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The Journal of Immunology, 2000, 164: 3627-3634.
Copyright © 2000 by The American Association of Immunologists

Anti-LFA-1 Therapy Induces Long-Term Islet Allograft Acceptance in the Absence of IFN-{gamma} or IL-41

Mark R. Nicolls2,*, Marilyne Coulombe2,{dagger}, Huan Yang{dagger}, Amy Bolwerk{dagger} and Ronald G. Gill3,{dagger}

* Division of Pulmonary Sciences and Critical Care Medicine, and {dagger} Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences University, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
mAb therapy directed against a variety of cell surface accessory molecules has been effectively utilized to prolong allograft acceptance in various models of tissue and organ transplantation. The purpose of this study was to determine whether transient therapy directed against the adhesion molecule LFA-1 (CD11a) was sufficient to induce donor-specific tolerance to pancreatic islet allografts. Anti-LFA-1 monotherapy was found to be efficacious in inducing long-term islet allograft acceptance in multiple donor-recipient strain combinations. Graft acceptance following anti-LFA-1 therapy was not simply due to clonal ignorance of donor Ags in that the majority of recipients bearing established islet allografts resisted rejection induced by immunization with donor-type APCs. Furthermore, donor-specific tolerance from anti-LFA-1-treated animals could be transferred to secondary immune-deficient animals. Taken together, these results indicated that transient anti-LFA-1 monotherapy resulted in donor-specific tolerance. In vitro, functionally tolerant animals retained normal anti-donor reactivity as assessed by proliferative, cytotoxic, and cytokine release assays that demonstrated that tolerance was not secondary to general clonal deletion or anergy of donor-reactive T cells. Finally, anti-LFA-1 treatment was effective in both IL-4-deficient and IFN-{gamma}-deficient recipients, indicating that neither of these cytokines are universally required for allograft acceptance. These results suggest that anti-adhesion-based therapy can induce a nondeletional form of tolerance that is not overtly dependent on the prototypic Th1 and Th2 cytokines, IFN-{gamma} and IL-4, respectively, in contrast to results in other transplantation models.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell adhesion is mediated by multiple receptor-ligand interactions that are critical in morphogenesis, wound healing, the integrity of solid tissues, and host defense (1, 2). These receptors include two adhesion molecule families: the integrin family and the Ig superfamily. LFA-1, a ß2 integrin, has three known ligands (ICAM-1, -2, and -3) that are members of the Ig superfamily (3). mAb therapy directed against LFA-1 has been utilized in various animal models and in human trials (reviewed in Ref. 4). Isobe et al. (5) demonstrated that transient-combined treatment with Abs directed against both LFA-1 and its ligand ICAM-1 triggered profound prolongation and tolerance to cardiac allografts. Other studies since have demonstrated the efficacy of anti-LFA-1 and/or anti-ICAM-1 therapy in experimental and clinical transplantation of islets (6, 7), kidney (8, 9), bowel (10, 11), liver (12, 13), bone marrow (14, 15), and heart (15, 16, 17).

The mechanism of graft prolongation following therapy targeting LFA-1 remains controversial. Anti-LFA-1 therapy could act by interfering with LFA-1-dependent processes such as cell motility, migration, and phagocytic responses (18). However, in addition to its influence on cellular morphology and trafficking, LFA-1 also facilitates T cell activation, possibly by lowering the threshold levels of Ag necessary for T cell activation (19). Thus, anti-LFA-1 therapy may increase the antigenic stimulus required for T cell activation. In addition, LFA-1/ICAM-1 interaction is implicated as a costimulatory signal for TCR-mediated activation of resting T cells (20, 21). It is also conceivable that Abs that engage LFA-1 induce differential signaling in addition to inhibiting T cell activation. For example, blocking LFA-1/ICAM-1 or LFA-1/ICAM-2 interactions in vitro can lead to increased Th2 cytokine (IL-4 and IL-5) production, suggesting that in vivo therapy could lead to similar immune deviation (22). Therefore, perturbing LFA-1 activity in vivo may have varied effects other than blockade of cell adhesion. Thus, the goal of this study was to determine the cytokine requirements and anti-donor status of mice tolerized to pancreatic islet allografts by anti-LFA-1 therapy in vivo.

