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The Journal of Immunology, 2003, 170: 853-860.
Copyright © 2003 by The American Association of Immunologists

IFN-{gamma} Production Is Specifically Regulated by IL-10 in Mice Made Tolerant with Anti-CD40 Ligand Antibody and Intact Active Bone

Dengping Yin1,*, Nadav Dujovny*, Lianli Ma1,*, Anncy Varghese*, JiKun Shen1,*, D. Keith Bishop{ddagger} and Anita S. Chong1,2,*,{dagger}

Departments of * General Surgery and {dagger} Immunology/Microbiology, Rush Presbyterian-St. Luke’s Medical Center, Chicago, IL 60612; and {ddagger} Departments of Surgery, and Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-{gamma} production that is donor specific, and a reduction in the frequency of IFN-{gamma}-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-{gamma} responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-{beta}, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-{gamma} response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-{gamma} production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-{gamma} levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-{gamma}. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-{gamma} production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite the development of new approaches and the clarification of the mechanisms of tolerance induction in murine models, transplantation tolerance remains an elusive clinical goal. Both central and peripheral mechanisms have been implicated in the induction of tolerance in mice. Central deletion of alloreactive cells has traditionally been thought to induce the most durable form of donor-specific tolerance. This type of tolerance requires the establishment of a chimeric state within the host, usually after the administration of donor bone marrow cells, which is responsible for deleting alloreactive cells in the thymus or bone marrow (1, 2). In the presence of this chimeric state, the host can accept donor organs without the need for immunosuppressive drugs (3). Although the approach of central deletional tolerance is conceptually simple, significant problems prevent its routine application in the clinic. These problems include the toxicity of the host conditioning that is generally necessary for consistent allogeneic marrow engraftment, and the occurrence of lethal graft vs host disease when HLA disparities are involved.

A number of approaches have been reported to induce peripheral tolerance; the most successful of these generally involves the partial inhibition of T cell function by blocking signal 1 or 2 (4, 5, 6, 7). These approaches are based on the basic science principle that inappropriate activation of T cells is not a neutral event, but induces prolonged hyporesponsiveness or anergy (8, 9, 10). Over the past decade, in vivo experiments have affirmed this principle, and inhibition of signal 2, with anti-CD40 ligand (CD40L)3 mAb and/or CTLA-4Ig, otherwise referred to as costimulation blockade, can result in prolonged allograft acceptance (6, 11, 12, 13, 14, 15). Dissection of the mechanisms by which costimulation blockade elicits allograft acceptance has led to many hypotheses, including the promotion of cytokine deviation, anergy, activation-induced cell death, and regulatory T cells (16, 17, 18, 19). There are varying degrees of support for each of these hypotheses, and it is possible that some or all of these events contribute to the induction or maintenance of tolerance.

Understanding the basis for tolerance is critical for the rational design of tolerance strategies and for the development of functional assays of tolerance for the clinic. Achieving these goals will depend on having a robust model of tolerance in which long-term allografts exhibit clinically acceptable standards of histology and function. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40L mAb treatment (250 µg/mouse daily from day 0–3, then q.o.d. from day 5–13 posttransplantation) to induce robust donor-specific allograft tolerance (20, 21). Tolerant mice accepted a second donor-specific heart (transplanted on day 60–90) and also donor-specific skin (transplanted on day 90–120), but rejected third-party skin (transplanted on day 90–120). More importantly, the histology of the transplanted allografts (as late as 270 days posttransplant) was normal, with minimal cellular infiltration and an absence of graft vasculopathy. Deposited alloantibodies in the allograft were also minimal, and allospecific B cell responses were tolerized (20, 21). Based on these early characterizations, we hypothesized that the combination of IAB and anti-CD40L mAb induces a robust model of allograft tolerance in which the basis of tolerance can be investigated. In this study, we report that tolerance induced by IAB and anti-CD40L mAb is associated with hyporesponsive IFN-{gamma} production that is actively regulated by IL-10.


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

C57BL/6, C3H, and BALB/c mice were purchased from Taconic (Germantown, NY) and maintained at the animal facility at Rush Presbyterian-St. Luke’s Medical Center. Spleen cells from IL-10-deficient BALB/c mice were a generous gift from A. Finnegan (Rush Presbyterian-St. Luke’s Medical Center). Heterotopic mouse hearts were transplanted into the abdomen of C57BL/6 recipients by anastomosing the donor aorta to recipient aorta, and the donor pulmonary artery to the recipient inferior vena cava. Second heart grafts were transplanted on day 60–90 post-first heart transplantation into the cervical area of the recipient by anastomosing the donor aorta to the recipient carotid artery, and the donor pulmonary artery to the recipient external jugular vein (end-to-side). The heart grafts were monitored daily until rejection; rejection was defined as complete cessation of pulsation. Some tolerant mice received C3H skin grafts at 90–120 days post-BALB/c heart transplant. Skin transplantation was performed by grafting full-thickness chest skin from C3H mice onto the flank of recipients, and securing with running 6-0 Ethicon sutures.

