More than 70,000 patients with end-stage renal disease in the United States are currently awaiting renal transplantation but fewer than 18,000 total renal transplants are performed each year (1). A significant barrier to transplantation for these patients is sensitization to human leukocyte antigen (HLA). For those patients who have developed anti-HLA antibodies through pregnancy, transfusion, and prior transplants, the wait times are significantly longer than for those patients who have not developed anti-HLA antibodies (2). These recipients also have more rejection episodes and decreased graft survival compared with nonsensitized recipients (3–5). It is especially difficult to find compatible donors for patients who are sensitized to HLA (6, 7).
Two desensitization regimens have evolved to decrease or eliminate the donor-specific antibodies thereby allowing for successful transplantation (8–13). These regimens are the high-dose IVIG protocol and the plasmapheresis (PP) plus low-dose IVIG protocol (11, 14–17). Our approach has focused on investigating the efficacy of the high-dose IVIG protocol. We have reported the NIH-funded multicenter placebo-controlled trial showed IVIG significantly lowered anti-HLA antibody levels (P<0.04) and improved rates of transplantation (P≤0.02) compared with placebo (15). This protocol and a similar one used at our center from 2000 to 2005 required a 4-month period for desensitization. Recently, we have reported an improved protocol using two doses of IVIG and rituximab that requires less time, is less costly, and is effective in improving rates of transplantation in HLA-sensitized patients (16).
Critical to the success of these desensitization protocols is the monitoring of antibody levels to determine efficacy of treatment, both pre- and posttransplantation. Acceptable levels of DSA that allow for successful desensitization must be determined as well as post-desensitization DSA levels that allow for successful transplantation that permit long-term graft function. Zachary et al. (17) have shown that the initial titer and specificity of the DSA are critical in determining the likelihood of successful desensitization. We have reported an in vitro approach to identifying patients who are likely to benefit from desensitization (18). Quantitative solid-phase antibody methodologies have allowed for a more defined approach to monitor the feasibility and efficacy of the desensitization protocols. Here, we report our findings to determine the strength of DSA that would allow for successful transplantation after desensitization treatment with high-dose IVIG and rituximab, to identify patients at higher risk for AMR, and to monitor changes in posttransplant antibody course, which may identify points for therapeutic intervention aimed at improving graft outcome.
MATERIALS AND METHODS
The desensitization protocol consisted of two doses of human polyclonal IVIG (2.0 g/kg, maximum dose 140 g) on days 0 and 30. Rituximab (1 g) was given on days 7 and 22 to reduce or eliminate the donor-specific flow cytometry crossmatch positivity. The transplanted patients received alemtuzumab (30 mg SQ ×1) as induction therapy immediately posttransplant (16). The maintenance immunosuppression consisted of prednisone 2 mg/Kg with a rapid taper to 5 mg/day by 2 weeks posttransplant, mycophenolate mofetil 500 mg twice daily, and tacrolimus to maintain a target level of 7 to 9 ng/mL for the first 3 months, 6 to 8 ng/mL for months 3 to 6, and 5 to 7 ng/mL after 6 months.This robust protocol has enabled us to transplant 80% of patients treated (16). Sera from 16 patients sensitized to HLA with DSA were tested pretreatment, pretransplant, and at several times posttransplant. Eight patients received deceased donor transplants, and eight patients received living donor transplants after desensitization therapy. There was a follow-up time of up to 6 months for all patients.
The flow cytometry crossmatch and complement-dependent cytotoxicity crossmatches (CDC) were performed as previously described (15, 19). Three-color flow cytometry crossmatches were performed according to the method of Bray et al. (19), using a FACScan cytometer (Becton Dickinson, San Jose, CA). T and B lymphocytes were stained with phycoerythrin-conjugated mouse monoclonal antibody specific for human CD3 and CD19, respectively. The presence of bound antibody was determined using a fluorescein isothiocyanate-conjugated (FITC) anti-human IgG (Jackson Immuno Research Laboratories, West Grove, PA). T-cell flow crossmatch results were expressed as mean channel shift (MCS) over background. T-cell crossmatches were considered positive at more than 50 MCS and B-cell flow crossmatches more than 100 MCS. B-cell crossmatches were not performed after the patient received rituximab.