Of particular interest is the contribution of various cytokines to the induction and/or maintenance of transplantation tolerance. Acutely rejecting tissues are typically characterized by elevated proinflammatory Th1-like cytokines (23, 24, 25), while transplantation tolerance has often been associated with reduced Th1 cytokines or the emergence of Th2 cytokines (26, 27, 28, 29, 30, 31). Although Th1 and Th2 cells have been postulated to explain allograft rejection and tolerance, respectively, this simple paradigm has been called into question (25). For example, Th2 cytokines (e.g., IL-4 and IL-10) can also be elevated in rejecting tissues (32, 33). The use of neutralizing Abs and the development of defined cytokine-deficient animals have allowed further examination of cytokine requirements for graft rejection and acceptance. Some studies implicate a requirement for IL-4 in allograft tolerance (33, 34, 35), while others indicate that this cytokine is not necessary for long-term graft acceptance (36, 37). More recent evidence unexpectedly has indicated that the Th1 cytokine IFN-{gamma} can be required for long-term skin and cardiac allograft survival induced by costimulation blockade (31, 38). These studies indicated that allograft acceptance depended on the presence of IFN-{gamma} in that either treatment of wild-type animals with anti-IFN-{gamma} mAb or grafting IFN-{gamma}-deficient recipients abrogated graft prolongation. Our present results indicate that short-term anti-LFA-1 therapy does lead to the induction of donor-specific tolerance and that long-term graft acceptance does not require either IL-4 or IFN-{gamma}. Thus, neither of these prototypical Th2 or Th1 cytokines are required for allograft survival in this model.


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

Male C57BL6/J (B6, H-2b), C3H/HeJ (C3H, H-2k), CBA/J (CBA, H-2k), and BALB/cByJ (BALB/c, H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Severe combined immune-deficient C.B-17 scid/scid (SCID, H-2d) mice were provided by L. Schultz and bred at the Barbara Davis Center Rodent Facility (Denver, CO). IL-4-/- and IFN-{gamma}-/- gene knockout BALB/c mice were also obtained from The Jackson Laboratory.

mAbs and treatment protocols

Animals received one of the following protocols: anti-LFA-1 (KBA; rat IgG2a, cell line generously provided by Dr. Ihara, Charlestown, MA), 100 µg/day i.p. on days 0–6 (day 0 being the day of transplantation); or control treatment with 100 µg/day i.p. of rat IgG (Sigma, St. Louis, MO) on days 0–6. KBA ascites was generated in SCID mice and quantitated using an isotype (rat IgG2a)-specific ELISA. Purified IgG2a standard, capture, and detection Abs were purchased from PharMingen (San Diego, CA). Where indicated, recipients were also treated with a short course of the depleting anti-CD8 mAb 2.43 (10 mg/kg days -1 through +2 relative to transplant), as previously described (39).

Islet transplantation

Islets were isolated from adult mouse pancreata by collagenase (Sigma type V) digestion (40) and Ficoll purification (41). The islets were handpicked and transplanted under the left kidney capsule utilizing one of two methods: (1) suspended in several microliters of host blood, as previously described (42), or (2) via silastic tubing. The latter method utilized an islet micrometer handmachined and generously provided by Dr. R. Rajotte (Edmonton, Alberta, Canada). With this method, islets were drawn up into PE-50 silastic tubing (Becton Dickinson, Sparks, MD) and concentrated by centrifugation of the tubing. The tubing was then inserted under the kidney capsule, and the micrometer was utilized to advance the islets. Results obtained using both methods were comparable. Mice rendered diabetic (a minimum of two consecutive blood glucose values = 20 mM) by the i.v. injection of 200–275 mg/kg streptozotocin (Calbiochem, La Jolla, CA) were used as allograft recipients. Diabetic recipients were subsequently grafted with 450 islets under the left kidney capsule, as described above.

Assessment of tolerance

Graft function was assessed by monitoring blood glucose weekly with a Medisense (Waltham, MA) blood glucose meter. At 60 days, BALB/c mice with functioning B6 allografts (blood glucose <10 mM) were challenged with 1 x 106 donor-type spleen cells i.p. Blood glucose was monitored two to three times/week following this challenge. Animals that resisted challenge (i.e., remained euglycemic) >30 days following immunization were considered tolerant animals. At this point, nephrectomy of the graft-bearing kidney was performed to definitively determine that euglycemia was graft dependent. Unchallenged mice that had allografts surviving for >100 days were also considered tolerant if their spleen cells were capable of transferring tolerance to SCID mice bearing recent donor-type islet allografts.

