in 0.2 ml of complete tissue culture medium for 6 days at 37°C (5% CO2 atmosphere). Con A blasts generated from SJL or BALB/c spleen cells (2 days in the presence of 2 µg of Con A/2 x 106 cells/ml) were labeled with 51Cr (NEN, Boston MA). A cell-mediated lysis assay was performed using variable numbers of MLR effector cells and 5 x 103 target cells in 96-well, V-bottom plates. 51Cr release was measured after 4-h incubation at 37°C. Results are expressed as specific lysis, calculated as follows: % specific lysis = 100 x ((experimental release – spontaneous release)/(maximum release – spontaneous release)). The SD of replicate values was consistently <10% of the mean.

Deletion of anti-H-2d T cells by anti-third-party CTLs

Spleen cells of 2C Tg H-2b mice, expressing TCR-{alpha}{beta} with specificity for H-2Ld mice (provided by J. Nikolic-Zugic, Sloan-Kettering Institute) were collected as described above. The cells (2 x 106/ml) were then stimulated with irradiated (20 Gy) BALB/c splenocytes (2 x 106/ml) in the presence of 20%, 10% or 2% cells of the veto anti-third-party CTLs originated from BALB/c or from FVB (background control) splenocytes. Cultures were incubated for 72 h, respectively, in 24-well plates. The deletion of Tg T cells was monitored by cytofluorometric analysis, measuring the level of 2C Tg cells, specifically stained by the 1B2 Ab, directed against the clonotypic anti-H-2Ld TCR.

Cytofluorometric analysis

FACS analysis was performed using a modified FACScan (BD Biosciences, Mountain View, CA). Fluorescence data were collected using 3-decade logarithmic amplification on 25–50 x 103 viable cells, as determined by forward light scatter intensity. Cells were stained with anti-CD8{alpha} (Ly-2)-FITC, anti-CD8{alpha} (Ly-2)-CyChrome, anti-CD8{alpha} (Ly-2)-allophycocyanin, anti-CD3{epsilon}-PE, anti-CD95 (Fas)-FITC (BD Pharmingen, San Diego, CA), and anti-CD4-Quantum Red ({sigma}). 1B2 biotinylated Abs (provided by J. Nikolic-Zugic, Sloan-Kettering Institute) was stained with R-PE-streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA).

Inhibition of the veto effect of CTLs by anti-CD8 mAb

Anti-third-party CTLs of DBA/2 origin were added to the MLR described above (C3H against irradiated DBA/2 splenocytes or 2C against irradiated DBA/2 splenocytes) at a final concentration of 2 or 10% respectively. Anti-CD8{alpha} mAb (provided by U. Hamerling, Sloan-Kettering Institute) directed against Ly-2.1 Ag, expressed selectively on the veto cells and not on the effector cells, was added to the MLR at different concentrations, as described in Results, and inhibition of the veto cells was monitored.

Detection of apoptotic cells

Annexin V-Cy5 was used to detect apoptotic cells. Cells were incubated in annexin V binding buffer (14, 15, 16) and supplemented with 5 µl of annexin V-Cy5. The cells were incubated at room temperature for 5 min in the dark, then washed in binding buffer. Positive cells were monitored by flow cytometry.

Fractionation of cell lysate

Cells were cultured in MLR for the indicated periods, and viable cells were harvested by Ficoll, resuspended in 1 ml of buffer C (10 mM KOH (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, and 0.5 mM DTT), incubated on ice for 30 min, and centrifuged for 10 min at 1,000 x g. The supernatant was then transferred to an ultracentrifuge tube (Sorvall, Newtown, CT) and centrifuged at 100,000 x g for 1 h. The remaining cytosolic supernatant was frozen. The pellet was washed in 1 ml of buffer D (20 mM KOH, 25% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA), left on ice for 30 min, and recentrifuged at 100,000 x g for 30 min. The pellet suspended in 200 µl of buffer F (10 mM Tris-HCl (pH 6.8), 0.1 M NaCl, and 1% SDS) represented the membrane fraction.

