Currently, a major limitation in organ transplantation is the necessity of broad-based immunosuppression to prevent allograft rejection. Potentially serious side effects of panimmunosuppression include opportunistic infection, drug toxicity, and malignancy. Donor-specific immunosuppression is an attractive alternative to the use of immunosuppressive drugs and their attendant complications. One approach to donor-specific immunosuppression is exposure of the recipient to donor major histocompatibility complex (MHC)* antigens in the peritransplant period. For instance, pretransplant donor-specific blood transfusions are capable of prolonging organ graft survival time (1). However, such an approach also risks sensitization of the recipient to donor MHC antigens.
More recently, purified MHC class I molecule preparations have been used to study the immunologic effects of donor-specific molecules. Sumimoto and Kamada have shown that continuous infusion of soluble donor MHC class I antigen can significantly prolong allograft survival, and removal of this soluble antigen from the serum abolishes its effect (2). Unfortunately, such experiments are limited by the low availability and purity of donor MHC class I antigens. MHC class I antigens are difficult to purify and the in vitro synthesis is hindered by the complexity of the molecule, making whole MHC molecule-based strategies difficult at this time. However, MHC peptide based strategies have also been investigated. Recently, Clayberger and Krensky have shown that bulk purified peptides may be useful for inducing donor-specific unresponsiveness. More specifically, there is evidence that peptides corresponding to the more polymorphic regions of the alpha-1 and alpha-2 domains of the MHC class I molecule block lysis by cytotoxic T lymphocyte (CTL) in an allele-specific manner (3). In contrast, in vitro data also suggest that peptides corresponding to less polymorphic regions of the MHC class I molecule, specifically residues 75-84 of the alpha-1 helix, may decrease CTL activity in a non-allele-specific manner (4). In followup in vivo studies, B7.75-84, a decamer corresponding to residues 75-84 of the human leukocyte antigen (HLA)-B7 alpha-1 helix, combined with cyclosporine induced unresponsiveness to heterotopic cardiac allografts in the Lewis-to-ACI strain combination (5). B7.75-84 alone had no significant effect on graft survival in this strain combination.
In the present study, we report that B7.75-84 alone significantly prolongs heterotopic cardiac allograft survival in the WF-to-ACI strain combination. This model allowed us to investigate the immunosuppressive properties of B7.75-84 in vivo without the interference of cyclosporine. We have found that B7.75-84 induces a nonspecific decrease in helper T lymphocyte (HTL) function as reflected in decreased IL-2 production in response to donor and third-party antigen and a diminished IgG antibody response to donor MHC class I antigen. This decrease in HTL function is associated with a donor-specific decrease in CTL killing activity.
MATERIALS AND METHODS
Animals and transplantation. Adult male, pathogen-free ACI (RT1.Aa), Wistar-Furth (WF-RT1.Au), Lewis (RT1.Al) and Fischer (F344-RT1.Alv1) rats weighing 150 to 250 g were used in these studies. Animals were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were maintained according to standard NIH guidelines. Cardiac allografts were performed between ACI, Lewis, F344, and WF strains. Recipients were treated daily with B7.75-84 10 mg/kg/day i.v. on day 0 to day 4 perioperatively. Heterotopic cardiac allografts were performed using a modification of the technique originally described by Ono and Lindsey (6). Donor hearts were transplanted to the recipient's abdomen by anastomosing donor and recipient aorta, and donor pulmonary artery to the recipients inferior vena cava. Grafts were palpated to assess rejection, and nonpalpable contractions prompted direct inspection at laparotomy. Graft rejection time was defined as the time at which no cardiac contractions were either palpable or visible by direct inspection at laparatomy. Grafts were checked daily and time to rejection was measured from the day of transplant to the day of rejection in whole-day increments.
Peptides. Peptides were synthesized at UCB Bioproducts (Belgium) using an automated peptide synthesizer using F-moc chemistry. Peptides were purified by preparative reverse phase HPLC and were shown to be >90% homogeneous by analytical reverse phase HPLC. Amino acid content was confirmed by amino acid analysis. The amino acid sequence of B7.75-84 is as follows: R-E-S-L-R-N-L-R-G-Y.
