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The Journal of Immunology, 2000, 165: 5580-5587.
Copyright © 2000 by The American Association of Immunologists

Role of STAT4 and STAT6 Signaling in Allograft Rejection and CTLA4-Ig-Mediated Tolerance1

Ping Zhou*, Greg L. Szot{dagger}, Zhong Guo*, Oliver Kim{dagger}, Gang He*, Jun Wang*, Michael J. Grusby, Kenneth A. Newell*, J. Richard Thistlethwaite*, Jeffrey A. Bluestone2,{dagger} and Maria-Luisa Alegre2,{ddagger}

Section of Transplantation, Departments of * Surgery and {dagger} Pathology, and {ddagger} Section of Rheumatology, Department of Medicine, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
STAT4-/- mice have impaired type 1 T cell differentiation, whereas STAT6-/- mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4-/-, and STAT6-/- recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-{gamma} in STAT4-/- recipients and undetectable levels of IL-4 and IL-5 in STAT6-/- mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4-/- mice. However, 14% of CTLA4-Ig-treated STAT6-/- mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6-/- mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6-/- recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6-/- mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigenic stimulation of naive T cells results in cytokine production, proliferation, and differentiation of T cells into effector lymphocytes. T cells differentiate into specific phenotypes depending on the TCR and costimulatory stimuli that they receive, and the soluble factors present in the inflammatory milieu during activation. T cells that predominantly secrete IL-2 and IFN-{gamma}, but no IL-4, have been termed type 1 T cells (Th1 for CD4+ cells and cytotoxic T (Tc)41 for CD8+ cells), and T cells that produce IL-4, IL-5, IL-6, and IL-10, but no IFN-{gamma}, are called type 2 cells (Th2 and Tc2 cells) (1).

Multiple studies have suggested that type 1 and type 2 lymphocytes play critically important roles in both acute rejection and induction of tolerance in allogeneic transplantation. It is widely proposed that Th1 cells promote allograft rejection and hinder tolerance induction, whereas Th2 responses favor a state of tolerance. These hypotheses come largely from the fact that, in multiple models, acute allograft rejection has been associated with the local presence of type 1 cytokine proteins or mRNA transcripts, whereas lack of rejection has correlated with the presence of type 2 cytokines (2, 3, 4). In addition, treatment with anti-IL-12 to prevent type 1 differentiation has resulted in prolonged allograft survival when acute rejection was dependent on indirect presentation of allopeptides or in the case of minor Ag-mismatched allografts (5). However, many recent studies have raised doubts about the validity of the Th1/Th2 paradigm (6, 7). Mice made genetically deficient in IL-2, IL-12, or IFN-{gamma} signaling have all been shown to reject various allografts and neutralization of IL-12 has correlated with improved graft survival in one model (8, 9, 10, 11). Conversely, transgenic expression of IL-4 in allografts or injection of a soluble form of IL-10 did not preclude acute rejection and instead accelerated graft loss (12, 13). Furthermore, mice deficient in IL-4 or IL-10 reject allografts (14, 15, 16, 17). However, such data must be interpreted in light of the redundant effects displayed by cytokine networks in vivo, as, in some cases, several cytokines can signal using the same intracellular pathway. Moreover, it remains controversial whether tolerance can be achieved in these different animals.

The generation of mice deficient in signal transducers and activators of transcription (STAT) genes has provided useful experimental models to explore this field, as signaling by all cytokines using a specific STAT pathway is abolished. STAT genes were discovered through the study of IFN signaling (18). Among STAT family members, STAT4 and STAT6 are key regulators of T cell differentiation involved in immune responses (19). The STAT4 gene knockout mice (STAT4-/-) are deficient in their ability to respond to IL-12 signaling and thus have impaired Th1 cell responses. Activated T cells and NK cells derived from STAT4-/- mice produce significantly less IFN-{gamma} and lymphotoxin in response to IL-12 stimulation than cells derived from wild-type mice (20). In contrast, under normal circumstances, STAT6-/- mice cannot develop Th2 cells in response to IL-4 (21). Thus, these two gene-knockout mouse strains provide a model to study the role of type 1 and type 2 lymphocyte interactions in allograft rejection and tolerance induction.