Western blot analysis

Cells were cultured in MLR for the indicated periods with various treatments. Cell lysates were prepared from these cultures (4 x 106 viable cells) and from 6 x 106 unstimulated (day 0) cells. Samples containing equal amounts of protein were separated by SDS-PAGE (4% stacking/7.5% resolving) and electroblotted to nitrocellulose. Immunoblotting was performed using anti-FLIP and anti-Bcl2, and thereafter cells were reacted with rabbit anti-mouseHRP Abs and visualized by chemiluminescent detection (ECL; Amersham Bioscience, Little Chalfont, U.K.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct evidence for a deletion-based mechanism: induction of annexin V expression on 2C effector T cells by veto CTLs

Early studies using the indirect functional assays indicated that the neutralizing activity of CD8+ veto T cells is probably mediated by apoptosis of the effector cells upon binding (1). The ability to monitor the fate of 2C responder cells by FACS enabled us in the present study to verify the role of apoptosis by measuring the levels of double-positive CD8+1B2+ cells in the presence or the absence of veto cells. As shown in Fig. 1, the percentage of these double-positive effector cells in a typical experiment was reduced from 58 to 17% within 72 h after incubation with CTL veto cells of H-2d origin, whereas no reduction was observed when veto CTLs of H-2s origin were used (Fig. 1A). Furthermore, triple staining with annexin V, CD8, and 1B2 showed that this pattern of deletion coincided with the induction of annexin V on H-2d-specific 2C effector cells. Thus, the level of triple-positive cells was increased upon interaction with H-2d veto CTLs from 3 to 40%, whereas only a marginal increase (to 9%) was observed when H-2s veto CTLs were added (Fig. 1B).



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  		FIGURE 1. Veto activity of anti-third-party CTLs: the role of apoptosis. A, Representative experiment monitoring 2c Tg effectors by FACS analysis in the absence (A) or the presence of nonspecific (B) or specific (C) veto CTLs. Veto activity was induced by adding 5% anti-third-party CTLs. B, Level of annexin V on 1B2/CD8-positive cells. Black indicates 2C cells alone, green shows 2C and nonspecific veto cells, and yellow indicates 2C and specific veto cells.

 
In three independent experiments, analysis of annexin expression within the 2C effector population as a function of time revealed that marked apoptosis of the effector cells was induced between 24 and 48 h after the addition of veto CTLs to the MLR (Fig. 2A). Maximal deletion of CD8+1B2+ effector T cells was exhibited 24 h later by the end of 72-h incubation with veto cells (Fig. 2B).



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  		FIGURE 2. Veto activity of anti-third-party CTLs: annexin expression on effector cells at different time points during MLR. Annexin expression is shown in the absence of veto cells ({blacksquare}) or in the presence of specific ({square}) or nonspecific ({cjs2112}) veto CTLs. A, Frequency of 1B2+CD8+ T cells positive for annexin V. B, Frequency of surviving 1B2+CD8+ T cells. The figures show the average ± SD of three experiments.

 
Deletion by veto CTLs is mediated by CD8 molecules on veto cells

The role of CD8 molecules on veto cells was previously indicated from functional studies using anti-CD8-blocking Abs. To distinguish between the CD8 molecules on veto CTLs and CD8 on responder T cells, we used veto cells that bear the CD8{alpha} Ly-2.2 allele (BALB/c background) and effector cells that are positive for the Ly-2.1 allele (C3H/Hej). In agreement with Sahambra et al. (2), the veto activity determined by 51Cr release assay after 6-day culture was completely blocked by the addition of mAbs (19/178) directed against the CD8{alpha} Ly-2.2 allele (Fig. 3A). Interestingly, only 50–60% inhibition was found when veto activity was assayed in the 2C Tg murine model by FACS analysis (Fig. 3B). Thus, the specific deletion of a substantial proportion of 2C CD8+ effector T cells by CTLs of H-2d origin may be independent of the interaction between CD8 on veto cells and the {alpha}3 domain of H-2 class I on the effector cells. As expected, the addition of Ab against CD8 partially inhibited the induction of annexin-positive cells by veto CTLs. Thus, although the percentage of such triple-positive cells was increased in three experiments to 40 ± 12%, it was only marginally elevated to 15 ± 4.4% in the presence of the Ab, as shown in Fig. 3C (representative experiment).