CTL and HTL limiting dilution assays (LDA). The CTL-LDA was performed as described in detail previously (7). For the HTL-LDA ACI lymph node cells were diluted and 12-well replicates of serially diluted cells were distributed into 96-well plates, together with 1 × 105 irradiated (2000 rads) WF or third-party (Lewis) splenocyte stimulators. After 3 days of culture, 100 μl of supernatant was removed from each well and placed in new wells with 8 × 103 IL-2-responsive CTLL-2 cells (8). After 18-hr the CTLL-2 cells were pulsed with 3H-thymidine for 4 hr, cells were harvested, and CPM was determined. A well was scored as positive if 3H-thymidine uptake was >3 standard deviations above the mean spontaneous release as described previously (9). The frequency of CTL and HTL precursors was calculated using maximum likelihood method as outlined by Derry and Miller (10). In addition to precursor frequency analysis, each point in an LDA was plotted on a graph of cell number versus% target lysis (effector CTL activity) or CPM (corresponding to HTL IL-2 production), and a linear regression line was drawn. This line indicates the relative CTL killing activity or IL-2 production in LDA cultures. This analysis was included since we have observed in other experiments that the CTL and HTL precursor activity between test groups can be the same, while the killing activity or IL-2 production in the effector lymphocyte populations can be different. Therefore, the data compiled reflects not only CTL and HTL precursor numbers, but also indicates effector cell activity in the LDA cultures. Linear regression lines were compared by weighted analysis of covariants.
Antibody assay. Anti-WF IgM and IgG antibody levels were measured using a modification of the method reported by Morris and Williams (11). Briefly, standard dilutions of rat serum in phosphate buffered saline with 0.5% bovine serum albumin (PBS/BSA) were performed, and 25 μl were added to duplicate 12 × 75 mm polystyrene tubes. WF-strain blood was collected in heparinized tubes, washed two times with PBS/BSA, and red blood cells were diluted to 10% (v/v). Diluted cells were added to each test tube in 25-μl aliquots, and the serum/cell mixture was incubated for 1 hr with either 125I-labeled goat antirat IgM or 125I-labeled goat antirat IgG (antibodies purchased from Jackson Immunoresearch, West Grove, PA) antibody at 300,000 cpm/tube. Antibodies were radiolabeled with [125I]Na (Amersham, Arlington Heights, IL) using the Iodo-gen iodinating reagent as specified by the manufacturer (Pierce, Rockford, IL). After incubation, cells were washed and cpm was determined. All incubations were at 4°C.
Statistics. Treatments were compared by weighted analysis of covariance. An unweighted analysis of covariance was first performed to obtain predicted values. The reciprocals of the predicted values were used as weights in the weighted analysis of covariance. These weights are appropriate for a Poisson error structure. All analyses were performed with SAS statistical software (SAS Institute Inc.) (12).
To investigate the effects of B7.75-84 on T cell function, a rat strain combination was selected in which B7.75-84 treatment perioperatively prolonged cardiac allograft survival without cyclosporine (Fig. 1). Lewis recipients of ACI cardiac allografts treated with B7.75-84 had a slight prolongation of graft survival time when compared with the nontreated control (mean survival time, M.S.T. of 7.2 vs. 6.4, respectively) (P= 0.04). All allografts in this strain combination rejected by day 8. B7.75-84 treated ACI recipients of F344 cardiac allografts had significant graft prolongation (MST=15.8) when compared with untreated controls (MST=8.3 days, P=0.0027) while all allografts rejected by day 18. ACI recipients of WF allografts treated with B7.75-84 had the most substantial graft prolongation (MST=29.6) when compared with untreated controls (MST=11.8 days, P=0.0023). Moreover, approximately one-half of B7.75-84-treated ACI recipients of WF cardiac allografts had functioning grafts >25 days.
We investigated the effects of B7.75-84 on HTL and CTL in the WF-to-ACI strain combination. To evaluate the effect of B7.75-84 treatment on the lymphocyte response, cervical lymph nodes were harvested on day 10 posttransplant from B7.75-84-treated ACI recipients of WF cardiac allografts. In HTL, CTL and antibody assays, B7.75-84 treated recipients with prolonged graft survival time (GST) (>25 days) were compared with treated recipients which rejected grafts early (12-15 days). In addition, lymphocytes from a normal, naive ACI rat were tested in each assay to establish a baseline response to donor cells. A HTL-LDA was performed to estimate HTL precursor frequency and measure IL-2 production against donor and third-party alloantigen. Results of HTL assays from one representative experiment of 3 performed are shown in Figures 2 and 3. Diminished HTL precursor frequency in animals with prolonged GST was seen in response to both donor and third-party (Lewis) antigen when compared with animals that rejected grafts early. Figures 2 and 3 also show diminished IL-2 production in response to donor and third-party (Lewis) antigen at day 10 in treated recipients with prolonged GST when compared with recipients which rejected grafts early.