IAB transplantation and immunosuppression

The knee joints containing the heads of tibiae and femorae from the hind legs of BALB/c mice were harvested and cleaned of connective tissue. Each knee joint was cut with scissors into six to eight small fragments, and the fragments of one to two knee joints were transplanted under the kidney capsule of each recipient mouse on the day of cardiac allograft transplantation. Each knee joint contains ~1–2 x 107 bone marrow cells. Anti-CD40L (MR1) was administered from the day of transplantation at a dose of 250 µg/mouse i.v. from day 0–3; then i.p., q.o.d., from day 5–13.

Antibodies

Anti-CD40L (MR1), neutralizing anti-IL-10 (JES5 2A5), anti-IL-4 (BvD4-D11), anti-IL-10R (IB1.3a), anti-CTLA-4 (UC10-4F10-11), depleting anti-CD4 (GK1.5), CD8 (2.4.3), and CD25 (7D4) mAbs were purified from protein-free culture supernatants, precipitated by 45% ammonium sulfate, and dialyzed in PBS. The protein concentrations were determined by spectrophotometry (OD280) and compared with a standard curve of BSA. The purity of the mAb preparation was determined by SDS-PAGE analysis to be >90% pure. All of the hybridoma clones were from American Type Culture Colleciton (ATCC, Manassas, VA), with the following exceptions: UC10-4F10-11 was a gift from M.-L. Alegre (University of Chicago) with permission from J. Bluestone (University of California, San Francisco, CA); neutralizing polyclonal rabbit anti-TGF-{beta} Abs were purchased from R&D Systems (Minneapolis, MN); while anti-TGF-{beta} mAb (A411) and human CTLA-4Ig were generous gifts from P. Heeger (Cleveland Clinic, Cleveland, OH) and R. Peach (Bristol-Myers Squibb, New York, NY), respectively. The anti-IL-10R (IB1.3a) hybridoma cells were purchased from ATCC with permission from K. Moore (DNAX, Palo Alto, CA). All neutralizing Abs were used in vitro at 50 µg/ml. PE-conjugated anti-CD4 and fluorescein-conjugated anti-V{beta}5, anti-V{beta}11, and anti-V{beta}8 mAbs were purchased from BD PharMingen (San Diego, CA).

Analysis of alloantibody titers

Alloantibody titers were determined by flow cytometry, as previously reported (1, 2). Briefly, 1/100 dilutions of mouse serum were incubated with BALB/c lymph node cells for 1 h at 4°C, then cells were washed and incubated with PE-conjugated anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA) or fluorescein-conjugated anti-mouse IgG (Southern Biotechnology, Birmingham, AL). The mean channel fluorescence of the stained samples was determined by flow cytometry (FACScan; BD Biosciences, Mountain View, CA).

In vitro cytokine production

Splenic cells were prepared by centrifugation on a cushion of lymphocyte separation medium (Cellgro, Herndon, VA) and suspended in HL-1 medium (BioWhittaker, Walkersville, MD). Responder (5 x 106/ml) and stimulator cells (BALB/c or C3H spleen cells irradiated at 30 Gy; 5 x 106/ml) were incubated for 72 h, and the supernatants were harvested. Anti-IL-10, anti-TGF-{beta}, anti-IL-4, anti-IL-10R, anti-CTLA4-Ig or isotype controls, and CTLA4-Ig were added at 50 µg/ml for the duration of the experiment. The levels of IL-2 and IFN-{gamma} in the supernatant were quantified using ELISA OptEIATM kits from BD PharMingen.

IFN-{gamma} and IL-4 ELISPOT assay

The IL-2, IL-4, and IFN-{gamma} ELISPOT assays were performed as previously published (22). Briefly, ELISPOT plates were coated overnight with anti-IL-2 (JES6-1A12; 8 µg/ml), anti-IL-4 (11B11; 8 µg/ml), or IFN-{gamma} (R46A1; 8 µg/ml), respectively (BD PharMingen). The plates were blocked with PBS/1% BSA, then splenocytes (5 x 105/well) were added with BALB/c or C3H stimulators (2 x 105/well; 30 cGy) in a total volume of 200 µl/well HL-1 medium. Anti-IL-10, anti-TGF-{beta}, anti-IL-4, anti-IL-10R, anti-CTLA4-Ig or isotype controls, and CTLA4-Ig were added at 50 µg/ml at the initiation of the experiment. After 24 h, the plates were washed with PBS/0.025% Tween and probed with biotinylated anti-IL-2, anti-IL-4, and anti-IFN-{gamma} mAbs (JES6-5H4, BVD-24G2, or XMG1.2, respectively). After 25 h, the plates were washed and incubated with alkaline phosphatase-conjugated anti-biotin (Vector Laboratories, Burlingame, CA) for 2 h. The plates were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich, St. Louis, MO), and the resulting spots were counted by a computer-assisted Immunospot image analyzer (Cellular Technology, Cleveland, OH).

