NONDEPLETING ANTI-CD4 MONOCLONAL ANTIBODY ENHANCES THE... : Transplantation (original) (raw)
Oral administration of antigen is a classical method of inducing antigen-specific peripheral tolerance (1). In addition to its physiological role of preventing systemic immune responses to ingested proteins, oral administration of autoantigens has recently been used to treat autoimmune diseases in animals and in humans (2). In the field of transplantation, oral administration of allogeneic cells has been shown to prevent sensitization by skin grafts and to modify the response of presensitized recipients resulting in acute rather than accelerated rejection of the graft (3). Evidence that oral administration of allopeptides can modify cellular immune responses has also been shown in a rat model (4). The demonstration that feeding of alloantigen prevented accelerated rejection in presensitized hosts suggests that this route of alloantigen delivery has the potential to induce immune suppression in nave recipients.
First, a donor recipient strain combination mismatched for a single MHC class I antigen was used to evaluate the efficacy of oral alloantigen delivery to induce graft prolongation. Hearts from CBK (H2k+Kb) transgenic mice (5), a kind gift of Professor A. L. Mellor (Medical College, Augusta, GA) were transplanted into CBA (H2k) recipients using microsurgical techniques (6). Graft function was followed by palpation of the grafted hearts at least three times a week. CBA mice rejected CBK cardiac grafts acutely with a median survival time (MST) of 13 days (Table 1; group 1).
Survival of CBK and (C57BL/10×CBA)F1 cardiac grafts in CBA recipientsa after oral administration of donor alloantigen either alone or in combination with nondepleting anti-CD4
Oral administration of 1×107 CBK splenocytes 15, 13, 11, 9, and 7 days before transplantation of a CBK heart resulted in modest prolongation of graft survival (Table 1; group 4, MST=42 days) compared to recipients fed with multiple doses of 1×107 syngeneic CBA splenocytes (Table 1; group 2, MST=12 days;P <0.001). A single oral delivery of 1×107 CBK splenocytes 7 days before transplantation of a CBK heart (Table 1; group 3) also resulted in graft prolongation compared with untreated controls (Table 1; group 1) (MSTs=19 and 13 days, respectively, P <0.05). Allograft survival between groups was compared by Mann-Whitney U test using StatView SE + Graphic (Abacus Concepts Inc., Cary, NC).
In an attempt to improve the graft prolongation achieved by oral delivery of alloantigen alone, a nondepleting anti-CD4 [YTS 177, hybridoma kindly provided by Professor H. Waldmann, Sir William Dunn School, Oxford, UK (7)] was included in the treatment protocol. In pervious studies using intravenous delivery of alloantigen it has been shown that the addition of a T cell modulating reagent such as anti-CD4 (8), CTLA4Ig (9), or anti-CD40L (10) can enhance the degree of specific unresponsiveness induced.
When the anti-CD4, YTS 177, (200 μg/dose) was given i.p. at the same time as the multiple oral doses of alloantigen the combined treatment (Table 1; group 6) was no more effective than oral delivery of alloantigen alone (Table 1; group 4) (MSTs=28 and 42 days, respectively); the difference in MSTs between the two groups was not statistically significant. Previous work from our laboratory has indicated that intravenous injection of alloantigen plus anti-CD4 is only effective when the antibody is administered before alloantigen challenge (11). Therefore, the treatment protocol was modified such that the nondepleting anti-CD4, YTS 177, was administered for 2 days before the first oral delivery of CBK splenocytes, i.e. 16 and 15 days before grafting. In marked contrast to mice treated with anti-CD4 at the same time as oral alloantigen delivery (Table 1; group 6), mice treated with anti-CD4 before oral treatment accepted their grafts indefinitely (>100 days) (Table 1; group 8). Mice treated with the YTS 177 antibody alone (Table 1; group 7) rejected their grafts acutely (MST=11.5 days). Histologically, the long-term surviving grafts were very similar to syngeneic grafts harvested at the same time point; no evidence of chronic rejection was detectable (Fig. 2). Our data suggest that modulation of the function of CD4+ T cells before oral exposure to alloantigen also has the capacity to promote oral tolerance.
Next, to determine if it was essential to feed recipients on multiple occasions, CBA mice were given a single dose of 1×107 CBK splenocytes orally 7 days before transplantation in combination with the nondepleting anti-CD4 (Table 1; group 10). The majority of the CBK grafts survived indefinitely after the combined pretreatment (MST >100 days) whereas oral delivery of alloantigen alone (Table 1; group 3) or YTS177 alone (Table 1; group 9) resulted in only modest prolongation of grafts (MSTs=19 and 29 days, respectively;Fig. 1a). Histologically, the long-term surviving grafts were indistinguishable from hearts transplanted into mice treated with multiple doses of alloantigen orally (Fig. 2). To test whether operational tolerance to H2Kb had been induced donor-specific (CBK) and third party (Balb/c) cardiac grafts were transplanted into recipients bearing a primary CBK graft (>100 days). Second CBK hearts but not third party Balb/c hearts survived for more than 50 days, suggesting that the operational tolerance to H2Kb had been induced (data not shown).
Oral administration of donor antigen in combination with nondepleting anti-CD4 can prolong the survival of class I mismatched and semi-allogeneic heart grafts indefinitely. A, CBK heart grafts; MHC class I mismatched. B, (C57BL/10xCBA)F1 heart grafts; MHC class I and II mismatched
Histology of cardiac grafts 100 days after transplantation, hemaoxylin and eosin staining. Left; CBK cardiac graft after multiple oral deliveries of alloantigen plus anti-CD4 (group 8) (Magnification x200). Middle; CBK cardiac graft after a single oral delivery of alloantigen plus anti-CD4 (group 10) (magnification x100). Right; (C57BL/10 x CBA)F1 cardiac graft after a single oral delivery of alloantigen plus anti-CD4 (group 14) (magnification x100).