In the present study, fully allogeneic C3H/HeN (C3H) cardiac grafts were transplanted into STAT4-/-, STAT6-/-, and wild-type BALB/c animals. Type 1 and type 2 T cell development was clearly disrupted in the STAT4-/- and STAT6-/- mice, respectively, even within the inflammatory sites of the allografts. However, acute allograft rejection was normal in both STAT4-/- and STAT6-/- mice. Administration of the CD28/B7 antagonist CTLA4-Ig significantly prolonged cardiac allograft survival in both the STAT4-/- and STAT6-/- mice. However, costimulatory blockade was less effective in STAT6-/- mice as a significant percentage (14%) of the grafts was lost between days 20 and 100 post transplant. Moreover, a high proportion of the STAT6 knockout (STAT6KO) allograft recipients rejected their grafts after 100 days. Thus, the results demonstrate that type 1 and type 2 lymphocytes and cytokines are not essential for either acute allograft rejection or the prolongation of graft survival following costimulation blockade. However, STAT6 signaling may be involved, at least in part, in long-term maintenance of tolerance.


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

STAT4-/-, STAT6-/-, and wild-type mice were back-crossed six generations onto the BALB/c (H-2d) background (22). C3H (H-2k), BALB/c, C57BL/6 (B6, H-2b), and BALB/c ByJSmn-Prkdcscid (BALB/C.SCID, H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed under laminar flow hoods in an environmentally controlled animal facility. During the course of these studies, there was an outbreak of mouse hepatitis virus in some of our colonies. However, similar results were observed under pathogen-free conditions.

Reagents

Murine CTLA4-Ig was a gift from Mary Collins (Genetics Institute, Boston, MA). Individual mice received 50 µg/mouse of murine CTLA4-Ig injected i.p. every other day for 14 days, beginning on the day of the transplant.

Cardiac transplantation

Abdominal heterotopic cardiac transplantation was performed using an improved technique from the one originally described by Corry et al. (23). Cardiac allografts were transplanted in the abdominal cavity, by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient’s aorta and vena cava, respectively. Cervical cardiac allografts were transplanted using the technique of Chen et al. (24). The cardiac graft was placed under the skin of the front side of the neck. The innominate artery of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient by interrupted stitches. The pulmonary artery of the donor heart was anastomosed end-to-end to the external jugular vein of the recipient with a continuous suture. The heartbeat of the allografts was assessed daily by palpation. The day of rejection was defined as the last day of a detectable heartbeat. Graft rejection or tolerance was verified in selected cases by autopsy and pathological examination. Samples were obtained from recipient mice sacrificed at different time points and were fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with hematoxylin-eosin. Acute rejection was scored from 0 to 3 by a pathologist blinded to the clinical rejection status of the heart (0, no infiltration; 1, scattered mild infiltration; 2, multifocal or diffuse infiltration; 3, multifocal or diffuse dense infiltration with myocyte death or necrosis).

Adoptive transfer of splenocytes

Spleens were harvested from control or transplanted mice and prepared into single cell suspension. Live cells were counted by trypan blue exclusion. An aliquot of cells was stained with anti-Thy1.2-FITC (PharMingen, San Diego, CA) and analyzed by flow cytometry using forward vs side scatter to define a gate of live cells. The number of T cells per sample was calculated by multiplying the percent Thy1.2+ cells obtained by flow cytometry analysis by the total number of live cells counted. An equivalent of 7 million T cells was resuspended into 150 µl PBS and injected into the tail vein of BALB/C.SCID recipients that had been transplanted 2–5 days previous with C3H or B6 cardiac allografts.

Immunohistochemistry procedure and quantitative analysis of CD4+ and CD8+ T cells

Grafts were removed, immediately frozen in liquid nitrogen, and embedded with OCT (Tissue-Tek, 4583, Bakura). The samples were sliced into 6-µm-thick sections at -20°C. Slides were incubated with a rat anti-CD8 IgG supernatant (neat) or a purified rat anti-CD4 IgG Ab (GK 1.5, 1 µg/ml) overnight at 4°C. After primary Ab incubation, the slides were rinsed and incubated with a secondary Ab (0.01 mg/ml biotinylated anti-rat IgG; Vector Laboratories, Burlingame, CA) at room temperature for 30 min. The Ag-Ab binding was detected using an avidin-biotin-HRP system (Vector Laboratories). The slides were immersed briefly in hematoxylin for counterstaining and evaluated under light microscopy by a pathologist blinded to the clinical rejection status of the heart. The numbers of CD4+ and CD8+ T lymphocytes were counted in three to four randomly chosen high-powered visual fields per section (x400 magnification). The fields with the highest concentration of mononuclear infiltration were chosen for the cardiac grafts that had only focal or patchy infiltration.

