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*Substance via MeSH
Medline Plus Health Information
*Islet Cell Transplantation
The Journal of Immunology, 2003, 170: 3015-3023.
Copyright © 2003 by The American Association of Immunologists

Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance 1

Nancy E. Phillips2,*, Thomas G. Markees2,*, John P. Mordes*, Dale L. Greiner*,{ddagger} and Aldo A. Rossini3,*,{dagger},{ddagger}

Departments of * Medicine and {dagger} Molecular Medicine, and {ddagger} Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of mice with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb to block CD40-mediated signaling uniformly induces donor-specific transplantation tolerance. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. The nature of the cellular mechanisms involved and the basis for the difference in survival of islet vs skin allografts are not known. In this study, we used CD40 knockout mice to investigate the role of CD40-mediated signaling in each component of the tolerance induction protocol: the DST, the graft, and the host. When CD40-mediated signaling was eliminated in only the DST or the graft, islet allografts were rapidly rejected. However, when CD40 signaling was eliminated in the host, ~40% of the islet allografts survived. When CD40 signaling was eliminated in the DST, the graft, and the host, islet grafts survived long term (>84 days), whereas skin allografts were rapidly rejected (~13 days). We conclude that transplantation tolerance induction in mice treated with DST and anti-CD154 mAb requires blockade of CD40-mediated signaling in the DST, the graft, and the host. Blockade of CD40-mediated signaling is necessary and sufficient for inducing islet allograft tolerance and is necessary but not sufficient for long-term skin allograft survival. We speculate that a requirement for regulatory CD4+ T cells in skin allograft recipients could account for this differential response to tolerance induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signaling through CD40 on APCs after its ligation with CD154 on T cells has a central role in the costimulation pathways that promote T cell activation (1, 2). CD40-mediated signaling leads to maturation of APCs, including dendritic cells, which are required for activation of naive T cells (3, 4). Dendritic cell maturation includes up-regulation of CD80/86 and secretion of cytokines such as IL-12 (5, 6, 7, 8). CD40-mediated signaling is believed to play a role in the pathogenesis of chronic inflammatory diseases, autoimmunity, graft vs host disease, atherosclerosis, and graft rejection (1, 2, 9, 10, 11, 12).

In vitro costimulation blockade has been shown to prevent naive T cell activation and to induce T cell nonresponsiveness (3, 13, 14, 15, 16). Translating this in vitro observation into in vivo model systems, several laboratories have documented that costimulation blockade leads to donor-specific transplantation tolerance (17, 18, 19, 20, 21, 22). Our costimulation blockade protocol uses a single donor-specific transfusion (DST)4 to activate the host alloreactive T cells plus a brief course of anti-CD154 mAb to block costimulation (17). This protocol leads to permanent islet allograft survival (23, 24), prolonged skin allograft survival in euthymic recipients (25), and indefinite skin allograft survival in thymectomized recipients (26). Deletion of recipient alloreactive CD8+ T cells and the presence of a regulatory population of CD4+ cells appear to be critical factors for the induction of prolonged allograft survival (26, 27, 28). The role of CD40 signaling on each component of the protocol is still largely unknown, as are the factors responsible for the differential survival of islet vs skin allografts.

The experiments reported here were designed to determine whether prevention of CD40-mediated signaling in the DST, graft, or host was necessary or sufficient for the induction of transplantation tolerance. The data show that prevention of CD40-mediated signaling in all three tissues was necessary and sufficient for the induction of permanent islet allograft survival, but that it was not sufficient for achieving prolonged skin allograft survival. The data suggest that some additional factor is required for prolonged survival of skin allografts in mice treated with costimulation blockade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
C57BL/6 (H2b), BALB/c (H2d), and FVB (H2q) mice were obtained from the National Cancer Institute (Frederick, MD). CD40 knockout (KO) mice on a segregating (C57BL/6 x 129) background (29) were kindly provided by H. Kikutani (Osaka University, Osaka, Japan) and crossed to BALB/c and C57BL/6 inbred mice for 7–12 generations. Mice were typed by PCR using DNA from ear punches and published primers (29) or by flow cytometry of peripheral blood using FITC-conjugated anti-CD40 mAb and PE-conjugated anti-CD45R (anti-B220) mAb. Heterozygous CD80 transgenic FVB mice (30), kindly provided by G. Feeman (Harvard Medical School, Boston, MA), were typed by flow cytometry of peripheral blood lymphocytes using FITC-conjugated anti-CD80 mAb.

All animals were certified to be free of Sendai virus, pneumonia virus of mice, murine hepatitis virus, min virus of mice, ectromelia, lactate dehydrogenase-elevating virus, mouse poliovirus, Reo-3 virus, mouse adenovirus, lymphocytic choriomeningitis virus, polyoma, Mycoplasma pulmonis, and Encephalitozoön cuniculi. They were housed in a specific pathogen-free facility in microisolator cages, given autoclaved food and acidified water, and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School and the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996).