In contrast to HTL precursor frequency, CTL precursor frequency was not different in B7.75-84-treated recipients with prolonged graft survival when compared with recipients which rejected grafts early (Fig. 4). However, CTL killing activity against donor alloantigen was reduced in lymphocytes of treated recipients with prolonged graft survival when compared with recipients that rejected grafts early. CTL killing activity against third-party (Lewis) antigen at day 10 was equivalent (p=0.65) in all treated recipients (data not shown). This pattern of donor-specific decreased CTL killing activity in B7.75-84-treated animals with prolonged graft survival was consistent in all 3 experiments. Table 1 summarizes the HTL and CTL results of four animals with prolonged graft survival using B7.75-84 alone, compared with 6 treated animals which rejected grafts early.
Antibody analysis by radioimmunoassay was performed at day 8 in all treated recipients. Figure 5 summarizes the IgG and IgM antibody response to donor MHC class I antigen in treated recipients and a normal, untreated ACI control. Serum from 3 of 4 recipients with prolonged graft survival showed no measurable IgG antibody response against donor antigen, while all treated recipients that did not show prolonged graft survival consistently demonstrated an IgG response. In contrast, graft recipients showed a strong IgM response against donor antigen, regardless of graft survival outcome. Figure 5 shows results from one representative experiment; 2 additional B7.75-84-treated recipients with prolonged graft survival showed IgM antibody levels near those of B7.75-84-treated recipients without graft survival prolongation (data not shown). Results from all experiments are summarized in Table 1.
It has been previously reported that administration of MHC class I-derived peptide combined with cyclosporine induces tolerance in rat heterotopic cardiac allograft models. However, the contribution of peptide to this effect is difficult to separate at a mechanistic level from the immunosuppressive effects of cyclosporine. In the present study, we were able to use B7.75-84 peptide treatment alone perioperatively to prolong heterotopic cardiac allograft survival in the WF-to-ACI strain combination. Treatment of ACI recipients of WF cardiac allografts with B7.75-84 at 10 mg/kg/day from day 0 to day 4 resulted in significant prolongation of allograft survival time. More importantly, approximately one-half of B7.75-84-treated recipients in this strain combination retained allografts longer than 25 days compared with treated recipients that rejected allografts at 12 to 15 days. With this model, we were able to investigate the immunosuppressive effects of B7.75-84 in vivo without the interference of cyclosporine. Furthermore, the WF-to-ACI strain combination allowed us to study the effects of B7.75-84 in a system in which donor and recipient were completely MHC-disparate. Prolongation of heterotopic cardiac GST with B7.75-84 treatment alone has been reported only in the Lew.1W to Lew.1A congeneic strain combination prior to this study (13).
Examining the CTL and HTL responses in B7.75-84-treated recipients with prolonged graft survival and those animals that rejected grafts early provided insight into the immunosuppressive mechanism of B7.75-84. B7.75-84 treated ACI recipients of WF cardiac allografts with prolonged graft survival showed a decreased HTL precursor frequency and IL-2 production when challenged with donor antigen, compared with animals that rejected grafts early. Antibody analysis of serum from treated recipients with prolonged graft survival generally revealed no measurable IgG response. In comparison, treated recipients that rejected grafts early showed a positive IgG response at the same time point. Analysis of the antidonor IgM levels in all treated recipients showed a positive response regardless of donor allograft outcome. IgG responses are generally considered HTL-dependent while IgM responses are not. Therefore, the decreased antidonor HTL responsiveness and lack of an antidonor IgG response in treated recipients with prolonged graft survival suggests that B7.75-84 influences the HTL response. Interestingly, a study by Piquet (14) showed that while the IgG response is HTL-dependent, HTL may also have some influence on the magnitude of the IgM response. This may explain the lower IgM response in 1 of 2 treated recipients that demonstrated a positive IgM response (Fig. 5).