Semiquantitative RT-PCR

Grafts were obtained at different time points and preserved in liquid nitrogen. Total RNA was isolated from portions of cardiac grafts using a RNeasy Mini Kit (Qiagen, Hilden, Germany). A total of 3–5 µg of total RNA was reverse-transcribed using the First Strand cDNA Synthesis kit (Amersham Pharmacia, Piscataway, NJ). PCR amplification was performed using 30-µl reaction mixtures containing reverse transcriptase mixtures: 20 mM Tris-HCI (pH 8.4), 50 mM MgCl2, 0.125 mM dNTP, 0.125–0.25 µm of each forward and reverse amplification primer, and 0.025 U/µl Taq Polymerase (Life Technologies, Gaithersburg, MD). The amplification primers shown in Table IGo were synthesized by Integrated DNA Technologies (Coralville, IA). The amplification protocol consisted of an initial denaturation at 94°C for 5 min. Each cycle of amplification included three steps in which the reaction was denatured at 94°C for 45 s, annealed at the temperature indicated (Table IGo) for 1 min, and elongated at 72°C for 2 min for a total of 20–30 cycles. The elongation stage of the final cycle was extended to 7 min.


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  		Table I. Amplification primers

 
For semiquantitative RT-PCR, two rounds of PCR amplification were performed. The starting amount of cDNA used for each sample in the first round of amplification was equalized based on the intensity of GAPDH bands detected after a trial PCR. The first round of PCR amplification was performed using primers for each cytokine and GAPDH. Three tubes (each containing 3 µl of first-round PCR products) were used for second-round PCR. For each cytokine and GAPDH, one tube was removed after 10, 15, or 20 cycles. PCR products for cytokines and GAPDH were compared by determining the intensity of the bands detected on 2% agarose gels using a Kodak Gel Photography System and Kodak ID software (Rochester, NY).

Analysis of cytokine mRNA by quantitative real-time RT-PCR

All real time quantitative RT-PCR were performed as described previously (25). Briefly, PCR amplification was performed in the ABI Prism 7700-sequence detection system (Perkin-Elmer, Foster City, CA). The cytokine probes were labeled with the fluorescent reporter dye 6-carboxyfluorescin at the 5' end of the oligonucleotide and a quencher dye 6-carboxytetramethylrhodamine attached at the 3' end. The 5' end of the GAPDH (housekeeping gene) probe was labeled with the fluorescent reporter dye TET and the 3' end was attached to 6-carboxytetramethylrhodamine. The primer and probe sequences were used as described by Overbergh, et al. (26). All reactions were performed using the TaqMan Universal PCR master mixture containing AmpliTaq Gold DNA Polymerase according the manufacturer’s recommendation (PE Applied Biosystems). The adjusted amounts of cDNA were used to quantify cytokine and GAPDH genes with specific primers. PCR conditions were as follows: 50°C for 2 min and 95°C for 10 min. Each amplification was performed for 15 s at 95°C and 1 min at 60°C for 40 cycles. PCR results were analyzed using the {Delta}{Delta}CT method according to the manufacturer’s instructions, with the concentration of target cDNA normalized to the amount of the housekeeping gene, GAPDH.

Statistical analysis

Heart graft mean survival time (MST), SE, and p values were calculated using the Kaplan-Meier/log rank test methods. Rejection grades were compared using the one-way ANOVA test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
STAT4 is not required for acute allograft rejection or induction of tolerance

Type 1 lymphocytes and cytokines are thought to play an important role in allograft rejection. Therefore, we examined whether a deficiency in STAT4 altered the kinetics of acute rejection of allogeneic heart grafts. As shown in Fig. 1GoA, all cardiac grafts placed abdominally in STAT4-/- mice were rejected promptly with a MST of 10 ± 1 days, as compared with 8 ± 1 days when allogeneic hearts were transplanted into wild-type mice. Histological studies confirmed severe rejection in all cardiac allografts (scored as grade 3) when compared with syngeneic control grafts (scored as grade 1 or 0; data not shown). Semiquantitative PCR for cytokine mRNA was performed on cardiac allografts at the time of rejection to verify that type 1 responses were deficient in these animals. Levels of expression of IFN-{gamma} mRNA transcripts were dramatically reduced in rejected allografts obtained from STAT4-/- recipients as compared with wild-type animals, whereas levels of IL-4 mRNA were similar in both groups (Fig. 1GoB). These results indicate that despite impaired up-regulation of IFN-{gamma} gene expression, STAT4-/- mice are competent to reject cardiac allografts.



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  		FIGURE 1. Cardiac allograft rejection and CTLA4-Ig-mediated tolerance occurs normally in STAT4-/- mice despite reduced type 1 cytokine. Cardiac allografts from C3H donor mice were transplanted in the abdominal cavity of wild-type BALB/c (n = 5) or STAT4-/- (n = 6) mice. The animals were left untreated or received CTLA4-Ig for 2 wk (50 µg every other day, injected i.p.). A, The percent of graft survival over time in the different groups. B, The intragraft expression of IFN-{gamma} and IL-4 mRNA transcripts, determined by semiquantitative RT-PCR. Grafts were harvested at the time of rejection, RNA was extracted, and results were normalized as fold induction over IFN-{gamma} (left axis) or IL-4 (right axis) gene expression in one syngeneic graft.