Abs and flow cytometry

FITC-anti-CD40 mAb (clone HM40.4), FITC-anti-CD80 mAb (clone 16-10A1), FITC-anti-H2-Kb mAb (clone AF6-88.5), PE-anti-CD45R mAb (anti-B220, clone RA3-6B2), PE-anti-H2-Kd (clone SF1-1.1), PerCP-anti-CD4 (clone RM4-5), APC-anti-CD25 (clone PC61), and biotinylated anti-CD86 (clone GL1) visualized using CyChrome-streptavidin were obtained from BD PharMingen (San Diego, CA). Appropriately labeled isotype controls (clones G235-1, G155-178, A110-1, and R35-95) for the different Abs were also obtained from BD PharMingen and were included in each experiment. Clone MR1 hamster anti-mouse CD154 mAb (31) was produced as ascites in scid mice and purified by protein A affinity chromatography (Amersham Pharmacia Biotech, Uppsala, Sweden). Ab concentration was determined by measurement of optical density and confirmed by ELISA as described (28). The concentration of contaminating endotoxin was determined commercially (Charles River Endosafe, Charleston, SC) and was uniformly <10 EU per mg of mAb.

Flow microfluorometry was performed as described (32). Briefly, single cell suspensions were labeled with Ab, washed twice, fixed in 1% paraformaldehyde, and analyzed on a FACScan or FACSCalibur (BD Biosciences, San Jose, CA). At least 2 x 104 events were analyzed for experimental samples. Forward angle and side scattering properties were used to exclude dead cells and gate lymphocytes. CellQuest (BD Biosciences) and FlowJo (TreeStar, San Carlos, CA) software were used for postacquisition analysis.

Preparation of donor spleen cells

Preparation of single cell suspensions of spleen cells, Ab-mediated depletion of T cells, and size fractionation by elutriation were performed as described (23, 25, 33).

Islet allograft transplantation

Recipient mice 8–16 wk of age were rendered diabetic by a single i.p. injection of streptozotocin (150 mg/kg). Diabetes was defined as a plasma glucose concentration >250 mg/dl on at least two different days. Plasma glucose concentration was measured using a Beckman II glucose analyzer (Beckman, Fullerton, CA). All diabetic animals were treated with s.c. timed release insulin pellets (one to two pellets per animal; Linbits, Linshin, Ontario, Canada) that were removed at the time of islet transplantation. Pancreatic islets were isolated by collagenase digestion (23, 32) and were transplanted at a dose of 20 per g body weight into the renal subcapsular space of chemically diabetic recipients. Grafts that did not reduce plasma glucose concentration to <250 mg/dl within 48 h were deemed technical failures and were excluded from analysis; the historical rate of technical failures in our laboratory is ~6% (34). Graft rejection was defined as recurrence of a plasma glucose concentration >250 mg/dl on two successive days. In the case of all islet recipients that were normoglycemic at the end of the period of experimental observation, graft function was confirmed by unilateral nephrectomy of the kidney bearing the transplant and documentation of the reappearance of diabetes. In selected animals, the presence of an intact functioning islet graft was also confirmed by immunohistochemistry documenting the presence of insulin-containing {beta} cells in the graft.

Skin allograft transplantation

Full thickness skin grafts 1–2 cm in diameter were obtained from the flanks of donor mice and transplanted onto the dorsal flanks of recipients as described (26). After bandages were removed on day 7, grafts were examined visually and tactilely three times weekly. Rejection was defined as the first day on which the entire graft surface appeared necrotic. Grafts adherent to the bandage or fully necrotic on day 7 were deemed technical failures and were excluded from analysis (25).

Transplantation tolerance induction procedures

Graft recipients were treated with a single DST and a short course of anti-CD154 mAb as described (23, 25, 26). Three variants of this protocol were used. In the first protocol, the DST consisted of 45–70 x 106 small, T cell-depleted, elutriated spleen cells given 7 days before transplantation. Anti-CD154 mAb was given i.p. at a dose of 0.25 mg immediately before the DST and twice weekly thereafter for a total of 14 injections (23). In the second and third protocols, the DST consisted of a single i.v. injection of 107 spleen cells given 7 days before transplantation. Anti-CD154 mAb was given i.p. at a dose of 0.25 mg for a total of either four doses (on days -7, -4, 0, and +4 relative to transplantation) or 14 doses (on days -7, -4, 0, and +4 and then twice weekly for 5 wk). The three protocols were evolved over time in our laboratory, and in general each was found to be comparable with respect to prolonging survival of skin and islet allografts in normal C57BL/6 recipient mice (23, 25, 35). The half-life of anti-CD154 mAb in C57BL/6 mice is ~16.5 days (28).