Adoptive transfer of tolerance

C.B-17 SCID mice, rendered diabetic with streptozotocin, were grafted with 450 islets from either donor B6 (H-2b), or third-party C3H (H-2k) mice, as outlined above. At 3–7 days after transplantation, recipient mice were injected i.p. with 3 x 107 unfractionated spleen cells from tolerant unchallenged BALB/c mice bearing C57BL/6 islet allografts (>100 days after transplant) or from naive age-matched controls. Weekly blood glucoses were monitored after immune reconstitution. Rejecting allografts were harvested after a minimum of two blood glucose readings >10 mM and examined histologically. The graft-bearing kidneys of mice with functioning allografts were removed 60 days postreconstitution, and the blood glucose of these nephrectomized animals was monitored for the return to hyperglycemic values.

Histology

At the conclusion of each study, kidneys bearing rejecting allografts or those removed by nephrectomy were fixed in 10% buffered Formalin. Paraffin sections were stained with hematoxylin-eosin and, in parallel sections, insulin granules were detected with aldehyde fuchsin or immunoperoxidase staining for insulin and glucagon. Tissue sections were examined to determine the degree of tissue damage and mononuclear cell infiltration of the graft.

T lymphocyte proliferation and cytotoxicity

The MLR was established by mixing 2 x 106/ml lymph node or spleen cell responders from BALB/c mice with 3 x 106/ml 2000 rad gamma-irradiated donor-type (B6) or third-party strain (C3H) splenic stimulator cells in a total of 0.2-ml cultures in 96-well flat-bottom plates. Cells, cultured in Eagle’s MEM supplemented with 10% FCS, 10-5 M 2-ME, and antibiotics, were incubated at 37°C in 10% CO2. Proliferative responses were determined by pulsing the culture wells with 1.25 µCi of 3H for 6 h, harvesting, and counting samples on a Micromedic Plus beta counter (Micromedic Systems, Horsham, PA). For CTL assays, primary MLCs were established by mixing 2 x 106/ml lymph node or spleen cell responders from BALB/c mice with 3 x 106/ml 2000 rad-irradiated B6 splenic stimulator cells in a total of 2 ml in 24-well flat-bottom plates. CTL activity in vitro was assessed by a standard 51Cr release assay, as described (39). Briefly, on day 5 of the primary MLR, limiting dilutions of effector T cells were incubated with 104 51Cr-labeled EL-4 (H-2b) or R1.1 (H-2k) tumor target cells for 4 h at 37°C in 10% CO2. Supernatants were harvested and 51Cr release detected on a Micromedic Plus gamma counter. Cytotoxic activity was expressed as a percentage of specific lysis.

Cytokine assays

Supernatant of anti-donor and anti-third-party MLCs utilizing tolerant and naive lymph node or spleen cells as responders was collected on days 1 through 5 of culture. Test supernatants were then assayed for IL-2, IL-4, IL-10, and IFN-{gamma} with a solid-phase enzyme immunoassay (ELISA). Specific recombinant cytokine standards capture and detection Abs (PharMingen) were utilized to estimate a standard curve for individual cytokines. Inactivation of IL-4 and IFN-{gamma} gene function in gene knockout animals was confirmed by performing IL-4- and IFN-{gamma}-specific ELISA. Specifically, the absence of IL-4 was confirmed using spleen cells from IL-4-/--deficient or control mice stimulated in primary allogeneic culture for 7 days in the presence of mouse rIL-4 (1 ng/ml; PharMingen) and anti-IFN-{gamma} (20 µg/ml; XMG, PharMingen). Blasts were washed three times in HBSS and then stimulated with Con A (2.5 µg/ml). Culture supernatants were collected at 24 h and assayed for IL-4 and IL-2 as positive controls. To confirm the absence of IFN-{gamma} in gene knockout animals, spleen cells from IFN-{gamma}-/- and control mice were stimulated for 72 h in the presence of 2.5 µg Con A/ml. Supernatants were analyzed for IFN-{gamma} and IL-2 at 24, 48, and 72 h. Neither IL-4 nor IFN-{gamma} was detected in supernatant from IL-4-/- and IFN-{gamma}-/- mice, respectively, but was present in supernatant derived from control animals. Importantly, both types of knockout animals proliferated well in culture with levels of IL-2 greater than or equal to control levels.