 
The use of the CD28/B7 antagonist, CTLA4-Ig, has been among the most effective means of preventing allograft rejection and inducing tolerance. Blockade of CD28 appears to block more efficiently naive and type 1 than type 2 T cell responses. Thus, tolerance induction may be altered in allograft recipients with impaired generation of type 1 T cell responses. Therefore, CTLA4-Ig-induced immunosuppression was examined in STAT4-/- mice. As shown in Fig. 1GoA, CTLA4-Ig treatment resulted in allograft acceptance for >100 days both in wild-type and STAT4-/- mice. In fact, the grafts retained function in wild-type and STAT4-/- animals for well over 150 days, indicating that the CTLA4-Ig effects were long-lasting in these animals. To determine whether the lack of rejection was due to graft adaptation or tolerance, four wild-type and three STAT4-/- mice were retransplanted at approximately day 100 with a second C3H heart vascularized to a cervical location. All second hearts were accepted for >68 days and up to 133 days, at which times the animals were sacrificed or died, indicating that graft tolerance to the first transplant was not due to graft adaptation (data not shown). In contrast, cervical allografts were rejected promptly by untreated mice (MST of 10 ± 1 days, n = 3, data not shown). Taken together, these findings support the conclusion that the capacity to generate type 1 T cell responses is not essential for allograft tolerance in this model.

To investigate whether lack of STAT4 signaling affected the subset of T cells present in the allografts, quantitative immunohistochemical analysis of CD4+ and CD8+ T cells was performed at the time of rejection. Although CD4+ T cells have been shown to be both necessary and sufficient for heart allograft rejection (27), CD8+ T cells were found to dominate the graft infiltrate in wild-type mice (G. L. Szot and M.-L. Alegre, manuscript in preparation). Similar CD4+ and CD8+ T cell infiltrates were observed in allografts from wild-type and STAT4-/- mice (Fig. 2Go). These findings indicate that impairment of type 1 T cell differentiation does not preclude generation of effector CD4+ and CD8+ T cells and does not prevent acute cardiac allograft rejection.



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  		FIGURE 2. Absence of STAT4 does not affect the quality or quantity of intragraft T cell infiltrate. A normal heart, a syngeneic graft at day 8, and cardiac allografts from wild-type and STAT4-/- recipients at day 8 were harvested, and infiltrating CD4+ and CD8+ T cells were analyzed by immunohistochemistry on frozen tissue sections. Sections were examined under light microscopy by a pathologist, and positive cells were counted in three to four high magnification fields (x400). Staining with anti-CD4 and anti-CD8 mAbs was compared with that obtained with rat control isotype used as a negative control. Grafts from three wild-type and two STAT4-/- mice were analyzed in the experimental groups. The counts per field for each T cell subset were pooled in each group, and the graph represents the mean + SD of those counts.

 
Lack of STAT6 does not result in acceleration of acute allograft rejection

Type 2 T cells produce cytokines known to down-regulate type 1 responses and may be important in suppressing allograft responses. T cells isolated from an ongoing immune response in STAT6-/- mice have been reported to produce little, if any, type 2 cytokines (21). To determine whether lack of STAT6 would influence the kinetics of acute allograft rejection, C3H donor hearts were transplanted into wild-type or STAT6-/- recipients. As shown in Fig. 3GoA, grafts were rejected at similar times in both strains of mice. To investigate whether absence of STAT6 prevented generation of type 2 cytokines in our model, mRNA from rejected grafts was analyzed by semiquantitative RT-PCR. Expression of IL-4 and IL-5 mRNA transcripts was undetectable in allografts from STAT6-/- mice (Fig. 3GoB). In contrast, cardiac grafts from wild-type animals revealed high levels of IL-4 and IL-5 gene expression, and grafts from both wild-type and STAT6-/- recipients exhibited high levels of IFN-{gamma}. Therefore, despite the lack of type 2 cytokines, there was no acceleration of graft loss in these animals. In addition, no significant difference was found in the numbers of CD4+ and CD8+ T cells infiltrating the grafts from STAT6-/- mice at the time of rejection, as compared with wild-type mice (Fig. 4Go).