The binding level of donor-specific antibody was determined by the multianalyte bead assay performed on the Luminex platform. The single antigen Luminex bead assay was standardized with Quantiplex beads (One Lambda, Inc., Canoga Park, CA), and results were expressed as standard fluorescence intensity (SFI). Briefly, 20 μL of sample serum were added to 5 μL mixed beads, HLA class I and class II single antigen Luminex beads (One Lambda, Inc.), and incubated in the dark for 30 min at room temperature. After washing with wash buffer, 100 μL goat anti-human IgG secondary antibody conjugated with R-phycoerythrin was added to the beads followed by a 30-min incubation in the dark at room temperature. After a wash step, the samples were read on the LABScan 100. Precalibrated Quantiplex beads were used to convert trimmed mean fluorescent values of test samples into SFI units. Final DSA assignment and binding level was analyzed through HLA Visual 2.2 software (One Lambda, Inc.).
Biopsies were performed for all recipients with suspected rejection episodes. The biopsies were graded using the Banff 97 classification (20). Antibody-mediated rejection was defined by C4d deposition (21). Delayed graft function was defined as the need for dialysis during the first week posttransplant.
We reported that patients undergoing this desensitization therapy showed a significant decrease in T-cell flow crossmatch results performed on serum samples obtained before the initiation of therapy and immediately pretransplant (212.75±95 to 148.9±97 MCS, P=0.02) (16). The posttreatment B-cell crossmatches and posttransplant T- and B-cell crossmatches are technically challenging to perform due to the presence of rituximab (anti-CD20) and alemtuzumab (anti-CD52) in the patients' sera. The use of the single antigen beads to monitor the pre- and posttransplant binding of class I- and II-specific DSA with the quantitative Luminex technique allows the ability to follow changes in DSA binding.
Figure 1 shows the relationship between the SFI obtained for the highest DSA bead and the MCS obtained for the T-cell flow crossmatch with the donor cells using the same serum sample. The flow crossmatches are considered negative at less than 50 MCS. After desensitization, 6 of the 16 patients had negative T-cell flow crossmatches (<50 MCS) with a concomitant mean binding of 8,805 SFI (range, 660–22,934) for the highest DSA bead. Five patients had T-cell flow crossmatches ranging from 78 to 192 MCS with mean DSA levels of 55,869 SFI (range, 15,680–166,942). No patients in either group experienced AMR. Five patients had T-cell flow crossmatches ranging from 242 to 267 MCS with mean DSA levels of 118,063 SFI (range, 45,684–214,667). Three of these patients experienced AMR. The mean MCS and DSA levels for patients with AMR were significantly higher than those of all patients without AMR (MCS: 248 vs. 102, P=0.02, SFI: 35,251 vs. 172,854, P<0.001). Nine patients had class II DSA at the time of transplant. The mean class II DSA level for the two patients with AMR was 234,483 SFI when compared with 60,896 SFI for those with no AMR (P=0.014). B-cell flow crossmatches were not assessed due to the interference of rituximab. All patients had negative CDC T-cell crossmatches with the exception of the patient 18117 with the T-cell flow crossmatch (222 MCS) who had a positive CDC T-cell crossmatch with undiluted serum that became negative at the 1:2 dilution. The average class I mismatch for all patients was 3.2 for HLA-A and B antigens. The average class II mismatch was 2.8 for HLA-DR and DQ antigens.
Posttransplant serum samples were obtained for all patients to follow DSA strength and specificity; long-term data has been obtained for 11 patients. Two major patterns were observed. For those patients without complications (n=7), DSA strength remained below 105 SFI and decreased to approximately 104 SFI by 5 to 8 weeks posttransplant for both class I and II DSA for all but one patient. Although third party decreased, it was not eliminated below 20,000 SFI in all cases within the 6-month follow-up time. For patients with AMR or delayed graft function (n=3), the DSA remained at more than 105 SFI or rebounded to that level. Most third party–directed antibody decreased but some did not decrease below the 105 SFI.