Cincinnati cytokine capture assay

The Cincinnati cytokine capture assay (CCCA) was used to measure serum levels of IFN-{gamma}, and IL-4 was performed as previously described (23). Mice were injected on the indicated postoperative days with 10 µg of the biotin-labeled anti-IFN-{gamma} or IL-4 mAb (R4-6A2 or BVD4-1D11) and bled 24 h later, and the sera were frozen at -70°C until use in ELISA. The cytokine/anti-cytokine mAb complexes in the serum were quantified in a standard ELISA. Briefly, 96-microwell plates were incubated overnight at 4°C with the appropriate nonneutralizing anti-cytokine mAb (AN-18 or BVD6-24G2.3) in PBS for 18–24 h. The plates were washed with PBS/0.025%Tween and blocked with PBS/1% BSA, and then samples of mouse sera were added at a 1/10 and 1/40 dilution. After 1.5 h at 27°C, the plates were washed and the avidin-HRP reagent was added to the wells. The plates were washed, and a 1:1 ratio of substrate A + B (BD PharMingen) was added to the wells. The reaction was stopped by the addition of 1 M H3PO4. Adsorbance (OD) was determined by an ELISA plate reader (Bio-Rad, Richmond, CA) at 450 nm. The cytokine concentrations were calculated by comparison against a standard curve of serially diluted preconjugated cytokine/anti-cytokine mAb complexes.

Immunohistochemical staining

Transplanted hearts were surgically removed and snap frozen in Tissue-Tek OCT (Sakura Finetek, Torrence, CA). Five-micron cryosections were cut serially, and one section of each tissue was stained with H&E for histologcal observation. The remaining sections were subjected to immunohistochemistry using the modified avidin-biotin peroxidase method. Briefly, microsections were fixed with cold acetone; endogenous peroxidase and Fc receptors were blocked with glucose-glucose oxidase and 5% goat serum, respectively. Primary Abs of anti-mouse IgM (R6-60.2), anti-mouse IgG (G1-6.5), anti-CD4 (H129-19), and anti-CD8{alpha} (53-6.7) were purchased from BD PharMingen. Biotinylated goat anti-mouse IgG was purchased from Jackson Immunochemicals (West Grove, PA), and HRP-conjugated streptavidin was purchased from Zymed Labs (South San Francisco, CA).

Statistical analysis

Statistical significance was determined utilizing an ANOVA using StatView (Abacus Concepts, Berkeley, CA) and a posthoc Student-Newman-Keuls test. A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Donor-specific IAB is necessary for induction of allograft tolerance

We have previously demonstrated that IAB synergizes with transient anti-CD40L mAb treatment (250 µg/mouse/day, i.v. daily from day 0–3; then i.p., q.o.d., from day 5–13) to induce allograft tolerance in the completely major and minor histocompatibility Ag-mismatched model of 129 x DBA/2 x C57BL/6 mice (H-2d x b) receiving C3H hearts and skin grafts (H-2k) (21). In this study, we extend those findings and demonstrate that IAB synergizes with anti-CD40L mAb to induce allograft tolerance in the completely major and minor histocompatibility Ag-mismatched combination of C57BL/6 mice receiving BALB/c hearts (Table I). In the absence of IAB, cardiac grafts are rejected in a mean time of 81 ± 11 days under anti-CD40L mAb treatment alone, while the cotransplantation of BALB/c IAB and anti-CD40L mAb resulted in significantly prolonged survival, with the majority of the grafts surviving for >137 days. Mice receiving IAB and anti-CD40L mAb with long-surviving grafts at 60–90 days accepted a second BALB/c heart graft for >49 days in the absence of additional immunosuppression. Alloantibodies remained low in tolerant mice receiving IAB and anti-CD40L, but were elevated in mice with rejected allografts (either untreated or anti-CD40L treatment only; Fig. 1D). Histology of the grafts revealed minimal lymphocytic infiltrate and no evidence of transplant vasculopathy as late as 137 days posttransplant (Fig. 1). In contrast, allografts rejected by mice treated with only anti-CD40L mAb at 60–70 days posttransplantation demonstrated significant levels of cellular infiltration, which is consistent with cellular rejection (data not shown). The effect of IAB was donor specific, as IAB from the recipient strain, C57BL/6, or third-party strain, C3H, did not result in enhanced prolongation compared with the group treated with anti-CD40L mAb alone (Table I; Student-Newman-Keuls, p > 0.05).


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  		Table I. Induction of long-term cardiac allograft survival with IAB cotransplantation and anti-CD40L mAb

 


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  		FIGURE 1. Histology of accepted BALB/c hearts on day 137 posttransplantation from C57BL/6 mice receiving BALB/c IAB and transient anti-CD40L mAb treatment (A). Immunohistochemistry revealed minimal CD4+ (B) and CD8+ T (C) cell infiltration in the accepted cardiac allografts. Alloantibody titers were elevated in untreated mice (None; day 6–7 posttransplant) and mice treated with only anti-CD40L (MR1; day 70–100 posttransplant) with rejected hearts (D). In contrast, alloantibodies remained low in tolerant mice receiving anti-CD40L and IAB (MR1 + IAB). Data are the mean of at least six mice/group and presented as means ± SEM.