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  		FIGURE 3. Lack of STAT6 does not affect acute cardiac allograft rejection. Grafts from C3H donor mice were transplanted into the abdominal cavity of wild-type (n = 5) or STAT6-/- (n = 6) recipients. A, The percent of graft survival over time in wild-type and STAT6-/- recipients. B, The levels of IL-4, IL-5, and IFN-{gamma} mRNA transcripts in the grafts at the time of rejection normalized as fold induction over expression in one syngeneic graft.

 


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  		FIGURE 4. T cell infiltrate in the grafts from STAT6-/- recipients is similar to that found in transplants from wild-type mice. A normal heart, a syngeneic graft at day 8, and cardiac allografts were harvested from wild-type (n = 3) and STAT6-/- (n = 2) recipients at day 8, and infiltrating CD4+ and CD8+ T cells were determined as described in Fig. 2Go. The counts per field for each T cell subset were pooled in each group, and the graph represents the mean + SD of those counts.

 
STAT6 is not required for the initial prolongation of graft survival induced by CTLA4-Ig but may be important for robust, long-term maintenance of tolerance

In several experimental models, the tolerogenic effect of CD28 blockade has been linked to the production of IL-4 and IL-10 inferring an important role for type 2 cytokines in CTLA4-Ig-mediated tolerance (28). Therefore, we examined the ability of CTLA4-Ig therapy to induce tolerance in STAT6-/- mice. Administration of a short-course of CTLA4-Ig to wild-type and STAT6-/- mice after cardiac transplantation resulted in prolongation of graft survival in all animals (Fig. 5GoA). Although most animals retained their grafts for >100 days, ~14% of the STAT6-/- mice rejected the heart allografts early. These results suggested that a deficiency in STAT6 signaling may have a modest impact on long-term allograft survival. Therefore, five CTLA4-Ig-treated wild-type and five CTLA4-Ig-treated STAT6-/- mice were continually monitored past 100 days. Cardiac allografts stopped beating between 103 and 125 days after transplant in three of the five STAT6-/- animals, whereas all wild-type animals retained their grafts for over 200 days (Fig. 5GoB). All five STAT6-/- animals were sacrificed at day 145 for immunohistochemistry assessment, cytokine analysis, and adoptive transfer studies.



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  		FIGURE 5. STAT6 may be involved in long-term maintenance of CTLA4-Ig-mediated tolerance. Hearts from C3H donor mice were transplanted in the abdominal cavity of wild-type (n = 8) and STAT6-/- recipients (n = 15), and all animals received i.p. injections of CTLA4-Ig (50 µg/2 days for 2 wk). A, All animals were examined for 100 days. B, In addition, five mice per group were monitored for a longer period of time. The two graphs represent the percent of survival of the grafts over time. The five STAT6-/- animals were sacrificed on day 145 for adoptive transfer experiments (see Fig. 8Go).

 
Immunohistochemistry was performed in long-term grafts harvested between day 100 and day 145 posttransplant in CTLA4-Ig-treated wild-type and STAT6-/- animals (two to five animals per group). In all cases, the grafts were infiltrated with CD4+ and CD8+ T cells. However, there was a trend toward increased numbers of infiltrating CD4+ and CD8+ T cells in grafts from CTLA4-Ig-treated STAT6-/- mice, as compared with grafts from CTLA4-Ig-treated wild-type mice (Fig. 6Go).



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  		FIGURE 6. Grafts from CTLA4-Ig-treated STAT6-/- animals have slightly higher numbers of infiltrating T cells than grafts from CTLA4-Ig-treated wild-type mice. Cardiac allografts from CTLA4-Ig-treated wild-type, rejector, and nonrejector STAT6-/- mice were harvested between day 100 and 145 and analyzed by immunohistochemistry for expression of infiltrating CD4+ and CD8+ T cells. The figure represents the mean + SD of counts in the different fields, as described in Fig. 2Go. Results from several mice were pooled in each group; grafts from CTLA4-Ig-treated wild-type (n = 5), nonrejector CTLA4-Ig-treated STAT6-/- (n = 3), and rejector CTLA4-Ig-treated STAT6-/- (n = 2).

 
Cardiac allografts from rejector CTLA4-Ig-treated STAT6-/- mice express higher levels of IFN-{gamma} mRNA than those from nonrejector recipients

Analysis of IFN-{gamma} gene expression was performed in allografts from CTLA4-Ig-treated rejector and nonrejector STAT6-/- mice using a quantitative real-time PCR assay. Levels of IFN-{gamma} gene expression were higher in grafts from CTLA4-Ig-treated rejector than nonrejector STAT6-/- mice (Fig. 7Go). Therefore, late rejection in CTLA4-Ig-treated STAT6-/- animals correlates with a trend toward an increase in IFN-{gamma} production.