Figure 2 illustrates representative results for patients without immune complication. Patient 18134 (Fig. 2a) received a kidney graft from her spouse that was mismatched for one HLA-A, two B, two DR antigens, and one DQ antigens. The patient's serum contained DSA to HLA-B38. At the time of transplant, the patient had class I DSA to HLA-B38 and the concomitant T-cell crossmatch was 171 MCS. Weak antibodies to HLA-Cw7, DR12, and DQ2 were also detected but remained below 104 SFI at all time points tested. The HLA-B38 binding decreased from 38,144 SFI pretreatment to 18,964 SFI at transplant and 863 SFI posttransplant. This patient has not experienced AMR and has stable creatinine levels.
Patient 15787 (Fig. 2b) received a deceased donor allograft mismatched for seven HLA-A, B, DR, and DQ antigens. The T-cell crossmatch at the time of transplant was 196 MCS. The patient's serum contained weak binding to HLA-A30 and stronger binding to HLA- DR4, 15, and DQ6 class II antigens. The binding to the class II antigens decreased posttransplant to levels less than 14,000 SFI for both DR beads and less than 1,000 SFI for the DQ bead; the binding to the HLA-A30 bead remained low at less than 1,000 SFI. The patient had third party–directed antibody to the public epitope HLA-Bw6 that remained above 105 SFI and showed no change subsequent to transplant (results not shown). The patient has not experienced AMR and has a stable creatinine level.
Figure 3 shows representative results for patients who experienced AMR or delayed graft function. Patient 18117 (Fig. 3a) received a deceased donor allograft mismatched for 3 HLA-A, B, antigens and no mismatches for HLA-DR and DQ antigens. The pretransplant crossmatches revealed strong binding for the T-cell flow crossmatch of 225 MCS and a weakly positive CDC crossmatch that was negative at the 1:2 dilution. These results were unexpected because none of the mismatched antigens were identified as being unacceptable based on the single antigen specificity analysis of the serum obtained posttreatment. The HLA typing information in UNOS DonorNet did not include typing information for HLA-Cw and DP. Subsequent typing in our laboratory revealed the graft was also mismatched for Cw2 and DP3 antigens, and the patient had antibodies to both antigens. The patient developed a rapid decrease in renal function on day 2 posttransplant and was treated with plasmapheresis X3 followed by repeat IVIG (2 g/kg) and rituximab (375 mg/m2) on day 10 posttransplant for presumed AMR. The creatinine levels started to drop but the antibody levels remained high at more than105 SFI. The antibody binding to the HLA-Cw2 bead decreased at 2 months posttransplant but is currently above 105 SFI. The creatinine levels remain stable (1.3 mg/dL) and a biopsy performed 60 days posttransplant showed no evidence for AMR.
Patient 17240 (Fig. 3b) received a deceased donor graft mismatched for 6 HLA-A, B, DR, and DQ antigens. The patient's serum contained antibody to the donor antigens: HLA-A26, B39, DR7, and DR13. The pretransplant T-cell flow cytometric crossmatch showed that 235 MCS and the CDC crossmatch was negative. Immediately posttransplant, the antibody levels started to decrease below the 105 SFI level. However, at 1 month posttransplant the levels started increasing with binding to the HLA-A26, DR7, and DR13 beads exceeding the 105 SFI level. Concomitant creatinine levels had not increased. Nonetheless, a biopsy was ordered and revealed C4d deposition. IVIG (2 g/kg) and rituximab (375 mg/m2) was given to the patient to preemptively treat the AMR episode. The patient now has stable creatinine (0.9 mg/dL) and no signs of acute rejection. There have been two graft losses. One patient experienced accelerated acute rejection (pretransplant crossmatch=266 MCS). One patient experienced late onset AMR (>6 months posttransplant) because of medication reduction and delayed transfer to the transplant center for treatment. For all patients entered into the protocol, the 1-year patient and graft survival rates are 100% and 94%, respectively.