 
Deletion is not the basis for tolerance induced by IAB and anti-CD40L mAb

A classic test for central deletion is a loss of V{beta}5- and V{beta}11-expressing CD4+ T cells in C57BL/6 mice with stable mixed chimerism of donor cells expressing I-E MHC class II molecules (22, 23). In BALB/c mice, V{beta}11+ and V{beta}5.1/2+ CD4+ T cells are normally deleted in the thymus as a result of their affinity for endogenous retroviral superantigens presented by I-E (24) (Fig. 2). C57BL/6 mice do not express I-E, and ~4–5% of CD4+ T cells are V{beta}11+, while 2–3% are V{beta}5+ (Fig. 2). We predicted that if BALB/c IAB mediated tolerance through central deletion, both V{beta}5+ and V{beta}11+ CD4+ T cells would be deleted in the tolerant mice. We observed no significant deletion of either V{beta}5+ or V{beta}11+ CD4+ T cells in the peripheral blood of tolerant mice receiving IAB and anti-CD40L mAb by day 60 or 100 posttransplantation (Fig. 2; Student-Newman-Keuls, p > 0.05). Similarly, there was no significant deletion of either V{beta}5+ or V{beta}11+ CD4+ T cells in the mice that rejected their grafts and received only anti-CD40L mAb (Student-Newman-Keuls, p > 0.05). Collectively, the absence of deletion of V{beta}5+ or V{beta}11+ CD4+ T cells in our model suggests that central deletion of alloreactive cells is unlikely to be the mechanism of tolerance in our model.



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  		FIGURE 2. Frequency of CD4+ cells expressing V{beta}11 and V{beta}5 (a) or V{beta}8 (b) in the peripheral blood, in naive C57BL/6 (B/6) or naive BALB/c (B/c) mice, IAB and anti-CD40L mAb tolerant mice (day 60 and 100 posttransplantation), or anti-CD40L mAb-treated rejecting mice (day 70–100 posttransplantation). Data are the mean ± SE of 5–20 individuals per group.

 
Tolerance is associated with a donor-specific suppression of IFN-{gamma} production in vitro

We next confirmed previous observations that splenic cells from tolerant C57BL/6 mice demonstrated a specific reduction in ability to produce IFN-{gamma} when cultured in vitro with donor-specific (BALB/c) stimulators (20, 21) (Fig. 3b). Positive controls were spleen cells from C57BL/6 mice treated with anti-CD40L mAb and rejected BALB/c hearts on days 60–80. Negative controls were spleen cells from naive C57BL/6 mice. IFN-{gamma} production by tolerant splenocytes stimulated by BALB/c splenocytes was significantly higher than that of naive C57BL/6 splenocytes, but significantly lower than that of positive control splenocytes (Student-Newman-Keuls, p < 0.05). There was no reduction in the production of IL-2 (Fig. 3a; Student-Newman-Keuls, p > 0.05), confirming that systemic deletion of donor-specific cells was not the mechanism of tolerance in our system. To test the specificity of this hyporesponsiveness, mice tolerant to BALB/c grafts were sensitized to third-party C3H skin grafts in vivo. Spleen cells from these mice sensitized with C3H skin grafts exhibited a primed IFN-{gamma} response to C3H stimulators (Fig. 3b), confirming that the defect in IFN-{gamma} production by splenocytes from tolerant mice was donor specific.



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  		FIGURE 3. IL-2 (a) and IFN-{gamma} (b) production by responder splenic cells from naive control C57BL/6 mice, IAB and anti-CD40L mAb tolerant mice (90–130 days posttransplantation), and anti-CD40L mAb-treated rejecting mice (day 90–100 days posttransplantation). Mice tolerant to BALB/c grafts were additionally sensitized to third-party C3H skin grafts and sacrificed on the day of rejection of C3H skin grafts, while mice that had rejected the BALB/c hearts did not receive C3H skin grafts. Responder spleen cells were cultured with medium (open bars) or stimulator cells (irradiated BALB/c (stripped bars) or C3H spleen cells (shaded bars)) for 72 h, then the supernatants were collected, and IL-2 and IFN-{gamma} were quantified by ELISA. All experiments were performed in triplicate, and data are the mean ± SE of 5–9 individuals per group. ELISPOT assays of IL-4 (c) and IFN-{gamma} (d) production by responder spleen cells from naive control C57BL.6 mice, IAB + anti-CD40L mAb tolerant mice (90–130 days posttransplantation), and anti-CD40L mAb-treated rejecting mice (day 90–100 days posttransplantation). Responder spleen cells were cultured with medium (open bars) or stimulator cells (irradiated BALB/c (stripped bars) or C3H spleen cells (shaded bars)) for 24 h, then the frequency of IL-4- or IFN-{gamma}-producing cells was enumerated by a computer-assisted Immunospot image analyzer. All experiments were performed in triplicate, and data are the mean ± SE of 7–10 individuals per group.