Detection of cells injected as a DST

One and 3 days after injection of donor cells, selected animals were killed and peripheral blood, spleen, and inguinal, axillary, and brachial lymph nodes were collected and processed into single cell suspensions. The lymph node cells were pooled. Expression of the activation marker CD86 on donor and recipient cells was measured by flow microfluorometry using PE-anti-H2-Kd, FITC-anti-H2-Kb, and biotinylated anti-CD86 visualized using CyChrome-streptavidin (BD PharMingen).

Statistics

Average duration of graft survival is presented as the median. Graft survival among groups was compared using the method of Kaplan and Meier (36); the equality of allograft survival distributions for animals in different treatment groups was tested using the log rank statistic (37). All p values <0.05 were considered statistically significant. Parametric data are shown as the arithmetic mean ± 1 SD. Comparisons of two means used t tests with independent estimates of variance; comparisons of three or more means used one-way ANOVA and the Scheffe procedure for a posteriori contrasts (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Blockade of CD40-mediated signaling in cells in the DST is required for transplantation tolerance induction

Anti-CD154 mAb prevents activation and up-regulation of CD86 on APCs in the DST. In developing our tolerance induction protocol based on DST and anti-CD154 mAb, we have hypothesized that blockade of CD40-CD154 ligation prevents up-regulation of CD80/86 on APCs, but it is not known whether anti-CD154 mAb exerts this effect on APCs in the DST, graft, or host. To begin the analysis of this problem, we first determined whether anti-CD154 mAb blocks the activation of cells in the DST after injection into allogeneic recipients.

C57BL/6 mice were injected i.v. with BALB/c spleen cells as a DST in the presence or absence of anti-CD154 mAb, and the percentages of donor-origin spleen cells and of activated (CD86+) donor-origin spleen cells were determined. As shown in Table I, in untreated recipients, the background expression of donor-origin H2-Kd+ cells was ~0.02% (Table I, groups 1 and 5, and Fig. 1A), and in a freshly prepared suspension of 1% BALB/c H2d+ donor spleen cells with 99% C57BL/6 spleen cells, ~45% of the BALB/c spleen cells expressed CD86 (Table I, group 4, and Fig. 1G).


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  		Table I. Expression of CD86 on donor cells in the spleen of allogeneic recipient micea

 


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  		FIGURE 1. Anti-CD154 mAb prevents up-regulation of CD86 on cells in the donor-specific transfusion. C57BL/6 mice (H2-Kb) were injected with medium alone (A), BALB/c (H2-Kd) DST (B and E), or DST plus anti-CD154 mAb (C and F). One day later, spleen cells were analyzed by flow microfluorometry for the expression of H2-Kd and CD86 as described in Materials and Methods. D and G, Representative flow histograms of mixtures of 1% BALB/c (H2d) and 99% C57BL/6 (H2b) freshly isolated spleen cells. All cells were labeled with anti-H2-Kb mAb (x-axis) and anti-H2-Kd mAb (y-axis). A–D, Representative histograms identifying populations of H2-Kd+ donor cells present in recipient H2-Kb spleens. The number of H2Kd+ cells that were also CD86+ is shown in E–G. Each histogram is representative of four to seven individual determinations performed as part of two independent experiments. The numbers in the histograms represent the percentage of donor-origin (H2-Kd+) cells (A–D) or the percentage of H2-Kd+ cells that coexpressed CD86 (E–G). For each experiment, 20–100,000 events were acquired.

 
One day after the injection of a BALB/c DST, ~0.25% of the spleen cells in C57BL/6 recipients were of donor origin (Table I, groups 2 and 3, and Fig. 1, B and C). In the absence of anti-CD154 mAb, the percentage of the donor-origin H2-Kd+ cells that were CD86+ (Table I, group 2, and Fig. 1E) was significantly higher than it had been in the freshly prepared cell suspension (Table I, group 4, and Fig. 1G; p = 0.003). The percentage of donor-origin cells expressing CD86 was significantly less in recipients treated with anti-CD154 mAb (Table I, group 3, and Fig. 1C) than in recipients given DST but no anti-CD154 mAb (Table I, group 2, and Fig. 1E; p < 0.001). This percentage was statistically similar to that detected in freshly isolated spleen cells (Table I, group 4, and Fig. 1G; p = NS).

The absence of an increase in activated donor-origin cells in allogeneic DST recipients treated with anti-CD154 mAb suggests that activation of cells in the DST requires CD40-mediated signaling. To determine whether this is the case, we next analyzed the expression of CD86 on BALB/c CD40-KO donor cells after injection into allogeneic C57BL/6 recipients. Again, 1 day after injection, ~0.25% of the spleen cells in C57BL/6 recipients of a BALB/c CD40-KO DST were of donor origin (Table I, groups 6 and 7). In contrast with the previous experiment, the percentage of donor-origin cells expressing CD86 was comparable in recipients of DST alone (Table I, group 6) and DST plus anti-CD154 mAb (Table I, group 7; p = NS), as well as in the freshly prepared spleen cell mixture (group 8; p = NS).