To investigate whether this protocol could also be used to induce unresponsiveness to a graft mismatched for a complete H2 haplotype (C57BL/10 x CBA)F1 mice were used as donors. A single oral delivery of 1×107 (C57BL/10xCBA)F1 splenocytes alone (Table 1; group 13) induced slightly modest prolongation of a (C57BL/10xCBA)F1 heart compared to the survival of F1 hearts in untreated control CBA mice (Table 1; group 11) (MST=11 and 8 days, respectively). A single oral delivery of 1×107 (C57BL/10 x CBA)F1 splenocytes plus YTS 177 (Table 1; group 14) was found to prolong the survival of a (C57BL/10 x CBA)F1 heart whereas YTS 177 alone (Table 1; group 12) did not induce prolonged survival (MST >100 and 8 days, respectively, Figure 1b). However unlike the long term surviving CBK hearts focal lymphocyte infiltration was observed in most of the F1 heart grafts (Fig. 2), suggesting that a mismatched for MHC class II may trigger the development of chronic rejection after oral treatment plus anti-CD4.
The route of alloantigen presentation after oral delivery may be different from that after i.v. injection. To examine the fate of the orally administered cells, CBK splenocytes were stained with the intracellular green dye CFSE, (Molecular Probes, Eugene, OR) before p.o. or i.v. administration and mesenteric lymph nodes and spleens were harvested at several time points thereafter (6, 12, 24, and 48 hr after administration). Fluorescent cells could not be detected by flowcytometry using a FACSort and CellQuest software (Becton Dickinson, Mountain View, CA) at any time point after oral delivery whereas they were clearly detectable in the spleen and lymph nodes after intravenous injection (data not shown). These findings show, not surprisingly, that allogeneic cells are more rapidly degraded after oral delivery, suggesting that indirect presentation is the dominant route of allorecognition in this setting.
To examine the impact of oral tolerance on the B cell compartment, alloantibody production was monitored. CBA mice were either left untreated or treated with two doses of anti-CD4, YTS 177, alone 8 and 7 days before transplantation of a CBK heart, oral administration of CBK splenocytes alone 7 days before grafting, or the combined antibody/splenocyte protocol. Sera were harvested one day before and 50 days after grafting and analyzed for the presence of alloantibodies reactive with the donor class I molecule H2Kb by FACS. Antibody production was not initiated by oral administration of alloantigen (Fig. 3). After transplantation the mean fluorescence intensities (MFI) of anti-Kb IgG1 and IgG2a in mice treated orally with allogeneic cells and/or anti-CD4, YTS 177, independently were markedly suppressed compared to those in untreated mice (Fig. 3). These data suggest that oral delivery of alloantigen can modify the response of B cells reducing the amount of alloantibody produced after transplantation.
Alloantibody production is inhibited by oral delivery of alloantigen. CBA mice were either untreated or pretreated with two doses of 200 μg nondepleting anti-CD4, YTS 177, 8 and 7 days before transplantation of a CBK heart (Table 1; group 9), a single oral administration (OA) of 1×107 CBK splenocytes on day −7 (Table 1; group 3), or both (Table 1; group 10). Sera were harvested from these mice 1 day before and 50 days after transplantation and analyzed for the presence of alloantibodies reactive with the donor class I molecule H2Kb by FACS. The 5×104 Kb transfected L cells were incubated with the recipient sera for 45 min on ice. Cells were washed three times, then incubated for an additional 45 min with biotinylated rat anti-mouse IgG1 (1:200 dilution; Serotec, Oxford, UK) and IgG2a (1:50 dilution; Serotec) followed by streptavidin-phycoerythrin (Southern Biotechnology Associates, Birmingham, AL). All samples were examined at the same time. Serum from syngeneic heart grafts was used as negative control. Results are shown as the mean of two mice±SD.
The exact mechanisms mediating the tolerizing effects of oral alloantigen are unclear. Orally administered cells were destroyed and degraded within 12 hr of administration, suggesting that alloantigens were most likely presented as peptides by recipient antigen- presenting cells present in the gut-associated lymphoid tissue, implicating the indirect pathway of allorecognition in the induction of unresponsiveness. Moreover, the evidence that alloantibody production was reduced in the mice pretreated orally with alloantigen suggests that T cell responding via the indirect pathway of allorecognition can inhibit rather than enhance the activity of B cells specific for Kb molecule. How indirect T cells influence the ability of effector cells including B cells to mediate graft rejection is unknown. Deletion of alloreactive cells seems unlikely as a relatively low dose of alloantigen, 1×107 CBK splenocytes, was required in our study. In other studies where deletion of antigen reactive cells after the induction of oral tolerance has been demonstrated a very high dose of soluble antigen was necessary (12).
Bystander suppression might be responsible as was originally described after oral administration of a low dose of myelin basic protein (MBP) (13). In this system cells from animals fed with MBP suppressed proliferation of an OVA-specific cell line across a transwell, but only when triggered by the fed antigen. The indirect and the direct pathways of allorecognition could be linked in a similar manner when oral tolerance is induced.
In this study, we have shown that oral alloantigen delivery can induce prolonged survival of cardiac grafts mismatched for a single MHC class I antigen, H2Kb. The fed alloantigens are most likely presented to recipient T cells by host APC via the indirect pathway in the specialized microenvironment of the gut. Triggering of indirect T cells specific for Kb allopeptides in the presence of a nondepleting anti-CD4 promoted the development of unresponsiveness to H2Kb in vivo.
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