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  		FIGURE 3. Veto activity of anti-third-party CTLs: role of CD8 molecules on the veto cells. Specific inhibition of veto activity at different concentrations of anti-CD8 Ab added on day 0 to the culture was analyzed by two assays. A, Average ± SD of five different experiments monitoring killing activity by Cr51 release assay. The veto effect was induced by adding 2% BALB/c anti-third-party CTLs on day 0 as described in Fig. 1. B, Average ± SD of four different experiments monitoring deletion of 2c Tg effectors by FACS analysis. Veto activity was induced by adding 10% BALB/c anti-third-party CTLs. C, Level of annexin V on 1B2/CD8-positive cells. Black indicates 2c cells plus veto cells, and green shows 2c cells, veto cells, and anti-Ly-2.1 Ab.

 
Role of Fas-FasL apoptosis

In agreement with previous studies, activation of the 2C effectors is associated with enhanced FAS expression. As shown in Fig. 4A, which depicts a typical MLR of 2C splenocytes against BALB/c stimulators, Fas expression on CD8+1B2+ T cells was enhanced at 48 h and reached maximal expression at 72 h (Fig. 4B).



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  		FIGURE 4. Fas expression on 2c Tg effectors during MLR as a function of time. Splenocytes from 2c Tg mice (H-2b) bearing transgene TCR specific against H-2d (1B2+) were stimulated in MLR against BALB/c splenocytes (H-2d). The frequency of 1B2+ Fas+ T cells was determined by FACS every 24 h. The figures show the average ± SD of five experiments.

 
In agreement with the data shown in Fig. 2, in which annexin expression was monitored at different time points after the addition of veto CTLs, addition of BALB/c anti-C3H/HeJ, but not SJL anti-C3H/HeJ CTLs, led to a marked deletion of the Fas+-activated CD8+ T cells (Fig. 5). To evaluate more definitively the role of Fas on the effector CD8+ T cells, we bred Fas-deficient mice (C3H.MRL-lpr) with 2C TCR Tg mice. F1 mice were bred again with C3H.MRL-lpr, and the F2 offspring were tested for the expression of the Tg TCR and FAS. F2 mice were then tested for their sensitivity to veto CTLs.



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  		FIGURE 5. Deletion of Fas-positive effector T cells by anti-third-party veto CTLs. Splenocytes from 2C Tg mice (H-2b) bearing transgene TCR specific against H-2d were stimulated in MLR against BALB/c splenocytes (H-2d). A, 2C cells before MLR culture; B, 2C cells stimulated in the absence of veto cells; C, 2C cells stimulated in the presence of nonspecific veto CTLs; D, 2C cells stimulated in the presence of specific veto CTLs. FACS analysis was conducted 48 h after the initiation of MLR.

 
As shown in Fig. 6, the addition of BALB/c anti-third-party CTLs to MLR culture in which 2C-lpr Fas-deficient splenocytes were stimulated against BALB/c produced markedly reduced veto activity compared with that exhibited when the same veto CTLs were added to control MLR cultures of the original 2C effector T cells.