We have reported here on the levels of DSA that allow for successful transplantation with a lower risk for AMR. With this desensitization protocol, approximately 63% of patients in this study were transplanted with a positive donor specific T-cell flow cytometry crossmatch. Important to facilitating the transplantation of the sensitized patients is the ability to accurately characterize the specificity and strength of the HLA antibodies (7, 17, 22) by solid-phase assays such as the Luminex assay used in this study. While implementing the newer solid-phase techniques and determining relevance of the results, we performed retrospective analysis where outcome was known. We found patients with DSA more than105 SFI in single antigen Luminex bead analysis and T-cell flow cytometry crossmatches more than 200 MCS were at higher risk for AMR, albeit reversible. We determined the cutoff values based on the immunosuppression protocol and desensitization protocol used at our center. Although these values can be used as guidelines, the relevant cutoff values for each center need to be based on the immunosuppression and desensitization protocols implemented at that particular center and that program's ability to handle high-risk patients. There are technical and immunosuppression protocol dependent factors that influence the cutoff values that must be established by each transplant center (23–27). Quantitative SFI and mean fluorescent index values (MFI) can be used as cutoff values by transplant programs. These values depend on the reagents used by the laboratory and may change slightly with each reagent lot number. Because not all beads have the same amount of HLA antigen captured on the bead, cutoff indicators may vary slightly from one antigen bead to the next.
We found that seven MCS values of pretransplant sera resulted in negative flow T-cell crossmatches (<50 MCS) with a mean SFI of 8,805 SFI. Three of these sera gave SFI single antigen bead values more than 104 SFI above the correlation curve. Certainly, single antigen beads can be coated with a higher density of antigen than that expressed on T cells thereby resulting in a positive binding but a negative flow crossmatch. Consideration must also be given to the possible influence of donor-specific factors such as statins on the expression of HLA that may affect the binding observed in the crossmatches (2). Thus, careful consideration of MCS and SFI or MFI values must be evaluated to determine unacceptable antigens for a virtual crossmatch, which identifies antigens that must be avoided in potential donors to achieve a negative flow crossmatch in solid organ transplantation (6, 27–29).
In our study, patients with donor specific T-cell flow crossmatches less than 200 MCS and donor antigen–specific bead binding less than 105 SFI did not experience early AMR. These results show that successful transplantation can be achieved with low levels of donor-specific antibodies. Our results are consistent with those of Zachary et al. (17) who reported that titer and specificity influenced the efficacy of the desensitization protocols and transplant outcome. In our study, the strength of the antibody appeared to be critical in determining the level at which successful transplantation could be accomplished. Further, levels of DSA more than 105 SFI indicated the need for close posttransplant monitoring of antibody level and specificity.
The posttransplant assessment of antibody course and specificity showed that patients without immune complication had similar antibody profiles. Patients with DSA levels below 104 SFI were at low risk for AMR and patients with DSA SFI between 104 and 105 with pretransplant crossmatches less than 200 MCS. Both DSA and third party antibody levels continued to decrease, eventually below the 104 level considered to be flow crossmatch negative (Fig. 2). These patients' antibody courses were monitored weekly for the first month posttransplantation and then monthly for 3 months. The creatinine levels did not indicate a change that required more frequent testing. These results are in contrast to those of Zachary et al. (17) who reported that rebound in antibody levels can occur subsequent to low-dose CMVIg and plasmapheresis desensitization therapy. Their protocol appears to require the presence of donor antigen in the form of the transplanted organ to maintain the elimination or reduction of DSA levels (17). With the robust desensitization and immunosuppression protocols used in this study, the creatinine levels continued to decrease, and monitored antibody levels showed no early rebound effect. Further, our desensitization protocol shows a persistent decrease in antibody levels before transplant, which affords applicability to deceased and live donor transplantation (11, 14, 16).
Patients with DSA more than 105 and donor-specific crossmatches more than 200 MCS were considered at high risk for AMR and warranted more frequent antibody level monitoring posttransplantation. Anticipating the antibody course of patient 18117 was particularly challenging because the deceased donor was not typed for the relevant donor-specific HLA-Cw2 or DP3 antigens. However, given the high level of pretransplant donor cell–specific flow cytometric crossmatch results in the apparent absence of unacceptable antigens, the donor cells were typed for the all HLA specificities. Thus, donor-specific antibodies to the Cw and DP antigens were identified quickly after transplant. There have been proposals to type all deceased donors for antigens other than HLA-A, B, and DRB1. Certainly, donors should be typed for HLA-DQ and DR51, 52, 53 because many patients make antibodies to those specificities and clinical relevance has been implicated (2). There have also been reports of the clinical relevance of antibodies to the antigen expressed at a low level such as HLA-DP (30–32); however, the cost effectiveness of typing all deceased donors for both antigen groups remains speculative. With a complete antibody specificity analysis, the presence of these antibodies can be determined pretransplant and retrospective typing of deceased donors for these antigen groups can be accomplished quickly at the transplant center. Rescue therapy can be anticipated and implemented if necessary. For living donors, complete HLA typing can be performed prospectively to allow for comprehensive interpretation of crossmatch results. In both cases, preemptive treatment can be anticipated.