 
IFN-{gamma}-ELISPOT assays were performed to test whether the depressed IFN-{gamma} response observed in vitro was due to a reduction in the frequency of IFN-{gamma}-producing cells. We observed that spleen cells harvested from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, had a lower frequency of IFN-{gamma}-producing cells when stimulated with BALB/c stimulators, compared with positive control splenocytes (Student-Newman-Keuls, p < 0.05). Positive controls were recipients of BALB/c hearts and treated with anti-CD40L mAb only; these mice were not sensitized to C3H skin. The reduced frequency of IFN-{gamma}-producing cells in tolerant spleens was donor specific, as the same splenocyte population exhibited a normally primed IFN-{gamma} response to third-party C3H stimulators (Fig. 3d; Student-Newman-Keuls, p < 0.05). The frequency of IL-4-producing cells in the spleens of tolerant mice was comparable to the frequencies of IL-4-producing cells responding to third-party C3H stimulators or from spleens of mice that had rejected BALB/c hearts after anti-CD40L mAb treatment only (Fig. 3c; Student-Newman-Keuls, p > 0.05). Thus, these observations collectively suggest that tolerance in our model is specifically associated with depressed IFN-{gamma} production.

Donor-specific suppression of IFN-{gamma} production in vitro is reversed by neutralizing anti-IL-10 mAbs

A number of recent reports have implicated IL-10 and TGF-{beta} in the maintenance of the hyporesponsive state and long-term surviving allografts in mice and humans (25, 26, 27, 28). In addition, IL-10, CTLA-4, and TGF-{beta} have been identified to be important for the elaboration of regulatory activities of CD4+CD25+ cells in murine models of autoimmunity and transplantation (27, 28, 29, 30, 31). We therefore tested whether the depressed IFN-{gamma} production by splenic cells from tolerant mice could be reversed by neutralizing Abs against IL-10, TGF-{beta}, and CTLA-4. Only neutralizing Abs against IL-10 were able to restore the IFN-{gamma} production in response to BALB/c stimulators in the 72-h bulk culture assay (Fig. 4a; Student-Newman-Keuls, p < 0.05). Neutralizing anti-TGF-{beta} polyclonal Abs and mAbs, anti-CTLA-4 mAbs, CTLA-4Ig, and anti-IL-4 mAbs (Fig. 4a, and data not shown; Student-Newman-Keuls, p > 0.05) did not restore IFN-{gamma} production.



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  		FIGURE 4. Effect of neutralizing anti-IL-10 (JES5 2A5), anti-CTLA4 (UC10-4F10-11) mAbs, or anti-TGF-{beta} polyclonal Abs on IFN-{gamma} (a) production in 72-h bulk cultures, or on IL-2 (b), IFN-{gamma} (c), and IL-4 (d) production in 24-h ELISPOT assays. Responder spleen cells from tolerant mice were cultured with medium, irradiated BALB/c (B/c), or C3H spleen stimulator cells. Neutralizing Abs were used at 50 µg/ml, and the rest of the experiments were performed as described for Fig. 3. Data are the mean ± SE of five to eight individuals per group.

 
A similar observation with anti-IL-10 mAbs on IFN-{gamma} production emerged from the 24-h ELISPOT assay. In the presence of anti-IL-10 mAbs, the frequency of IFN-{gamma}-producing cells, stimulated by irradiated BALB/c splenocytes, was significantly elevated (Student-Newman-Keuls, p < 0.05), and restored to levels comparable to those of the positive control with C3H stimulators (Fig. 4c; Student-Newman-Keuls, p > 0.05). Similar results were observed with anti-IL-10R mAbs (Fig. 5). No restoration of IFN-{gamma} production was observed with neutralizing anti-TGF-{beta} polyclonal Abs or mAbs, anti-CTLA-4 mAbs, CTLA-4Ig, or anti-IL-4 mAbs (Figs. 4c and 5). The effect of anti-IL-10 mAbs on the IFN-{gamma} response was specific, as it did not enhance the frequency of IL-2- nor IL-4-producing cells (Fig. 4, b and d). These data demonstrate that the ability to induce IL-10 production was present in the tolerant mice and not elicited upon in vitro culture.



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  		FIGURE 5. IL-10-regulating IFN-{gamma} production is produced by tolerant responder spleen cells. Responder spleen cells from tolerant mice were cultured with medium, irradiated wild-type BALB/c, or IL-10-deficient BALB/c (IL-10Ko) spleen cells, as described in Materials and Methods. The frequency of IFN-{gamma}-producing cells was enumerated by a computer-assisted Immunospot image analyzer. Anti-IL-10R (IB1.3a; shaded bars), but not neutralizing anti-IL-4 (BVD4-1D11; stripped bars), mAb was able to restore IFN-{gamma} production. All experiments were performed in duplicate, and data are the mean ± SE of four individuals per group.

 
The IL-10 that is responsible for the suppression of IFN-{gamma} production could be produced by either the tolerant splenocytes themselves, or by the stimulator spleen cells under the influence of the tolerant spleen cells. To test these possibilities, tolerant spleen cells were stimulated with BALB/c spleen cells from IL-10-deficient mice. As demonstrated in Fig. 5, anti-IL-10R mAb was able to restore IFN-{gamma} production in the tolerant spleen population when IL-10-deficient BALB/c stimulators were used. Collectively, these observations suggest that the IFN-{gamma} response was not specifically deleted in tolerant mice, but was actively suppressed by IL-10 produced by recipient cells.