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  		FIGURE 6. Veto activity of anti-third-party CTLs: role of Fas. A, Representative experiment in which Fas levels expressed by the effector CD8 T cells are depicted after 72-h MLR cultures consisting of original 2C (A) or 2C-lpr (B) responders stimulated against BALB/c. The levels of CD8+1B2+ cells in MLR of 2C-lpr against BALB/c before (C) and after (E) addition of specific veto CTLs are contrasted with the levels of CD8+1B2+ cells in MLR culture of the original 2C with added veto CTLs (D). B, Role of Fas in the inhibitory activity of different potential veto cells. The veto activity was tested at a 1:10 veto:responder cell ratio. {square}, Anti-third-party CTLs; {blacksquare}, anti-third-party CD4 cells; {cjs2112}, activated NK cells; {cjs2117}, activated B cells. MLR cultures consisting of 2C responders stimulated against BALB/c in the presence of specific (A) or nonspecific (B) veto cells were compared with MLR cultures with 2C-lpr responders stimulated against BALB/c in the presence of specific (C) or nonspecific (D) veto cells.

 
Regulatory role of intracellular FLIP in effector cells

It has been shown that in lymphocytes and myeloid cells, death receptor-induced apoptosis is controlled by FLIP and not by the Bcl-2 protein family (17, 18, 19). Thus, it was of interest to investigate whether FLIP is preferentially down-regulated upon interaction of effector cells with veto cells.

To address this question, the responder cells were separated by MACS at different time points after initiation of culture with veto cells. The purified cells were lysed, the cell lysates were separated on SDS-acrylamide gel, and the levels of FLIP or Bcl2 were determined by immunoblotting. As shown in Fig. 7, upon interaction with stimulator cells, FLIP expression was markedly up-regulated, reaching maximal values at 24–48 h. However, these levels decreased between 48 and 72 h. In contrast, the expression of Bcl2 did not change throughout the entire culture period. (Fig. 8).



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  		FIGURE 7. FLIP expression in 1B2+CD8+ cells as a function of activation. 2C effectors were stimulated against BALB/c splenocytes. Every 24 h, 1 x 106 cells were harvested, lysed, and fractionated. Cytosolic fraction from 250,000 cells (35 µg per gel) was separated on SDS-PAGE and hybridized with anti-Flip Ab. A, Average ± standard deviation of four different experiments monitoring the level of Flip on IB2+CD8+ cells at different time points. B, Representative experiment of SDS-PAGE hybridized with ani-Flip Ab. C, Representative experiemnt of SDS-PAGE hybridized with anti-Bcl2 Ab.

 


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  		FIGURE 8. Mechanism of action of veto CTLs: working hypothesis. Upon recognition of class I on the veto cell by TCR on the effector T cell, Fas is up-regulated, allowing for FasL on the veto CTL to induce apoptosis. However, inhibitors such as FLIP, which are up-regulated within the first 24 h, can protect the activated effector T. The high affinity afforded by the interaction between CD8 on the veto cell and class I on the effector cell enables prolonged association until FLIP is down-regulated (48–72 h) and apoptosis can take place.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of TCR Tg mice affords a unique tool for the investigation of deletion-based mechanisms in immune tolerance induction. This approach has been extensively used in the context of thymic selection as well as the induction of T or B cell peripheral tolerance. In our two adjunct papers we validated the 2C TCR Tg mouse model for the study of veto CTLs by showing that several attributes of these cells found in this model are comparable to those previously found by indirect functional assays. The latter approach has indicated that the inhibition of effector cells is probably dependent on CD8 molecules on the veto cells interacting with the {alpha}3 domain of H-2 class 1 on the effector cells (1). Furthermore, by using knockout mice, the role of FasL on veto CTLs in conjunction with CD8 has been advocated (4, 8).

However, only by using TCR transgene staining has it become possible to follow the fate of effector cells in MLR and demonstrate their deletion upon addition of the appropriate veto CTLs presenting the specific H-2 recognized by the effector Tg CD8+ T cells. In addition, triple-staining of the effector cells with Abs against the TCR transgene, CD8, and annexin V has enabled us, for the first time, to follow the induction of apoptosis in a specific manner upon addition of veto cells. This specific induction of apoptosis can be blocked by anti-CD8 Ab directed against the allelic form present exclusively on veto cells. However, it is worth noting that although previous studies as well the present study showed full inhibition of effector CTLs, as measured by Cr release assay, we found only partial inhibition of the apoptosis of 2C effectors by veto cells. This discrepancy might be due to the different study population. Thus, whereas the former effector population is confined to the CTL precursors, the 2C CD8+ cell population expressing the TCR transgene comprises a broader and more heterogeneous cell population. It is likely that in about half these cells the affinity displayed by the TCR as well as other adhesion molecules is sufficient to lead to apoptosis independently of CD8 expression.