Statistics

Mann-Whitney U tests and/or Fisher’s Exact tests were used to compare graft survival between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-LFA-1 is efficacious in prolonging islet allograft survival in several strain combinations

In a murine cardiac allograft model, Isobe et al. (5) demonstrated long-term survival and donor-specific tolerance using a combination of Abs to LFA-1 and ICAM-1. In that study, indefinite graft acceptance required targeting of both of these molecules. However, our own pilot studies indicated that anti-LFA-1 monotherapy was actually superior to combined therapy with anti-ICAM Abs at inducing long-term islet allograft survival (data not shown). Thus, all subsequent experiments used anti-LFA-1 monotherapy to facilitate islet allograft prolongation. A 7-day course of anti-LFA-1 therapy led to long-term islet allograft survival (>100 days) in all three strain combinations tested (Fig. 1Go): BALB/c->CBA, C57BL/6->BALB/c, and BALB/c->C57BL/6. By contrast, the majority of animals treated with a control rat IgG Ab rejected their allografts within 3 wk of grafting, a result indistinguishable from untreated control animals (Fig. 1Go). However, despite the same dosing regimen for each strain combination, the percentage of animals with long-term functioning grafts at 100 days posttransplant varied somewhat between strains. While LFA-1 therapy led to long-term islet allograft survival in 89% of CBA recipients bearing BALB/c grafts (Fig. 1GoA), and in 82% of BALB/c mice bearing B6 grafts (Fig. 1GoB), results were less pronounced in B6 recipients, in which only 38% of BALB/c grafts survived >100 days (p < 0.01; Fig. 1GoC). Determination of the clearance of the rat KBA IgG2a anti-LFA-1 Ab in treated recipients did not indicate a dramatic difference in the pharmacokinetics between BALB/c and B6 mice. Also, increasing the dose of anti-LFA-1 to 200 or 300 µg/day or giving a more protracted course of Ab therapy did not lead to a higher rate of long-term allograft acceptance in B6 recipients (data not shown), suggesting that B6 animals are inherently more resistant to anti-LFA-1-induced allograft prolongation than the other strains tested. Thus, while anti-LFA-1 therapy demonstrated efficacy in all strains tested, there were significant strain-specific differences observed. A recent study by Trambley et al. has strongly implicated CD8 T cells as contributing to the relative resistance of B6 mice to allograft tolerance (43). However, our own pilot studies using a combination of anti-LFA-1 and anti-CD8 did not result in significant prolongation of BALB/c islet allografts in B6 recipients. Combination therapy using anti-LFA-1 plus anti-CD8 mAbs resulted in long-term survival (>100 days) in three of six recipients, a result that does not differ from data shown in Fig. 1GoC.



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  		FIGURE 1. Anti-LFA-1 monotherapy is efficacious in several strain combinations. Four hundred and fifty islets were grafted beneath the kidney capsule of streptozotocin-induced diabetic recipients in the indicated strain combinations: A, BALB/c (H-2d)-bearing CBA (H-2k) islets (KBA vs rat IgG-treated groups, p < 0.02); B, B6 (H-2b)-bearing BALB/c (H-2d) islets (KBA vs rat IgG-treated groups, p < 0.0001); and C, BALB/c (H-2d)-bearing B6 (H-2b) islets (KBA vs rat IgG-treated groups, p < 0.0001). Recipients were treated with 100 µg/day of either anti-LFA-1 (KBA, {circ}) or rat IgG (•) on days 0–6 relative to transplantation. Euglycemia (blood glucose values <10 mM) was indicative of allograft survival, and islet graft rejection was indicated by a minimum of two consecutive blood glucose values above 10 mM. Removal of the graft-bearing kidney in recipients of islets functioning >100 days resulted in hyperglycemia, demonstrating that normoglycemia was graft dependent.

 
Anti-LFA-1-induced islet allograft acceptance is not due to T cell clonal ignorance

A key question was whether anti-LFA-1 mAb therapy simply blocked cellular adhesion and/or lymphocyte trafficking to the graft. If this were so, then graft prolongation could be explained by immunologic ignorance (44), meaning that the host was neither sensitized nor tolerized by the allograft. To test this possibility, a cohort of anti-LFA-1-treated BALB/c recipients bearing B6 islet allografts was immunized with 106 donor-type B6 spleen cells as a source of immunogenic APCs ~60 days posttransplantation (Table IGo). Although this immunization triggered rejection of the established graft in some animals, the majority of animals maintained functioning allografts despite donor APC immunization indicating that long-term allograft acceptance was not simply due to ignorance of the allograft. Most B6 recipients with long-term (>100 days) functioning BALB/c islet allografts also resisted graft rejection when immunized with donor-type BALB/c spleen (Table IGo), demonstrating a similar property in this strain combination. In contrast, we have previously found that this dose of donor APCs can consistently trigger acute rejection of APC-depleted islet allografts in nontolerant animals (45, 46). Finally, histology of tolerated grafts universally demonstrated focal mononuclear accumulations adjacent to the allograft, further indicating that the host was not simply ignorant of the grafted tissues (Fig. 2Go).