Anti-CD154 mAb plus DST fails to prolong islet allograft survival if CD80 transgenic APCs are used as DST. Our first result suggested that failure of costimulation blockade to block CD80/86 expression and activation of APCs in the DST would preclude the induction of prolonged allograft survival. To test this inference, we used CD80 transgenic FVB (H2q) mice. The transgenic expression of CD80 was under the control of the IgM promoter, leading to high levels of CD80 expression on B cells in the spleen cell inoculum used for DST (30).

Chemically diabetic C57BL/6 mice were injected i.v. with 45–70 x 106 small, T cell-depleted, elutriated spleen cells as a DST and simultaneously were injected i.p. with 0.25 mg of anti-CD154 mAb as described (23). DST was prepared from wild-type or CD80 transgenic FVB donors. Anti-CD154 mAb injections continued twice weekly for a total of 14 injections or until the islet allograft was rejected. Seven days after injection of DST and the first injection of anti-CD154 mAb, the chemically diabetic mice were transplanted and islet allograft survival was documented by monitoring blood glucose levels.

Consistent with previous observations (23), monotherapy with anti-CD154 mAb led to prolonged (>3 wk) islet allograft survival in ~40% of recipients (Table II, group 1). Median survival time of the grafts was 17 days. As expected, treatment of islet allograft recipients with anti-CD154 mAb plus wild-type FVB donor B cells as a DST led to prolonged islet allograft survival in all mice (Table II, group 2; median survival time (MST) = 82 days). In contrast, treatment with anti-CD154 mAb plus DST obtained from CD80 transgenic mice reduced the duration of islet allograft survival (Table II, group 3; MST = 8 days). Only 2 of 20 grafts survived >100 days.


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  		Table II. Survival of FVB islet allografts in C57BL/6 recipients treated with anti-CD154 mAb plus a wild-type or CD80 transgenic DSTa

 
No evidence of chimerism in recipients of allogeneic DST and anti-CD154 mAb. These results suggest that anti-CD154 mAb acts at least in part by preventing the activation of DST APCs, but it could be argued that the role of the anti-CD154 mAb and DST in tolerance induction is to create a state of chimerism in the recipient. To exclude this possibility, we next determined whether anti-CD154 mAb prolongs the survival of a DST in allogeneic recipients. The percentage of donor cells in spleen, lymph nodes, and peripheral blood was measured 1 and 3 days after transfusion of 107 BALB/c cells into C57BL/6 recipients. Consistent with the results in Table I, donor cells comprised ~0.2% of the total cells in all three tissues on day 1 (Table III). By day 3, the level of donor cells in all three tissues fell to background levels in both control mice and mice treated with anti-CD154 mAb (Table III).


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  		Table III. Anti-CD154 does not prolong the survival of allogeneic spleen cellsa

 
Blockade of CD40-mediated signaling in cells of the DST is necessary but not sufficient for the induction of transplantation tolerance

CD40-KO cells as DST do not prolong islet allograft survival in normal recipients in the absence of anti-CD154 mAb. The observation that anti-CD154 mAb prevents up-regulation of CD86 on cells in the DST suggested that blockade of CD40-mediated signaling could be the necessary and sufficient key to the tolerogenic activity of DST. To test this possibility, we used CD40-KO cells as DST to determine whether they could induce long-term islet allograft survival in the absence of anti-CD154 mAb. T cell-depleted spleen cells for use as DST were prepared from BALB/c CD40-KO mice or their heterozygous CD40-positive littermates. Recipients were chemically diabetic C57BL/6 wild-type mice that were given a DST 7 days before transplantation of wild-type BALB/c islets. In both groups, islet graft rejection occurred very rapidly (Table IV, groups 2 and 3), with kinetics comparable to those observed in untreated recipients (group 1; p = NS).


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  		Table IV. Islet allograft survival in mice treated with CD40+/- or CD40-KO DSTa

 
In a second experiment, T cell-depleted, elutriated BALB/c (Table IV, group 4) or BALB/c CD40-KO (group 5) splenic B cells were used as the DST, but again, duration of islet allograft survival was brief and similar to that observed in untreated recipients (p = NS). In a third experiment, higher (70 x 106; Table IV, group 6) and lower (10 x 106; group 7) doses of T cell-depleted, elutriated BALB/c CD40-KO splenic B cells again failed to prolong islet allograft survival beyond the duration observed in untreated C57BL/6 mice (p = NS).