The goal of posttransplant monitoring is to predict graft outcome such that preemptive therapy can be initiated to prevent or thwart any immune injury to the graft. In the case of patient 18117, intervention did result in a transient decrease in antibody levels. However, DSA returned to pretransplant levels with no evidence of AMR. Previous studies indicate that antibodies to class I and class II HLA-DRB1 and DQ antigens decrease more rapidly than antibodies to antigens expressed at a lower level such as DR51–53 (33, 34). The DSA Cw and DP in this case would also be considered to be expressed at a low level. In a clinically quiescent graft, these antigens may not be expressed at levels detectable by antibodies. The biopsy results to date do not show C4d deposition, which is consistent with a quiescent state of accommodation (35–37). Posttransplant antibody assessment for patient 17240 showed an increase in antibody specific for HLA-DR13 at 4 weeks posttransplant. Although there was no significant increase in the creatinine or any other clinical indication of acute rejection, the patient was treated with IVIG. Subsequent biopsy results showed C4d deposition. This approach shows that an increase in DSA preceded any rise in creatinine and illustrates the potential to treat implement antirejection treatments preemptively in an attempt to assuage deterioration of the graft.
Low levels of DSA are known to persist after desensitization. Gloor et al. (38) reported that DSA was detected by flow cytometric crossmatch and solid-phase antibody assays for 4 months posttransplantation in most patients in their study. The persistence of DSA did not correlate with AMR or early graft loss for these patients. In our study, DSA levels decreased posttransplantation to levels not expected to be detected by flow cytometric crossmatches for most of the patients. However, for three patients, antibody levels remained high at levels that probably would be detected by the flow cytometric crossmatch. The level of expression and distribution of the target HLA is known to correlate with the ability of the antibody to be eliminated. Zachary et al. (17) reported that 75% of class I and 60% of HLA-DRB1 and DQ-specific class II antibodies were eliminated. Antibodies to HLA-DR51, 52, and 53, which are expressed at much low levels on the cell surface, were not as readily eliminated. They also reported that recipients with DSA detected only by flow cytometric methods were not at an increased risk for AMR.
The three patients in our study with DSA levels more than 105 experienced early immune complication but currently do not have evidence of AMR despite high DSA levels. One patient has DSA to antigens expressed at low levels on the cell surface (HLA-Cw and DP); however, the other patients have antibody to HLA-DRB1 and DQ encoded antigens. Some level of DSA may not be detrimental and may even be beneficial (36–37, 39, 40). Accommodation has been described in ABO incompatible allograft transplantation for grafts that maintain normal function in the presence of low level ABO-specific antibodies. Immune regulation, whether evidenced by the presence of T-regulatory cells or changes in the Th1 versus Th2 components, may decrease inflammation resulting in production of antiapoptotic proteins. Other reports show that class I DSA may result in signaling cascades that include survival proteins in endothelial cells (41–44). The mere presence of DSA may not pose a risk to the graft and may actually enhance survival. Nonetheless, increasing levels of DSA do appear to precede AMR. Thus, defining the level of DSA and the potential risk to the graft is challenging and not straightforward.
In summary, these results show a critical strength of antibody can be determined subsequent to desensitization therapy that will allow for successful transplantation. More than 60% of patients in our study were transplanted with a positive donor T-cell flow cytometric crossmatch. Higher levels of DSA associated with AMR can be identified allowing for focused posttransplant monitoring and anticipated preemptive therapy.
The authors acknowledge Andrea Selby for preparation of manuscript, the HLA Laboratory team for their technical expertise, and the Transplant Immunotherapy Program team for their excellent patient care and support.
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