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  		Table I. Anti-LFA-1-treated allograft recipients resist graft rejection induced by immunization with donor APCs

 


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  		FIGURE 2. Histological assessment of islet (I) allograft survival in various strain combinations following anti-LFA-1 therapy. A, Donor-type B6 islets placed beneath the kidney capsule of diabetic anti-LFA-1-treated BALB/c mice with infiltrating mononuclear cells (MNC) >100 days posttransplantation. B, Donor-type B6 islets are rejected in rat IgG-treated BALB/c hosts. C, Donor-type B6 islets in C.B-17 SCID mice are accepted after reconstitution with spleen cells from tolerant anti-LFA-1-treated BALB/c hosts bearing B6 grafts. D, Third-party C3H islets are rejected after reconstitution with spleen cells from tolerant anti-LFA-1-treated BALB/c hosts bearing B6 grafts. E, Donor-type B6 islets are accepted (>100 days posttransplant) in anti-LFA-1-treated IL-4-/- BALB/c hosts. F, Donor-type B6 islets are accepted (>100 days posttransplant) in anti-LFA-1-treated IFN-{gamma}-/- BALB/c hosts.

 
Anti-LFA-1-induced allograft acceptance can be adoptively transferred to SCID mice in a donor-specific manner

The finding that the majority of hosts resisted allograft rejection despite active immunization with donor-type APCs suggested an alteration of anti-donor reactivity. We then used adoptive transfer experiments to determine whether this change was due to donor-specific tolerance in anti-LFA-1-treated BALB/c mice bearing long-term functioning B6 islet allografts. Streptozotocin-induced diabetic immunodeficient C.B-17 scid (H-2d) mice were transplanted with either donor-type B6 or third-party C3H islet allografts and then were used as adoptive transfer recipients of either control or putatively tolerant BALB/c spleen cells. When SCID recipients were reconstituted with spleen cells from BALB/c mice bearing B6 islet allografts >100 days, the majority of donor-type B6 islet allografts were accepted for >60 days, while third-party C3H grafts were rejected with normal kinetics (Fig. 3Go). By contrast, animals reconstituted with naive spleen cells rejected the majority of both B6 and C3H islet transplants, respectively. Both B6 and C3H islet allografts functioned for >100 days in nonreconstituted SCID recipients, illustrating the immune-deficient status of SCID hosts (data not shown). Given that anti-LFA-1-treated animals resisted rejection when immunized with host APCs and were able to specifically transfer this state to secondary SCID mice, long-term allograft acceptance was considered to be a result of donor-specific tolerance.



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  		FIGURE 3. Anti-LFA-1-induced allograft tolerance can be adoptively transferred to SCID mice. Tolerance was induced in BALB/c mice (H-2d)-bearing B6 (H-2b) islet allografts with 100 µg of anti-LFA-1 administered i.p. on days 0–6 relative to transplantation. Animals with functioning grafts >100 days posttransplant or age-matched control BALB/c mice were used as spleen cell donors for adoptive transfer. Streptozotocin-induced diabetic C.B-17 scid mice (H-2d) received 450 donor-type (B6, H-2b) or third-party strain (C3H, H-2k) islet allografts beneath the renal subcapsular space. Within 3–7 days of grafting, SCID mice were reconstituted with an i.p. injection of 3 x 107 tolerant (open symbols) or age-matched naive (filled symbols) BALB/c spleen cells. Allograft survival (euglycemia) was monitored with frequent blood glucose testing, and graft rejection was indicated by a minimum of two consecutive blood glucose values = 10 mM. The transfer of donor-specific tolerance was demonstrated as tolerant cells resulted in the maintenance of donor-type grafts (6/7 surviving) and the rejection of third-party islets (1/11 surviving; donor-type vs third party, p < 0.0065). No survival differences were noted in B6 (2/12) or C3H (0/4) allografts in mice reconstituted with naive spleen cells (p = 1).

 
Anti-LFA-1-induced allograft tolerance is not associated with demonstrable clonal deletion or anergy