Combined therapy with a DST comprised of CD40-KO cells and anti-CD154 mAb prolongs skin allograft survival. The previous result suggested that anti-CD154 mAb must exert some effect in addition to blockade of CD40-mediated signaling in the DST to prolong allograft survival. To test this hypothesis, we combined a BALB/c CD40-KO DST with anti-CD154 mAb using wild-type C57BL/6 skin allograft recipients. Skin grafts were used in these experiments because up to 40% of islet grafts will exhibit long-term survival in recipients treated with anti-CD154 mAb monotherapy (23). Skin allograft survival requires both DST and anti-CD154 mAb (25, 26), and was therefore the appropriate system to test this hypothesis. Wild-type C57BL/6 mice were divided into three groups and transplanted with BALB/c wild-type skin allografts. One group received no other treatment. The other groups were given four injections of 0.25 mg of anti-CD154 mAb on days -7, -4, 0, and +4 and also were transfused with 107 spleen cells as a DST obtained from BALB/c CD40-KO or BALB/c wild-type donors on day -7 relative to skin transplantation on day 0.

Untreated C57BL/6 mice rapidly rejected BALB/c skin allografts (MST = 8 days; n = 5). C57BL/6 mice given BALB/c wild-type DST plus anti-CD154 mAb had prolonged skin allograft survival (MST = 77 days; n = 14; p < 0.0001). Skin allograft survival on C57BL/6 mice given anti-CD154 mAb plus DST from BALB/c CD40-KO mice (MST = 63 days; n = 9) was similar to that of mice given wild-type DST (p = NS). These data document that, in addition to blocking CD40-mediated signaling in cells of the DST, prevention of CD40-mediated signaling is required in either the graft or host for prolongation of allograft survival.

Blockade of CD40-mediated signaling is required in cells in the DST, the graft, and the host for optimal islet allograft survival

CD40-KO islet allografts are rapidly rejected by wild-type recipients. The above results demonstrate that blockade of CD40-mediated signaling in the DST is necessary but not sufficient for the prolongation of allograft survival. We next tested the hypothesis that prevention of CD40-mediated up-regulation of costimulatory molecules in islet grafts, presumptively on passenger leukocytes, would be sufficient for prolonging islet allograft survival. Passenger leukocytes are known to be important in the activation of the host immune system for rejection of islet allografts (39, 40, 41, 42). Chemically diabetic C57BL/6 wild-type mice were transplanted with islets obtained from BALB/c CD40-KO donors. Rejection of CD40-KO islets, however, was rapid (Table V, group 1; MST = 14 days).


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  		Table V. CD40-KO DST prolongs CD40-KO islet allograft survival in CD40-KO recipientsa

 
Blockade of CD40-mediated signaling in the host, in the absence of a DST, induces prolonged islet allograft survival in some but not all recipients. Our results to this point document that blockade of CD40-mediated signaling in neither the DST nor the graft is by itself sufficient to prolong islet graft survival. We next asked whether blockade of CD40-mediated signaling only in host cells would be sufficient for inducing prolonged islet allograft survival. Chemically diabetic C57BL/6 CD40-KO recipient mice were transplanted with either BALB/c wild-type islets or BALB/c CD40-KO islets. In contrast with the short survival of BALB/c CD40-KO islets in C57BL/6 wild-type recipients (Table V, group 1), 3 of 8 BALB/c wild-type islet allografts survived >50 days in C57BL/6 CD40-KO recipients (Table V, group 2) and 6 of 14 BALB/c CD40-KO islet allografts survived >83 days in C57BL/6 CD40-KO recipients (Table V, group 3). This level of graft survival is comparable to that which can be achieved by the use of anti-CD154 mAb monotherapy in the absence of a DST (23).

Anti-CD154 mAb does not enhance islet allograft survival in CD40-KO recipients given CD40-KO islets. We next hypothesized that treatment of CD40-KO recipients of CD40-KO islets with anti-CD154 mAb would further increase the number of islet allografts that survive indefinitely. This hypothesis was based on reports suggesting that anti-CD154 mAb can modulate the activity of activated CD4+ T cells that express CD154 (43, 44). To test whether anti-CD154 mAb would affect host T cells in the absence of CD40-mediated signaling, chemically diabetic C57BL/6 CD40-KO mice were transplanted with BALB/c CD40-KO islets and were given four injections of 0.25 mg of anti-CD154 mAb on days -7, -4, 0, and +4 relative to transplantation. As shown in Table V, 6 of 13 recipients had extended islet allograft survival for >84 days (Table V, group 4). These data are similar to islet allograft survival attained in the absence of anti-CD154 mAb (Table V, group 3) and suggest that anti-CD154 mAb mediates its effect in our tolerance induction protocol by blocking CD40-CD154 interaction and not by direct modulation of activated alloreactive CD4+ T cells that express CD154.