Tolerance is associated with a donor-specific suppression of IFN-{gamma} production in vivo

A deepening understanding that the immune response in vivo may be anatomically partitioned raises the possibility that investigations of ex vivo responses using splenic populations may not accurately reflect the in vivo situation. Reinhardt et al. (32) reported that memory T cells persisted as two separate populations: a small population in the lymph nodes that produced the IL-2, and a larger one in the nonlymphoid tissues that produced IFN-{gamma} upon rechallenge. In our model, it is possible that IFN-{gamma}-producing cells were sequestered in nonlymphoid tissues in tolerant mice and thus were not detected in the splenic populations used in the in vitro assays. Therefore, we tested whether tolerant mice exhibit suppressed IFN-{gamma} production in vivo. We used the newly developed CCCA assay to quantify IFN-{gamma} production in vivo over a 24-h period. In naive mice, we observed a dramatic increase in in vivo IFN-{gamma} production following challenge with donor-specific splenocytes (20 x 106, i.p.) or allogeneic hearts (Fig. 6, a and c). Peak IFN-{gamma} levels were 9.5 ng/ml on day 4–5 following BALB/c heart transplantation, and rapidly returned to baseline at the time of rejection (Fig. 6a), suggesting that the production of IFN-{gamma} during allograft rejection is tightly regulated. Peak IFN-{gamma} levels were 2.3 ng/ml on day 3–4, but remained elevated for up to 13–14 days following splenocyte immunization (Fig. 6c).



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  		FIGURE 6. In vivo production of IFN-{gamma} in naive C57BL/6 mice challenged with a second BALB/c heart (a) or immunized i.p. with 20 x 106 BALB/c spleen cells (c). Data are presented as the concentration of Ab/IFN-{gamma} complexes formed over a 24-h period at the indicated time posttransplantation or immunization. There were two to six individual mice for each time point. In vivo production of IFN-{gamma} over 24 h in control mice (empty bars) that received a first BALB/c heart, or tolerant C57BL/6 mice (filled bars) challenged on day 60–90 posttransplantation with a second BALB/c heart (b). In vivo production of IFN-{gamma} over 24 h in control mice (empty bars), or tolerant C57BL/6 mice (filled bars) immunized i.p. with 20 x 106 BALB/c spleen cells (d). Data are the mean ± SE of 4–10 individuals per group.

 
We challenged tolerant mice challenged with BALB/c splenocytes or second BALB/c hearts on day 60–90 after the first transplantation. The amount of IFN-{gamma} produced was measured before, and on days 3–4 and 6–7 after immunization with BALB/c splenocytes, and on day 3–4 after transplantation of BALB/c hearts. Tolerant mice challenged with BALB/c hearts or splenocytes made significantly reduced or undetectable IFN-{gamma} (Fig. 6, b and d). No IL-4 or IL-10 production was detected in tolerant or rejecting mice (data not shown). These data confirm our in vitro data that tolerance induced by IAB and anti-CD40L mAb is associated with a donor-specific suppression of the IFN-{gamma} response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported that the cotransplantation of IAB and anti-CD40L mAb induces allograft acceptance, and have formally demonstrated that IAB and anti-CD40L mAb induce tolerance because a second donor-specific heart is accepted. A unique feature of this model is that long-term allografts exhibit minimal cellular infiltration, and there are generally no signs of chronic rejection in the allograft even when examined as late as 240 days posttransplantation (20, 21). CTL responses were not detected in the spleen of tolerant mice (data not shown). This observation, together with the absence of or minimal T cell infiltration in the tolerant grafts, suggests an absence of primed CTL responses in our tolerant mice. The absence of obvious signs of inflammation and rejection in the allografts in our model provided a unique opportunity to examine the immunological features of a model of robust allograft tolerance.

Our previous studies revealed that IAB can reconstitute T and B cells in Rag-2-deficient mice, and that tolerance induced by IAB and anti-CD40L mAb in immunocompetent mice is associated with donor-cell microchimerism (21). We report in this study that the contribution of IAB to tolerance is donor specific, as cotransplantation of syngeneic or third-party allogeneic IAB was unable to synergize with anti-CD40L mAb to induce long-term allograft survival. However, two independent tests indicated that the tolerance we observe is not due to central deletion; there was no significant deletion of V{beta}5- and V{beta}11-expressing CD4+ T cells in the peripheral blood, and normal IL-2 production was observed with spleen cells from tolerant recipients. It is likely that insufficient hemopoietic cells emerged from the IAB in immunologically intact recipients to reshape the peripheral T cell repertoire and to induce central donor-specific tolerance. Our observations contrast with those of Bingaman et al. (33), who reported deletional tolerance when IAB was transplanted under the cover of anti-CD40L mAb and CTLA-4Ig, in the absence of vascularized hearts. The differences between our observations and those of Bingaman et al. might be due to the specific combination of mouse strains they used (C57BL/6-to-C3H), to their use of human CTLA-4Ig in addition to anti-CD40L mAb, or to the type of bone graft used. The ability to induce robust tolerance in the absence of central deletion, as observed in our system, is consistent with recent concepts that thymic negative selection alone cannot completely control self-reactive T cells, and that peripheral regulatory mechanisms are necessary (34).