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  		FIGURE 7. Levels of IFN-{gamma} in grafts from CTLA4-Ig-treated STAT6-/- mice. Cardiac allografts from CTLA4-Ig-treated nonrejector (n = 2) or rejector (n = 3) STAT6-/- mice were harvested on 145 day and levels of expression of IFN-{gamma} mRNA were assessed in each individual graft by quantitative real-time RT-PCR, as described in Materials and Methods. Results are expressed as fold induction over expression in one syngeneic control graft.

 
Adoptive transfer studies support a role for STAT6 in the long-term maintenance of tolerance induced by CTLA4-Ig

Splenocytes from CTLA4-Ig-treated STAT6-/- rejector or nonrejector animals were pooled in each group, analyzed by flow cytometry, and an equivalent of 7 x 106 T cells was adoptively transferred into BALB/C.SCID recipients transplanted with donor C3H or third party B6 hearts. As shown in Fig. 8GoA, mice receiving splenocytes from the rejector CTLA4-Ig-treated STAT6-/- recipients rejected both donor and third party cardiac allografts. This indicates that the graft loss in the original recipient correlates with the presence of cells in the spleen capable of mediating acute rejection of a fresh graft. In addition, C3H hearts were rejected with slighly faster kinetics than B6 hearts or than C3H hearts by SCID mice transfused with splenocytes from naive STAT6KO mice (Fig. 8GoD), suggesting that spleens from rejector CTLA4-Ig-treated STAT6KO mice contained cells primed to C3H Ags. In contrast, adoptive transfer of splenocytes from the nonrejector CTLA4-Ig-treated STAT6-/- mice resulted in acute rejection of third party but not of donor cardiac grafts (Fig. 8GoB). Similarly, animals receiving splenocytes from CTLA4-Ig-treated wild-type mice rejected third party but not donor grafts (Fig. 8GoC), whereas BALB/C.SCID mice injected with splenocytes from untreated wild-type and STAT6-/- mice rejected both C3H and B6 cardiac allografts (Fig. 8GoD). Taken together, these data indicate that tolerance is not permanent in all CTLA4-Ig-treated STAT6-/- mice.



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  		FIGURE 8. CTLA4-Ig treatment fails to induce long-lasting tolerance in a subset of STAT6-/- recipient mice. Five CTLA4-Ig-treated STAT6-/- mice were sacrificed at 145 days following transplantation. Three had lost their grafts between 103 and 125 days post transplant, whereas two recipient mice retained beating grafts at the time of sacrifice. Spleens were harvested in all animals and pooled as belonging to the rejector or nonrejector group. As controls, spleens from CTLA4-Ig-treated BALB/c wild-type mice were harvested on day 100 posttransplant, and spleens from unmanipulated BALB/c and STAT6-/- mice were used. A single cell suspension was prepared, and a cell-equivalent of 7 x 106 T cells was transfused into the tail vein of syngeneic BALB/C. SCID recipient animals transplanted 2–5 days previous with either donor C3H or third party B6 hearts. Graft survival was monitored in the different groups following the adoptive transfer, considered as day 0. A, Graft survival in mice transferred with cells from rejector STAT6-/- recipients. BALB/C.SCID recipients received B6 (n = 3) or C3H hearts (n = 3). B, The results from adoptive transfer from nonrejector mice (B6, n = 3; C3H, n = 2). C, Graft survival following adoptive transfer of cells from CTLA4-Ig-treated BALB/c wild-type mice (B6, n = 5; C3H, n = 5). D, The data obtained in recipients transfused with cells from untreated BALB/c (C3H, n = 4; B6, n = 3) or STAT6-/- (B6, n = 2; C3H, n = 3) animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of cytokines in the induction of transplantation tolerance has been largely studied in the context of the Th1/Th2 paradigm. The dogma states that Th2 lymphocytes favor tolerance by producing the anti-inflammatory cytokines IL-4 and IL-10, whereas Th1 lymphocytes facilitate rejection and may hinder tolerance induction by secreting IL-2 and IFN-{gamma}. However, recent studies have raised doubts about the Th1/Th2 paradigm. The data presented in this study suggest that the kinetics of cardiac allograft rejection are unaffected by the marked reduction of either type 1 or type 2 cytokines, but the presence of STAT6, or type 2 cytokines, may play a role in the long-term maintenance of tolerance.