Blockade of CD40-mediated signaling in DST, host, and graft induces permanent islet allograft survival in the absence of anti-CD154 mAb. The results to this point suggested that optimal islet allograft survival may require blockade of CD40-mediated signaling in all three components of the system: DST, host, and graft. We tested this using chemically diabetic C57BL/6 CD40-KO mice that were given 107 BALB/c CD40-KO spleen cells as a DST and that were transplanted 7 days later with BALB/c CD40-KO islets. All of the islet allografts survived to the end of the experimental observation period (83–85 days; Table V, group 5). Islet allograft survival after the addition of anti-CD154 mAb to this protocol (Table V, group 6; MST = >85 days) was similar to that obtained in the absence of anti-CD154 mAb treatment (group 5; p = NS).

Islet allograft survival in CD40-KO recipients of donor-specific CD40-KO islets does not permit survival of subsequent donor-specific CD40-KO skin allografts. Having demonstrated that blockade of CD40-mediated signaling in DST, host, and graft leads to permanent islet allograft survival, we asked whether the tolerant state in these animals would be permissive to skin allograft survival. Permanent islet allograft survival in this setting could be due to deletion of alloreactive T cells in the host or to other mechanisms such as ignorance or regulation (17). The animals generated in groups 3 and 5 of Table V provided the opportunity to determine whether the tolerant state was based on the deletion of alloreactive cells. Five C57BL/6 CD40-KO recipients of long-term-surviving BALB/c CD40-KO islet allografts (83–85 days) were grafted with BALB/c CD40-KO skin allografts. One animal died after surgery, but in the four remaining mice, both the healed-in islet grafts and the new skin allografts were rejected within 21 days after skin grafting, suggesting that mechanisms other than deletion are responsible for islet allograft survival.

Blockade of CD40-mediated signaling in cells in the DST, the graft, and the host is not sufficient for long-term skin allograft survival

Prevention of CD40-mediated signaling in the graft and host is not sufficient for prolongation of skin allograft survival. Prevention of CD40-mediated signaling in the donor graft and in the host is sufficient to induce long-term survival in ~40% of islet allografts (Table V). To test whether this was also true for skin allografts, we transplanted C57BL/6 CD40-KO skin grafts onto BALB/c CD40-KO recipients. Unlike islet allografts transplanted using the same protocol (Table V), all CD40-KO skin allografts were rapidly rejected (Table VI, group 1; MST = 11 days).


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  		Table VI. CD40-KO DST does not prolong CD40-KO skin allograft survival in CD40-KO recipientsa

 
Prevention of CD40-mediated signaling in the DST, graft, and host is not sufficient for prolongation of skin allograft survival. Prevention of CD40-mediated signaling in DST, host, and graft is sufficient to induce long-term survival in 100% of islet allografts (Table V). To test whether this was also true for skin allografts, we transplanted C57BL/6 CD40-KO skin grafts onto either BALB/c CD40-KO or BALB/c heterozygous littermate recipients. All mice were transfused with a CD40-KO DST. Again, unlike the islet allograft model system (Table V, group 5), BALB/c CD40-KO DST only marginally prolonged the survival of BALB/c CD40-KO skin allografts in C57BL/6 CD40-KO recipients (Table VI, group 2; MST = 11 days). Treatment with anti-CD154 mAb therapy did not improve skin allograft survival (Table VI, group 3). Considering all mice in which CD40-mediated signaling was blocked in DST, host, and graft (with or without anti-CD154 mAb treatment), only 3 of 27 skin allografts survived >50 days.

In contrast, skin allograft survival 50 days was achieved in five of six wild-type heterozygous littermate recipients given CD40-KO DST, anti-CD154 mAb, and CD40-KO skin allografts (Table VI, group 4).

C57BL/6 CD40-KO mice are relatively deficient in CD4+CD25+ T cells. These results clearly demonstrate a difference in survival between allogeneic islets and skin in a completely CD40-deficient model system, but the reason is unclear. It could be due to the presence of a costimulatory pathway in skin, but not islets that can bypass the requirement for CD40-CD154 interaction for T cell activation (45). Such a costimulatory pathway could be more important in CD40-KO mice than in wild-type mice. Alternatively, it could be due to an active regulatory process. We (26, 28) and others (15, 21, 22, 46, 47, 48, 49, 50, 51, 52, 53) have shown that CD4+ T cells, presumably "regulatory" CD4+ T cells, are required for tolerance induction and maintenance. BALB/c CD40-KO mice are reported to have a deficiency of CD4+CD45RBlow and CD4+CD25+ cells (54), phenotypes associated with regulatory T cell populations in mice (55, 56, 57).

To begin to examine the regulatory hypothesis, we quantified the relative percentage of CD4+CD25+ splenic T cells in the C57BL/6 CD40-KO mice used as recipients in these studies. We observed that 7.5 ± 2.4% of C57BL/6 splenic CD4+ T cells express CD25 (n = 6), whereas fewer C57BL/6 CD40-KO splenic CD4+ T cells are CD25+ (4.6 ± 0.9%; n = 6; p < 0.03). Representative histograms of C57BL/6 and C57BL/6 CD40-KO spleen cells stained for CD4 and CD25 are shown in Fig. 2.