Understanding how IAB induces tolerance requires a deeper understanding of how the tolerant state is maintained. In light of the absence of central T cell deletion, we hypothesized that the contribution of IAB to tolerance is not equivalent to classic models of tolerance induced by bone marrow transplantation (3, 35). We had previously reported a correlation between the occurrence of microchimerism, i.e., presence of donor cells in the peripheral blood as detected by PCR analysis, and tolerance induced by IAB and anti-CD40L mAb in the C3H-to-GT-Ko model (21). However, we were unable to demonstrate that a microchimeric state was essential and contributed directly to the tolerant state in that model. In this study, we analyzed for the occurrence of microchimerism in the current BALB/c-to-C57BL/6 model using the same molecular markers as in the C3H-to-GT-Ko model. However, we were not able to establish an association among IAB transplantation, graft acceptance, and microchimerism in the peripheral blood of tolerant recipients in the current model. This observation suggests a number of possibilities: first, that the level of detection of microchimerism was not sufficiently sensitive; second, that peripheral blood was not the ideal site to sample for microchimerism; or third, that microchimerism is not mechanism by which IAB contributes to tolerance. If the third possibility is correct, we speculate that the contribution of IAB to the development of a tolerant state may resemble the effects of donor-specific transfusion (4, 19). Studies to further define the mechanism of tolerance and the contribution of IAB are ongoing.

Ex vivo analyses of splenocytes from tolerant and rejecting C57BL/6 recipients revealed that, when cultured with BALB/c stimulators, rejection was associated with a primed IFN-{gamma} response, while tolerance was associated with a depressed IFN-{gamma} response. The same tolerant mice, primed by C3H skins, were able to exhibit primed IFN-{gamma} responses in vitro to third-party, C3H alloantigens, suggesting that suppression of IFN-{gamma} response was allo-Ag specific. The selective inhibition of IFN-{gamma} response in our model contrasts with a more generalized inhibition of in vitro immune responses in other models of tolerance. For instance, Hara et al. (28) reported that their model of tolerance was mediated by CD45RBlowCD4+ T cells. Their in vitro studies revealed that the CD45RBlowCD4+ T cells from tolerant mice were universally unresponsive to alloantigen, as measured by in vitro T cell proliferation and IL-2 and IFN-{gamma} production (28). The in vitro observations of Hara et al. resemble those seen with the classical T regulatory cells implicated in controlling autoimmunity (30, 31, 36, 37, 38, 39). Classical CD25+CD4+ T regulatory cells are anergic to stimulation via their TCR in vitro, and are also able to suppress the in vitro proliferation and IL-2 and IFN-{gamma} production of CD4+CD25- T cells, as well as the activation of CD8+ cells (31, 36, 40, 41, 42, 43, 44, 45). Our observations of the specificity of the hyporesponsiveness and that depletion of CD4+ or CD25+ T cells did not restore the IFN-{gamma} response (data not shown) are consistent with the conclusion that the mechanism of tolerance in our model differs from those mediated solely by classical CD4+CD25+ regulatory cells.

It is now apparent that in vivo immune responses are anatomically separated and that the spleen or lymph node may be preferentially depleted of effector, IFN-{gamma}-producing cells. Reinhardt et al. (32) reported that immune priming occurs in the peripheral lymphoid organs, and that effector cells rapidly migrate to sites of infection. Using immunohistology to visualize Ag-specific CD4+ T cells, they reported that naive CD4+ T cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes. After exposure to Ag under inflammatory conditions, the T cells proliferated in the peripheral lymphoid organs, but migrated out by day 11 to the lungs, liver, gut, and salivary glands. Over 21 days, the total number of Ag-stimulated T cells decreased, eventually leaving two stable populations of memory cells: one in the lymph nodes that produced the IL-2, and a larger one in the nonlymphoid tissues that produced IFN-{gamma} upon rechallenge. Based on this information, we reasoned that it was critical to test whether the depressed IFN-{gamma} response we observed ex vivo is also observed in tolerant mice in vivo. We observed that tolerant mice exhibited a profoundly muted IFN-{gamma} response to BALB/c hearts or splenocytes compared with nontolerant mice. To our knowledge, these are the first formal demonstrations that alloreactive IFN-{gamma}-producing cells are not sequestered in nonsplenic sites in tolerant mice and that IFN-{gamma} hyporesponsiveness, measured ex vivo or in vivo, consistently correlates with allograft acceptance.