In addition to the role of CD8, the use of 2C Tg mice deficient in Fas (by breeding between the two strains) clearly demonstrates the importance of Fas-FasL killing for the veto reactivity. These results are in accordance with blockade of the veto effect by FasFc.

A major intriguing question related to Fas-FasL killing is how the effector T cells become susceptible to such killing, considering the expression of inhibitory molecules that are also up-regulated upon T cell stimulation. To address this question, the TCR Tg effector T cells were purified by magnetic beads at different time points of the MLR and analyzed for the expression of relevant apoptosis inhibitors. Thus, the present finding that down-regulation of FLIP coincides with the optimal time point for annexin V expression, indicates that prolonged interaction between effector T cells and veto cells might be necessary for efficient killing of the former cells. Interestingly, in accordance with previous reports that in lymphocytes the Bcl-2 protein family is not involved in the control of death receptor signaling (17, 18, 19), we found no changes in the expression levels of Bcl-2 throughout the relevant 72-h period.

Taken together, it is tempting to propose a working hypothesis (Fig. 9) for the mechanism of action according to which, the role of CD8 molecules on veto cells might be simply associated with providing the necessary additional avidity required to maintain prolonged interaction between the effector T cells and the veto CTLs until effective Fas-FasL apoptosis can be induced.

An alternative explanation for the role of CD8 molecules might be indicated from recent insights made during study of the so-called "competence to die," which is induced in chronically activated T cells. Thus, very recently it has been suggested that upon continuous activation of the effector T cells, redistribution of Fas into lipid rafts could account for increased susceptibility to Fas-FasL apoptosis (20, 21, 22). Future studies could reveal whether such a distribution also occurs upon interaction with veto CTLs and, if so, whether it could be mediated through the interaction between CD8 molecules on the veto CTLs and H2 molecules on the effector T cells.

Interestingly, although numerous reports have demonstrated several different CD8+ veto cells, some other phenotypes have been described. In particular, recent studies suggested that hemopoietic progenitor cells (23, 24, 25) and early myeloid cells are also endowed with veto activity (26, 27, 28, 29, 30). In such cells that do not express FasL or CD8, it has been indicated that other death molecules, such as TNF-{alpha}, might exhibit a function similar to that displayed by FasL. In line with this mode of action, it is likely that other adhesion molecules might afford the extra affinity provided by CD8 on veto CTLs.

Clearly, the induction of apoptosis of effector cells must be tightly regulated in vivo; therefore, it is most likely controlled by more than one pathway. Our present data, which are based mainly on Fas knockout mice, demonstrate the role of a particular critical death molecule such as FasL, but they cannot rule out the involvement of several other important key molecules, each of which could make a major contribution without which the integrated decision to die will not be selected. Preliminary results with gene microarrays, comparing effector TCR Tg that have been exposed to normal stimulation with those triggered by specific veto cells, indicate that the veto signal involves a variety of intracellular pathways, the triggering of which can be blocked by anti-CD8 Abs. Additional analysis of up- or down-regulated genes induced by other types of veto cells might point out those genes that are most relevant to the induction of apoptosis by veto cells in general.


   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 in part by National Institutes of Health Grant CA49369 and grants from E. Drake and the Gabriella Rich Center for Transplantation Biology Research. Back

2 Address correspondence and reprint requests to Dr. Yair Reisner, Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. E-mail address: yair.reisner{at}weizmann.ac.il Back

3 Abbreviation used in this paper: Tg, transgenic. Back

Received for publication March 25, 2004. Accepted for publication September 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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