To determine whether tolerance was secondary to the induction of generalized clonal deletion or anergy of donor-reactive cells, anti-donor proliferative responses, cytotoxic reactivity, and cytokine release were assessed in vitro. Relative to naive animals, spleen cells from tolerant animals exhibited control levels of anti-donor (B6) responses in MLR and CTL assays (Fig. 4Go, A and B), demonstrating the presence and activity of donor-reactive T cells in tolerant mice. Anti-donor proliferative and cytotoxic responses were also comparable with responses directed against third-party (C3H) stimulator cells (data not shown). Similar results were found for anti-donor responses from lymph node cells from tolerant and naive BALB/c mice (data not shown). These findings in long-term tolerant animals were also noted for anti-LFA-1-treated BALB/c mice that had been immunized with donor-type (B6) APCs 60 days posttransplantation (data not shown). The finding of normal levels of in vitro donor reactivity in tolerant animals suggests that generalized clonal deletion or anergy is not required for tolerance and that this state is more consistent with an active mechanism of regulatory tolerance in vivo. To determine whether a demonstrable cytokine deviation had occurred in the anti-donor response, tolerant and naive BALB/c spleen cells were tested for anti-donor cytokine release of IL-2, IL-4, IFN-{gamma}, and IL-10 in vitro. To ensure that the peak responses of cytokines were detected, culture supernatants were assayed for cytokine production from days 1 through 5 of MLC. Although absolute levels of cytokines varied between experiments, donor-reactive T cells continued to produce control levels of IL-2 and IFN-{gamma} (Fig. 4GoC) and failed to produce significant levels of either IL-4 or IL-10 (not shown) in five separate experiments. Thus, no obvious cytokine deviation could be detected in donor-reactive T cells in vitro as defined by the cytokines examined.



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  		FIGURE 4. Anti-LFA-1-induced tolerant spleen cells exhibit normal anti-donor MLR and CTL reactivity in vitro. Spleen cells were harvested from either anti-LFA-1-treated BALB/c mice bearing B6 islet allografts for >100 days or age-matched control BALB/c mice. A, Proliferative responses. In MLR cultures, 2 x 105 BALB/c spleen cells were incubated with 3 x 105 gamma-irradiated B6 stimulator cells. Cultures were pulsed with 1.25 µCi of [3H]thymidine 6 h before harvesting. Anti-donor (B6, H-2b) proliferative responses of tolerant ({circ}) and naive (•) spleen cells are shown on days 3, 4, and 5 of culture. B, CTL responses. After 5 days of primary culture with B6 stimulators, CTL activity of tolerant ({circ}) and naive (•) BALB/c spleen cells was measured in a standard 4-h 51Cr release assay using EL-4 (H-2b) target cells. MLR and CTL data are representative of several replicate experiments. Error bars indicate the SE of triplicate or quadruplicate wells. Similar proliferative and cytotoxic responses were obtained with tolerant lymph node cells and with third-party strain (C3H, H-2k) stimulators. C, IFN-{gamma} production. MLC was established from tolerant or naive animals against donor-type (B6) or third-party (C3H) stimulator cells and assayed for cytokine release on days 1 through 5 of culture. Data shown are the mean ± SEM of peak IFN-{gamma} release (day 4).

 
Anti-LFA-1-induced allograft prolongation does not require either IL-4 or IFN-{gamma}

Because in vitro assessment of cytokine deviation did not reveal a cytokine profile different to control animals, cytokine knockout animals were used as hosts to delineate requirements for anti-LFA-1-induced graft prolongation. To this end, mice lacking the prototypic Th1 cytokine IFN-{gamma} or Th2 cytokine IL-4 were used as allograft recipients. Prior studies have shown that IL-4 is important in some models of allograft tolerance (33, 34, 35), but not others (33, 36, 37), and that IFN-{gamma} is required for allograft prolongation in model systems utilizing anti-costimulation therapy (31, 38). Our results indicated that anti-LFA-1 therapy was effective in both IL-4-/- and IFN-{gamma}-/- BALB/c recipients. Four of five BALB/c IL-4-/- mice grafted with B6 islets and treated with anti-LFA-1 had functioning allografts at >100 days (Fig. 5GoA). Also, in contrast to previous models (31, 38), IFN-{gamma} was not required for allograft prolongation in that eight of eight BALB/c IFN-{gamma}-/- mice grafted with B6 islets and treated with anti-LFA-1 had functioning allografts at >100 days (Fig. 5GoB).