STAT4-/- mice rejected cardiac allografts in a normal time frame. The remarkably low expression of IFN-{gamma} in STAT4-/- mice indicated that IFN-{gamma} was not required for cardiac allograft rejection in this model. It cannot be ruled out that very low levels of IFN-{gamma} were sufficient. However, in support of this hypothesis, STAT-1-/- mice on a DBA/2 background (H-2d) were capable of rejecting C3H cardiac allografts with similar kinetics as wild-type animals (MST 6 ± 1 days, three mice per group; data not shown). STAT1-/- mice have abolished signaling by type I and type II IFNs (29), and have recently been shown to lack tumor rejection capacity, for which IFN signaling appears to be essential (30). Similarly, IFN-{gamma}-/- mice retain allograft rejection capacity in an islet transplant model in mice (9).

Generation of type 2 T cell responses appears intact in STAT4-/- animals, as levels of IL-4, IL-5, and IL-10 (not shown) in the cardiac allografts are similar to those in grafts from wild-type controls. IL-5 is a chemoattractant and growth factor for eosinophils, that can play a role in acute allograft rejection in certain settings (31, 32). In fact, lack of IFN-{gamma} signaling in a model of islet allografts resulted in augmented levels of IL-5 and increased eosinophil infiltration in the grafts. Therefore, it was possible that the mechanism of cardiac allograft rejection by STAT4-/- mice in our model was different from that of wild-type mice, as a result of the markedly reduced levels of IFN-{gamma}. However, histology of the rejecting grafts from these animals did not reveal increased numbers of eosinophils infiltrating the grafts (data not shown). Rather, a massive T cell infiltrate with predominant numbers of CD8+ T cells was observed, similar to that found in the transplants from wild-type recipients, suggesting that the mechanisms of acute allograft rejection in STAT4-/- and wild-type animals were comparable. The exact mechanism by which rejection of cardiac allografts is performed by CD8+ T cells is unclear, but may depend on direct lysis via perforin/granzyme and/or Fas-dependent pathways. Both pathways may be involved in acute allograft rejection, as Abs blocking Fas have been shown in vitro to prevent lysis of allogeneic cells by T cell-lines generated from human renal transplants (33). In addition, recipient mice deficient in perforin were reported to have delayed rejection of MHC class I mismatched cardiac allografts (34). Taken together, these results support the notion that IL-12 signaling and IFN-{gamma} are not required for the generation of effector T cells, or the recruitment of T cells to the graft site.

Lack of STAT4 signaling did not affect the induction of tolerance by CTLA4-Ig. In fact, in grafts analyzed at 100 days post transplantation, the numbers of CD8+ T cells infiltrating the transplants were similar in CTLA4-Ig-treated wild-type and STAT4-/- mice, and reduced as compared with infiltrates found on day 7 in untreated animals (data not shown). In addition, similar levels of IL-4, IL-10, and IL-5 gene expression were found in CTLA4-Ig-treated wild-type and STAT4-/- animals (data not shown). These results suggest that IL-12 signaling and high levels of IFN-{gamma} production are not required for CTLA4-Ig-induced tolerance. This supports other studies in which IFN-{gamma} was not required for the induction of tolerance. For example, long-term islet allograft acceptance induced by anti-LFA-1 mAb was not dependent on IFN-{gamma} as the mAb remained effective in IFN-{gamma}-/- mice (17). However, a recent report using IFN-{gamma}-/- mice has identified IFN-{gamma} as an important cytokine for the induction of tolerance following CD28/B7 blockade in another murine cardiac allograft model (35). The reason for the different results in our study are unclear, but it remains possible that the low levels of IFN-{gamma} mRNA gene expression detected in the grafts from STAT4-/- recipients in our system may be sufficient to permit tolerance induction by CTLA4-Ig treatment. Alternatively, IFN-{gamma}-/- mice may have other compensatory mechanisms that alter tolerance induction in certain settings.

We conclude that STAT4 signaling is not required for acute cardiac allograft rejection and CTLA4-Ig-mediated tolerance induction, and that impaired type 1 differentiation does not affect these responses. However, other pathways may contribute to the generation of Th1 and Tc1 T lymphocytes. Indeed, IL-12 and IL-18 have been reported to favor type 1 T cell differentiation (36), and recent evidence indicates that T cell differentiation into Th1 lymphocytes can occur in the absence of STAT4 signaling (37). Therefore, whether rejection and tolerance can occur in the complete absence of type 1 T cell differentiation remains to be investigated in mice deficient in all the pathways that can generate type 1 T cell responses.