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  		FIGURE 2. C57BL/6 CD40-KO mice are relatively deficient in CD4+CD25+ T cells. Representative histograms show expression of CD25 on CD4+ spleen cells from C57BL/6 (B and D) or C57BL/6 CD40-KO mice (A and C). Cells were stained for CD4 and CD25 (A and B) or CD4 and an isotype control (C and D). Histograms were first gated on CD4 and forward side scatter (FSC). Then the CD4 subset was selected and gated for CD25 and forward side scatter. Flow histograms are representative of six individual determinations in two separate experiments. Overall means for the entire experiment are given in Results; 50,000 events were acquired for each analysis

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The survival of islet and skin allografts in mice is greatly prolonged by costimulation blockade based on the administration of a DST and a brief course of anti-CD154 mAb (23). The anti-CD154 mAb is injected at the time of DST, blocks CD40-CD154 interaction, and prevents CD40-mediated signaling. The MR1 anti-CD154 mAb used in our protocol has a half-life of ~16 days and is present in the circulation at high levels at the time of grafting (28). Given the design of the protocol, the anti-CD154 mAb can potentially block CD40-mediated signaling in three compartments: DST, graft, and host. However, which of these is critical for induction of graft survival is not known. In this study, we document that blockade of CD40-mediated signaling in all three components is necessary and sufficient to induce permanent islet allograft survival and is necessary but not sufficient for the induction of prolonged skin allograft survival. The latter observation suggests that induction of prolonged skin allograft survival by costimulation blockade must require mechanisms in addition to the blockade of CD40-mediated signaling.

Host alloreactive T cells can be activated by direct alloantigen presentation by APCs in the donor graft or by indirect alloantigen presentation by host APCs (58, 59). In our protocol, we use spleen cells as DST. This transfusion contains dendritic cells, macrophages, and B lymphocytes, all of which, when activated, can present alloantigen directly to the host immune system (14, 60, 61). Our data document that blockade of CD40-mediated signals on donor APCs in the DST is required for the induction of tolerance. This result supports the hypothesis that blockade of the direct Ag presentation pathway is critical for tolerance induction. Consistent with this hypothesis, we observed that the use of CD40-KO cells as the DST prevented up-regulation of CD86 and that these cells functioned efficiently in our protocol. In contrast, constitutive expression of CD80 on cells in the DST prevented tolerance induction. Prevention of CD40-mediated activation of APCs in the DST is clearly a requirement for the induction of prolonged allograft survival in our model system. However, there is one report that anti-CD154 mAb does not prevent the up-regulation of CD86 on resting B cells injected into allogeneic recipients (62). The reason for this discrepancy in outcomes is not obvious, but we suggest that the timing of the analysis in relation to cell injection may be critical. Resting CD40-KO B cells have been found to up-regulate CD86 transiently when presenting Ag to naive transgenic CD4+ T cells in vitro (63).

The data we have generated here and previously (17) suggest that the DST functions to present alloantigen in the absence of costimulation. Others have suggested that the duration of survival of the cells in the DST may be important for tolerance (64). Our data document, however, that the survival of DST in recipients in the absence or presence of anti-CD154 mAb is short. It could also be argued that the function of the DST is to establish chimerism. We (32, 65) and others (66, 67, 68, 69) have documented that establishment of hematopoietic chimerism using costimulation blockade leads to a form of donor-specific tolerance. This form of tolerance relies on the induction of "macrochimerism" (32, 66). However, we document that by day 3, donor DST cells constituted <0.02% (detection limits of our assay) of cells in the recipient spleen, even in the presence of anti-CD154 mAb. Although "microchimerism" at a level of <0.02% has been proposed as a mechanism for tolerance (70), the concept remains controversial (71). All data generated by our laboratory to date (26) suggest that DST does not generate any form of chimerism.

Like a DST, an allograft itself has the potential to present alloantigen directly. Both islets and skin harbor populations of APCs, and both grafts can activate host alloreactive T cells directly. In skin, a population of potent APCs termed Langerhans cells can be activated by CD40-mediated signaling (72). In islets, "passenger leukocytes" provide APC function and are thought to be capable of initiating islet allograft rejection (40, 42). However, our data again document that prevention of CD40-mediated signaling in an islet allograft is not sufficient to prevent rejection. Prevention of CD40-mediated signaling in skin allografts, even when achieved in a completely CD40-KO model system, is insufficient for achieving even modest graft survival.

Specifically with respect to islet grafts, we have used CD40-KO mice to document that prevention of CD40-mediated signaling in the graft recipient is sufficient to induce prolonged survival of ~40% of grafts. This rate of success is similar to what we have achieved previously using anti-CD154 mAb monotherapy for the transplantation of islet allografts (23, 24, 73). Furthermore, treatment of CD40-KO recipients of CD40-KO islet grafts with anti-CD154 mAb did not enhance this survival. These data suggest that anti-CD154 mAb primarily blocks host CD40-mediated signaling and that survival of most islet allografts requires an additional component. We demonstrate that the additional component is the DST.