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  		FIGURE 5. Long-term islet allograft acceptance can be induced in the absence of IL-4 or IFN-{gamma}. A, The majority of streptozotocin-induced diabetic BALB/c or BALB/c IL-4 knockout animals given 7 daily doses of anti-LFA-1 at 100 µg/day maintained functioning B6 (H-2b) islet allografts for more than 100 days. BALB/c IL-4 knockout mice treated with 100 µg of rat IgG/day on days 0–6 rejected B6 allografts as rapidly as wild-type, rat IgG-treated BALB/c recipients (KBA vs rat IgG treatment groups, p < 0.0001). B, BALB/c IFN-{gamma} knockout recipients maintained long-term functioning B6 allografts when treated with anti-LFA-1, but not with control rat IgG (p < 0.0005), indicating that IFN-{gamma} is not required for anti-LFA-1-induced long-term islet allograft survival.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from this study indicate that short-term mAb therapy directed against LFA-1 (CD11a) can lead to indefinite islet allograft acceptance and the induction of donor-specific tolerance. As such, these data are consistent with other models of heart (5) and islet (7) transplantation in which anti-LFA-1 therapy can result in allograft tolerance. A key question centers on how transient perturbation of LFA-1 results in durable graft acceptance, a complex issue given the multifaceted role of this molecule in immune responses. Although LFA-1 has been implicated as a costimulatory molecule (20, 21), several studies have emphasized the role of LFA-1 as an adhesion molecule with the primary effect of enhancing Ag recognition by reducing the threshold level of Ag required for T cell activation (19, 47). Alternatively, blocking LFA-1 may also inhibit leukocyte migration and so limit cell-cell interactions between donor and recipient cells. Thus, LFA-1 may primarily influence "signal one" presentation rather than more conventional costimulatory signals, such as those delivered by B7/CD28 interactions (47). If anti-LFA-1 therapy does decrease Ag recognition during the peritransplant period, then one potential mechanism of graft acceptance may be simple ignorance of the donor Ags such that the host is neither immunized nor tolerized to the allograft, analogous to transgenic models of peripheral Ag presentation (44). However, our results do not support this hypothesis in that most anti-LFA-1-treated animals resist rejection of established islet allografts following active immunization with donor-type APC and, importantly, transfer donor-specific tolerance to secondary recipients bearing islet allografts capable of triggering rejection by naive T cells. This later result is consistent with results by Nishihara et al. (7), who found that anti-LFA-1 therapy resulted in transferable tolerance to islet allografts. The finding that long-term established allografts display accumulations of mononuclear cells provides further evidence that the transplant is not merely ignored by the host.

Although anti-LFA-1 therapy induced long-term graft acceptance in all strains tested, this treatment was notably less effective in B6 recipients. The resistance of the B6 strain to allograft tolerance appears to be a relatively common finding (43). B6 and B10 mouse strains are known to have CD4-independent CD8 alloresponses that differ from many other strains, including BALB/c (48). This raises the possibility that such helper-independent CD8 T cells may contribute to the relative resistance of B6 mice to anti-LFA-1 therapy. In support of this concept, Trambley et al. (43) recently found that CD8 T cells were responsible for the lack of efficacy of costimulation blockade in B6 recipient mice. These investigators have recently shown that the addition of anti-CD8 treatment to anti-costimulation therapy dramatically prolonged skin allografts in B6 mice. However, our own pilot studies have not yielded similar results with anti-LFA-1 therapy. That is, the combination of anti-CD8 and anti-LFA-1 treatment did not result in significantly improved graft survival in B6 recipients relative to anti-LFA-1 therapy alone. We should also note that in vitro studies indicate that anti-LFA-1 treatment does directly inhibit CD8 alloresponses, unlike anti-CD40L Abs, which preferentially inhibit CD4 responses (our unpublished observations). Thus, while our data are similar to others suggesting that the B6 strain is particularly resistant to allograft-tolerizing protocols, our current results do not directly implicate CD8 T cells as playing a primary role in this attribute of B6 mice regarding anti-LFA-1 therapy.

The finding that the anti-LFA-1-treated recipients bearing long-term functioning islet allografts are not ignorant of donor Ags and the observation that there is no apparent deletion or anergy of donor-reactive T cells leave the possibility of an immune deviation of donor-reactive cells, possibly through altered donor cytokine production. Unmodified graft rejection is usually associated with Th1-like cytokines (23, 24, 33), although Th2 cytokines (e.g., IL-4 and IL-10) can also be elevated in rejecting grafts (32, 33). In contrast, allograft tolerance is often characterized by decreased expression of IL-2 and IFN-{gamma} and/or elevated Th2 cytokines (26, 27, 28, 29, 30, 31). There is both direct and indirect evidence that LFA-1 signals may influence T cell cytokine profiles. LFA-1 signaling has been implicated as directly promoting Th1-like responses and/or inhibiting Th2 immunity (22, 49). Thus, a potential consequence of anti-LFA-1 therapy may be the direct inhibition of IFN-{gamma} and corresponding induction of IL-4 production by donor-reactive T cells. Alternatively, anti-LFA-1 therapy may indirectly lead to Th2 immunity through a reduced strength of Ag signal. Thus, LFA-1 inhibition may mimic a low Ag concentration that previously has been shown to elicit Th2 production in a TCR transgenic model (50). However, despite these predictions, we did not detect an apparent Th1/Th2 deviation in donor-reactive T cells. Donor-specific responses maintained a Th1-like response in vitro despite the induction of donor-specific tolerance in vivo. Furthermore, the finding that long-term graft acceptance occurred in IL-4-/- mice indicated that this prototypical Th2 cytokine was not required for tolerance, a result consistent with some previous results (36, 37), but contrary to others implicating IL-4 as being necessary for allograft tolerance (33, 34, 35). It must, of course, be noted that other putatively regulatory cytokines such as IL-13 and TGF-ß not assessed in the current study should be considered as potentially playing a role in graft acceptance. However, given the limitations of the responses and cytokines measured, our results do not support a demonstrable Th1/Th2 cytokine deviation of donor-reactive T cells as a contributing factor to tolerance induction in this model.