STAT6-/- mice can reject their grafts with normal kinetics, as compared with wild-type animals in the absence of detectable levels of IL-4 and IL-5 mRNA transcripts in the transplant. Rather, rejection correlates with a massive infiltrate of CD8+ T cells, comparable to that in wild-type recipients. This indicates that type 2 T cell responses are not required for cardiac allograft rejection. Moreover, administration of CTLA4-Ig could significantly prolong cardiac allograft survival in STAT6-/- mice indicating that some function of CD28/B7 blockade is preserved in the absence of STAT6 signaling. The mechanism by which CTLA4-Ig results in long-term cardiac allograft acceptance is not fully elucidated, but it has been suggested that prevention of CD28 signaling can result in reduced proliferation of activated T cells and increased apoptosis of allospecific T lymphocytes (6). In our model, administration of CTLA4-Ig resulted in reduced numbers of T cells infiltrating the grafts of STAT6-/- and wild-type recipients 100 days post transplantation, as compared with the infiltrate observed on rejecting grafts of untreated animals on day 7 (compare T cell numbers in Figs. 6Go and 4Go).

Finally, tolerance induced by CD28/B7 blockade appears less robust in STAT6-/- animals than in wild-type or STAT4-/- mice, as some cardiac allografts were rejected between day 103 and day 125 in CTLA4-Ig-treated STAT6-/- but not in wild-type or STAT4-/- mice. This may be due to the absence of generation of type 2 T cell responses. Perhaps type 2 T cells can act as suppressor cells of other more cytotoxic cell-types, or the whole T cell response may shift to a nonaggressive type 2 environment in the presence of CTLA4-Ig in wild-type mice. Alternatively, STAT6, IL-4, or IL-5 signaling may be directly or indirectly important for the transcription of genes necessary for long-term maintenance of tolerance, irrespective of T cell differentiation. For example, activation of STAT6 has been shown to induce up-regulation of GATA3 and c-maf and to down-regulate expression of IL-12R{beta}2 chain (38). It is conceivable that STAT6 may regulate other genes involved in the maintenance of tolerance.

Adoptive transfer of splenocytes from untreated and CTLA4-Ig-treated mice demonstrated that spleens from rejector CTLA4-Ig-treated STAT6-/- animals contained cells capable of initiating rejection of donor hearts in secondary recipients. This indicates that the original STAT6-/- recipient mice were not tolerant to donor hearts, despite having retained the grafts for over 100 days. In contrast, wild-type CTLA4-Ig-treated animals do not reject cardiac allografts in our experience, even at very late time points (several months), and transplantation of a second cardiac graft in a cervical location of the original recipient results in permanent acceptance of both donor grafts, a landmark of tolerance. Although these experiments could not be performed in our STAT6-/- mice because of the limited numbers of mice available, the adoptive transfer experiments suggest that a subset of the CTLA4-Ig-treated STAT6-/- mice may be tolerant.

Why CTLA4-Ig does not induce long-lasting tolerance in some STAT6-/- mice is unclear. However, there was a trend toward increased number of T cells infiltrating the grafts from CTLA4-Ig-treated STAT6-/- animals as compared with CTLA4-Ig-treated control mice. This increased number of T cells may contribute to graft loss in a subset of the STAT6-/- mice following CD28/B7 blockade. However, the number of CD8+ T cell-infiltrating grafts from nonrejector CTLA4-Ig-treated STAT6-/- mice was even higher than that from rejector and wild-type recipients. This finding perhaps suggests that nonrejector STAT6-/- mice may have rejected their grafts as well if kept alive for a longer period. In addition, expression of IFN-{gamma} mRNA was higher in grafts from rejector than nonrejector CTLA4-Ig-treated STAT6-/- recipients. Although this is probably not the only factor leading to rejection, it clearly correlates with it. The process of rejection is probably a complicated multifactorial operation involving activation and differentiation of alloreactive T cells, migration into the graft, and effector function. Increased numbers of CD4+ and CD8+ T cells infiltrating the grafts and higher levels of IFN-{gamma} may contribute to tipping the immune system in the delicate balance between tolerance of the graft and graft rejection.

In conclusion, the notion that immune deviation will effect both graft rejection and tolerance induction must be reconsidered based on the findings reported in these and other studies.


   Acknowledgments
 
We thank Terri Li for excellent technical help with the histological studies, and Ingrid Rulifson for reviewing the manuscript.


   Footnotes
 
1 This work was supported by the National Institutes of Health Grant AI29531. Back

2 J.A.B. and M.-L.A. are co-senior authors for this study. Back

3 Address correspondence and reprint requests to Dr. Maria-Luisa Alegre, University of Chicago, 5841 South Maryland Avenue, Room N07N, MC0930, Chicago, IL 60637. Back

4 Abbreviations used in this paper: Tc, cytotoxic T; C3H, C3H/HEN; B6, C57BL/6; MST, mean survival time; BALB/C.SCID, BALB/c ByJSmn-Prkdcscid; STAT6KO, STAT6 knockout. Back

Received for publication June 16, 2000. Accepted for publication August 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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