Our data argue that the mechanism of tolerance in CD40-KO recipients of permanent CD40-KO islet allografts is the induction of a state of "ignorance" (62, 74, 75) and not permanent deletion of host alloreactive T cells (76, 77, 78). This interpretation is supported by our observation that recipients with intact long-term islet grafts challenged with donor-specific skin grafts 1) reject their healed-in islet allografts and 2) rapidly reject the skin allografts. Although we have previously shown that a primary function of the DST in our protocol is to delete host alloreactive CD8+ T cells (27, 28), new alloreactive T cells developed in the thymus in our long-term graft recipients and, if activated, are capable of rejecting both islets and skin. We interpret these results to suggest that prevention of CD40-mediated signaling is necessary and sufficient for the induction of a "functional" but not a permanent "deletional" tolerance to islet allografts.

Our skin graft results, however, suggest that this tissue responds differently to costimulation blockade induction using DST plus anti-CD154 mAb. Prevention of CD40-mediated signaling in the DST, graft, and host, which led to long-term allo-islet survival, was not sufficient to prolong skin allograft survival. We interpret this result to suggest that additional mechanisms must be preventing skin allograft survival.

One such possible mechanism is activation of the potent APCs in skin (the Langerhans cells), in the absence of CD40-mediated signaling. It is known that the immune system incorporates mechanisms that can bypass the requirement for CD40-mediated signaling and activate host T cells (45, 79, 80, 81). This is particularly evident in CD154-KO mice exposed to viral infection. Several studies have documented that virus can be cleared in the absence of CD40-CD154 interaction and CD40-mediated signaling (12, 82). Whether other costimulatory pathways active in the skin of CD40-KO mice can initiate dendritic cell maturation and host T cell activation cannot be determined from the present data.

A second mechanism could involve the nature of the Ags present on the graft. It could be argued that it is easier to achieve tolerance to islet-specific Ags than it is to skin-specific Ags, but data from other laboratories argue that this is not the case (83). A third possibility is the relative number of APCs in the skin compared with that in islets. Our data do not address either of these possibilities.

A fourth possible mechanism to explain skin allograft rejection in the CD40-KO model system is the lack of a regulatory cell population important in the induction and maintenance of tolerance. We have previously shown that CD4+ T cells are required for tolerance induction (26) and that they are involved in graft maintenance by preventing the activation of alloreactive CD8+ T cells (28). In other model systems of transplantation tolerance, CD4+, CD4+CD25+, and CD4+CD45RBlow cells have been found to be important in transplantation tolerance (15, 21, 22, 46, 47, 48, 49, 50, 51, 52, 53, 84). Interestingly, BALB/c CD40-KO mice reportedly have a deficiency of CD4+CD45RBlow and CD4+CD25+ cells (54). The present data confirm this relative deficiency of this potential regulatory population in the C57BL/6 CD40-KO mice used as recipients. Our data suggest that a key difference between islet and skin allografts undergoing tolerance induction may be a relative dependency of skin allograft survival on a population of host regulatory CD4+ T cells that are deficient in CD40-KO mice. This possibility is currently under investigation in our laboratory.

In summary, our results document that prevention of CD40-mediated signaling in the DST, graft, and host is critical for the induction of prolonged allograft survival using the combination of DST and anti-CD154 mAb. Prevention of CD40-mediating signaling in DST, graft, and host is necessary and sufficient for the induction of tolerance to islet allografts, but it is not sufficient for the induction of prolonged skin allograft survival. We speculate that an absolute requirement for regulatory CD4+ T cells in skin allograft recipients could account for this differential response to costimulation blockade using DST and anti-CD154 mAb.


   Acknowledgments
 
We thank Linda Paquin, Elaine Norowski, and Linda Leehy for their technical expertise in preparing and transplanting the islets of Langerhans for these experiments.


   Footnotes
 
1 This work was supported in part by Grant AI42669 and Institutional Diabetes Endocrinology Research Center Grant DK52530 from the National Institutes of Health, Grant DK53006 jointly funded by the National Institutes of Health and the Juvenile Diabetes Research Foundation, and Grant 1-2002-396 from the Juvenile Diabetes Research Foundation. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Back

2 N.E.P. and T.G.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Aldo A. Rossini, Diabetes Division, University of Massachusetts Medical School, Two Biotech, 373 Plantation Street, Suite 218, Worcester, MA 01605. E-mail address: aldo.rossini{at}umassmed.edu Back

4 Abbreviations used in this paper: DST, donor-specific transfusion; KO, knockout; MST, median survival time. Back

Received for publication October 22, 2002. Accepted for publication January 8, 2003.


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