A rather unexpected recent finding is the observation that IFN-{gamma} may play an important role in the generation of allograft tolerance. This concept is supported by studies indicating that IFN-{gamma}-deficient animals are refractory to allograft tolerance and/or that neutralizing Abs to IFN-{gamma} can prevent allograft tolerance (31, 38). Thus, IFN-{gamma}, commonly considered as a hallmark proinflammatory molecule associated with destructive allograft immunity, appears to also play a role in the regulation of the response. Unlike these other studies, however, we found that anti-LFA-1 therapy was as effective at inducing long-term islet allograft acceptance in BALB/c IFN-{gamma}-/- recipients as in wild-type animals. As such, the present results differ from the previous studies and further indicate that IFN-{gamma} is not universally required for allograft prolongation. This result is not merely a peculiarity of pancreatic islet transplantation, in that we have found that BALB/c IFN-{gamma}-/- animals are indeed resistant to the prolongation of islet allografts following anti-CD4 therapy (unpublished observations). Therefore, there appear to be both IFN-{gamma}-dependent and IFN-{gamma}-resistant approaches to allograft prolongation.

Currently, the mechanism of graft prolongation following therapies targeting LFA-1 remains unclear. Therapeutic perturbation of varied immune regulatory molecules may lead to reactivity unexpected based on predicted functions of the targeted molecule. Rather than preventing donor recognition as might be predicted based on the role of LFA-1 in cell adhesion and Ag recognition, anti-LFA-1 treatment appears to result in an altered donor reactivity in vivo. Conversely, costimulation blockade of CD80/86 might be expected to result in clonal anergy (51). However, islet allograft acceptance induced by CTLA4-Fc has been shown to involve active regulatory tolerance in vivo (52) rather than clonal anergy. Other studies using costimulation blockade to facilitate graft prolongation indicate a requirement for CTLA4 ligation (31, 53), suggesting a role for differential signaling in graft prolongation rather than complete inhibition of costimulatory molecules. Thus, the mechanism of induced allograft tolerance may result from secondary effects that are not readily apparent based on the role of the targeted molecule. One speculative hypothesis is that seemingly divergent approaches to achieving peripheral tolerance to allografts actually permit a recapitulation of self-tolerance rather than directly inducing allograft tolerance. That is, what several interventions may have in common is the blockade of destructive allograft immunity, allowing the generation of a peripheral regulatory response as seen in normal self-tolerance (54, 55). An example of this concept is the tolerance that spontaneously develops in response to APC-depleted islet allografts (56, 57). In this case, such treated islet allografts survive indefinitely in the absence of any host immunosuppression. However, recipients gradually develop a form of CD4-dependent donor-specific tolerance (57), indicating that the persistence of the allograft is sufficient to tolerize the recipient independent of other inductive strategies. Thus, it is intriguing to consider the possibility that many disparate approaches to achieving peripheral allograft tolerance may be mechanistically similar to the generation of tolerance to extrathymic self Ags. We are currently pursuing studies to determine whether anti-adhesion-based therapy results in active regulatory responses to islet allografts in vivo as seen in other models of induced allograft tolerance or self-tolerance.


   Acknowledgments
 
We thank Tony Valentine, Leslie Bloomquist, and Philip Pratt for excellent technical assistance.


   Footnotes
 
1 This work was supported by Grant DK33470 from the National Institutes of Health. Back

2 M.R.N. and M.C. had equivalent contribution to this study. Back

3 Address correspondence and reprint requests to Dr. Ronald G. Gill, Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences University, 4200 East 9th Avenue, Box B-140, Denver, CO 80262. E-mail address: Back

Received for publication November 21, 1999. Accepted for publication January 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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