Our results indicated that treatment with B7.75-84 caused a decrease in CTL killing activity in treated recipients with prolonged graft survival. CTL precursor frequency was not affected in any of the treated recipients. Cuturi et al. recently reported that graft infiltrating lymphocytes in cardiac allografts in B7.75-84-treated recipients displayed low levels of cytotoxicity but a normal CTL proliferative response to donor antigen (13). One explanation of our data could be a nonspecific suppression of HTL cytokine production by B7.75-84, resulting in a decrease in CTL killing activity. In support of this hypothesis, T cell-mediated allograft rejection is at least partially dependent upon the interaction between lymphokine secreting HTL and effector CTL. Furthermore, Bishop et al. have shown in a murine cardiac transplant model a decrease in stimulated CTL in mesenteric lymph node after treatment with anti-CD4 monoclonal antibody (15).
In our model, a similar pattern of decreased HTL IL-2 production was seen in response to donor (WF) and third-party (Lewis) alloantigen. Therefore, perioperative treatment with B7.75-84 resulted in a nonspecific reduction in the peripheral HTL response. However, our results showed a donor-specific decrease in CTL killing activity while the precursor frequency was unaffected. It has been postulated that donor-specific immunosuppression or unresponsiveness is dependent on a T cell help deficit at the time of original exposure to donor antigen (16,17). Hall et al. reported that in a rat cardiac allograft model, cyclosporine treatment induced donor-specific unresponsiveness characterized by a HTL deficit (18). Therefore, it is plausible that administration of an immunosuppressive agent capable of a nonspecific reduction of HTL function at the time of original exposure to donor antigen could induce donor-specific immunosuppression. Similar to the nonspecific effects of cyclosporine on HTL function, the nonspecific inhibition of HTL that we observed with B7.75-84 treatment may have resulted in donor-specific immunosuppression of CTL.
The ability of B7.75-84 to enhance the immunosuppressive effects of cyclosporine has been seen in our data (unpublished observations) and other reports. Clayberger et al. demonstrated an increased rate of tolerance induction in rat cardiac allografts in rats treated with B7.75-84 and cyclosporine on day 0 to 4 when compared with cardiac allografts in rats treated with cyclosporine alone (5). While cyclosporine has multiple effects on T cell function (19), one prominent mechanism of action of cyclosporine is through diminished HTL IL-2 production. It is possible that the immunosuppressive synergy seen with cyclosporine and B7.75-84 is the result of blocking IL-2 production via alternate pathways.
It is intriguing that the B7.75-84 peptide, which is derived from a human MHC class I molecule, is immunosuppressive in rats. Interestingly, experiments performed in our lab (unpublished observations), along with previous reports (5), indicate that peptides corresponding to the same regions of the rat MHC class I molecule have no immunosuppressive effect in a rat cardiac allograft model. Therefore, the effects of B7.75-84 appear to be non-allele-specific. It is unlikely that B7.75-84 exerts its effects through a T cell receptor (TCR) inhibition mechanism, since those peptides that inhibit CTL effectors are also active on TCR-negative NK cells (13,20). This non-allele-specific, non-TCR-dependent inhibition of the immune response indicates that B7.75-84 may exert its effects through binding of a cell surface molecule. Recently, Krensky and coworkers (21) found that HLA-derived peptides bind to cell surface molecules expressed on T lymphocytes and this binding is associated with an increase in intracellular Ca++. It is possible that the nonspecific action of B7.75-84 on helper T cells is mediated by binding to these cell surface molecules. Although there is no evidence to date that suggests that B7.75-84 is processed via the indirect pathway by antigen presenting cells, peptide presented in this manner may also be suppressive to HTL. Regardless of the precise mechanism of action of B7.75-84, mounting evidence for the immunosuppressive properties of HLA-derived peptides suggests that investigation is warranted into their potential applications to organ transplantation.
Acknowledgment. We thank Dr. Dennis Heisey for his assistance in statistical analysis.
Abbreviations: CTL, cytotoxic T lymphocyte; GST, graft survival time; HLA, human leukocyte antigen; HTL, helper T lymphocyte; LDA, limiting dilution assays; MHC, major histocompatibility complex.
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