The muted IFN-{gamma} response to donor alloantigen in tolerant mice could reflect a loss in IFN-{gamma}-producing cells as a result of immune deviation, deletion, or anergy. A second possibility is that IFN-{gamma}-producing cells are present in tolerant mice, but are actively suppressed. Ex vivo analysis revealed that spleen cells from tolerant mice exhibited primed IL-4 responses that were comparable to spleen cells from C57BL/6 mice that had been treated with anti-CD40L mAb alone, and that rejected their grafts. In addition, neutralizing anti-IL-4 mAbs were unable to reverse the in vitro hyporesponsiveness, which further suggested that immune deviation to Th2 cells per se is unlikely to be the mechanism of tolerance. The possibility that active suppression is a mechanism of tolerance in our model is consistent with an increasing body of literature showing that graft acceptors exhibit active regulation of alloreactivity. Cobbold et al. (46) first described allo-specific T regulatory cells following the passive transfer of splenocytes from tolerant mice into naive mice, preventing rejection of allogeneic hearts. VanBuskirk et al. (47) reported that long-term allograft acceptance was associated with an inability to mount donor-reactive delayed-type hypersensitivity (DTH) responses, and the ability to inhibit bystander third-party Ag-DTH responses. Subsequent studies revealed that DTH responses could be independently uncovered with anti-TGF-{beta} and/or anti-IL-10 Abs (25). Significantly, similar IL-10- and TGF-{beta}-dependent immune regulation of DTH responses was also observed with PBLs from transplant patients who had accepted allografts in the absence of immunosuppression (26). More recently, Wood and her colleagues (27, 28) have isolated a subset of CD4+ cells, coexpressing CD45RBlow and/or CD25+ from mice made tolerant with anti-CD4 and donor-specific spleen cells. They found that these cells could inhibit rejection initiated by CD4+CD45RBhigh or CD4+CD25- cells. The regulatory activities of these CD4+CD45RBlow and CD4+CD25+ T cells in vivo were also dependent on IL-10 and CTLA-4, but independent of IL-4 (27, 28). IL-10 and TGF-{beta} are cytokines with well-documented anti-inflammatory and immunosuppressive activity, while CTLA-4 can down-regulate T cell responses. Thus, the hypothesis that production of IL-10 and TGF-{beta} and that the expression of CTLA-4 represent distinct mechanisms by which CD4+CD25+ regulatory T cells mediate graft acceptance has quickly become accepted as possible bases of regulatory tolerance.

Hyporesponsive IFN-{gamma} production in mice made tolerant with IAB and anti-CD40L mAb was reversed by anti-IL-10, but not anti-TGF-{beta} or anti-CTLA-4, mAbs. The effect of anti-IL-10 mAb on the IFN-{gamma} response was specific, as it had no effect on in vitro IL-2 and IL-4 production. Involvement of IL-10 as an active regulator of alloreactivity is consistent with other investigations of tolerance (25, 26, 27, 28, 48). However, in contrast to their observations, inhibition of TGF-{beta} or CTLA-4 had no effect. At this stage, it is unclear whether the differences reflect different assay systems, different mechanisms of tolerance, or both. We used in vitro IFN-{gamma} production as our assays of immune function and tolerance, while the ex vivo DTH assay and CD4+CD25--mediated skin rejection were used in the other reports (25, 26, 27, 28, 48). In addition, our ex vivo experiments are primarily testing regulation by the direct pathway, without specific analysis of the indirect pathway. Thus, it is possible that an entirely different mechanism is regulating the indirect pathway, and this mechanism may predominate in vivo and in their systems. Third, IFN-{gamma} is produced primarily by CD8+ > CD4+ cells in our system (Chong, unpublished data), whereas the effector cells are CD4+ cells in the previously reported systems. Preliminary data suggest that different assay systems can unveil different mechanisms of regulation (Orosz, Bishop, and Chong, unpublished data). Additional experiments are ongoing to define the basis of tolerance induced by IAB and anti-CD40L.

In summary, we have identified a model of allograft tolerance induced by IAB and anti-CD40L mAb in which allograft hearts exhibit normal histology, and demonstrated that tolerance is not due to deletional mechanisms, but is associated with active suppression of IFN-{gamma} production. In this tolerance mode, proliferative responses, IL-2 production, and IL-4 priming occur normally. Active suppression of IFN-{gamma} production is mediated by IL-10, but not by TGF-{beta}, CTLA-4. These data suggest that the mechanism of allograft tolerance in our model may differ from the recently described regulatory mechanisms mediated by CD4+CD25+ T cells.


   Acknowledgments
 
We thank Drs. Ian Boussy and Charley Orosz for helpful discussions, and Debra Mares for proofreading the manuscript. We thank Dr. Fred Finkelman for assistance in developing the CCCA assay, and Drs. Peter Heeger and Anna Valujskikh for assistance in the cytokine ELIPSOT assays and the loan of the Immunospot image analyzer.


   Footnotes
 
1 Current address: Section of Transplantation, Department of Surgery, The University of Chicago, Chicago, IL 60637. Back

2 Address correspondence and reprint requests to Dr. Anita S. Chong, Section of Transplantation, Department of Surgery, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: achong{at}surgery.bsd.uchicago.edu Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; CCCA, Cincinnati cytokine capture assay; DTH, delayed-type hypersensitivity; IAB, intact active bone. Back

Received for publication May 6, 2002. Accepted for publication November 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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