The immune response to an allograft remains the major obstacle to successful transplantation despite modern immunosuppressive therapy. Lymphocyte depletion at the time of organ transplantation by total lymphoid irradiation or antibodies against lymphocyte cell-surface molecules may successfully induce tolerance in organ transplantation (1,2). Campath-1H is a humanized CD52-specific monoclonal antibody that depletes lymphocytes in humans and reduces the need for maintenance immunosuppression after renal transplantation (3). A recent study reported that all patients who received perioperative Campath-1H treatment without any maintenance immunosuppression developed reversible rejection episodes within 1 month, despite laboratory evidence of profound lymphocyte, especially T-cell, depletion. The mechanism of this type of acute rejection is not well known (4).
This study (1) characterized human leukocyte antigen (HLA) antibody production after renal transplantation in patients treated with Campath followed by sirolimus monotherapy and (2) determined the correlation between HLA antibody production and serum creatinine levels.
MATERIALS AND METHODS
Patient Selection and Immunosuppressive Protocol
Patients were enrolled under an institutional review board-approved protocol at the University of Wisconsin-Madison after informed consent. The protocol was also conducted under Food and Drug Administration surveillance through an investigational new drug to S.J.K. for off-label use of Campath-1H. Patients consented to biopsies of their kidney transplant at 6 months and 12 months, and to blood leukocyte population monitoring by flow cytometry. Primary cadaveric and living-donor adult renal transplant recipients (ages 18–60 years) were selected on the basis of the following criteria: current panel reactive antibody (PRA) less than 10%, historical peak PRA less than 25%, and body mass index less than 32. Patients with a 0 antigen mismatch were excluded, as were patients who were cytomegalovirus seronegative but had a cytomegalovirus seropositive donor. Donor kidneys from non–heart-beating donors were excluded, as were kidneys from donors older than 55 years or kidneys preserved for more than 36 hr. A negative National Institutes of Health crossmatch test was required before transplantation. Control patients were no older than 65 years of age and received cadaveric or living-donor kidneys. Those patients who were HLA identical to their donor were excluded as controls.
Campath-1H, a humanized anti-CD52 monoclonal antibody, was provided by Millennium Pharmaceuticals (Cambridge, MA), and later Ilex, Inc. (San Antonio, TX). A dose of 20 mg was administered intraoperatively on the day of transplant (day 0), and a second dose of 20 mg was given the day after transplantation (day 1). Thirty minutes before the Campath-1H infusion, the patients were administered 500 mg of methylprednisolone. Sirolimus (Rapamune, Wyeth, Philadelphia, PA) was administered at a dose of 2 mg orally starting on the day after the transplant. Doses were adjusted to achieve blood levels in the 8 to 12 ng/mL range. No other immunosuppressive medications were given, unless patients experienced rejection.
Screening of Human Leukocyte Antigen Class I- and II-Specific Antibodies by LATM Enzyme-Linked Immunosorbent Assay
Lamba Antigen Tray-Mixed (LATM) was used as directed by the manufacturer’s instructions (One Lambda) to identify immunoglobulin (Ig)G HLA class I- and II-specific antibodies. Diluted test serum (10 μL of 1:2) was added to the HLA class I- and II-specific wells in duplicate. Positive and negative quality control reagents were also added to the appropriate wells. Test and control sera were removed from the LATM trays after a 60-min incubation at 22°C by hand flicking. The trays were then washed twice with buffer before 10 μL diluted anti-human IgG alkaline phosphatase-conjugated antibody was added to each well. The tray was incubated for another 40 min at 22°C. After two further washes, 10 μL colorimetric enzyme substrate BCIP (Blue Phos; Kirkegard and Perry Laboratories) was added and allowed to develop at 37°C. The reaction was stopped after 10 to 15 min by adding 5 μL of stop reagent to each well. The assay was read at 630 nm with a Bio-Tek ELX 800 enzyme-linked immunosorbent assay (ELISA) tray reader and One Lambda computer software. Results equal to or greater than twice the value for the mean of the negative controls were determined to be positive.
Determination of Specificity of Human Leukocyte Antigen Class I and II Antibodies by FlowPRA-Single Antigen or Lamba Antigen Tray-Single Antigen
The assay was performed according to the manufacturer’s instructions (One Lambda) by using a set of the HLA bead groups. Each group (5 μL), which contained eight different antigen-coated beads, was incubated with 20 μL human serum for 30 min at 20°C to 25°C. The beads were washed three times with wash buffer, pelleted by centrifugation at 9000g for 2 min, and then incubated for 30 min with 100 μL fluorescein isothiocyanate-conjugated F(ab)2 fragment of goat antihuman IgG (Fc λ fragment specific, 1:100 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA). The beads were washed three times with 1 mL wash buffer and fixed with 0.5 mL fixing solution. The green and yellow fluorescence of 5,000 events were then analyzed on a flow cytometer. Antibody levels were expressed as linear values V (V=10D(C/M), D:decades (4), C=channel shift, M=1024).
This assay was used as directed by the manufacturer’s instructions (One Lambda) to identify the specificity of HLA antibody. Diluted test serum (10 μL of 1:3) was added to the single HLA antigen-coated wells (1 antigen/well), and positive and negative quality control reagents were also added to the appropriate wells. Test and control sera were removed from the trays after a 60-min incubation at 22°C by hand flicking. The trays were then washed twice with buffer before 10 μL diluted anti-human IgG alkaline phosphatase-conjugated antibody was added to each well. The tray was incubated for another 40 min at 22°C. After two further washes, 10 μL colorimetric enzyme substrate BCIP (Blue Phos; Kirkegard and Perry Laboratories) was added and allowed to develop at 37°C. The reaction was stopped after 10 to 15 min by adding 5 μL of stop reagent to each well. The assay was read at 630 nm by using a Bio-Tek ELX 800 ELISA tray reader and One Lambda computer software. Results equal to or greater than twice the value for the mean of the negative controls were determined to be positive.
Immunostaining for the C4d complement component was performed in patients with graft dysfunction. A three-step immunofluorescence technique with monoclonal antibody 10–11 (Biogenesis, Brentwood, NH) was used following the protocol by Collins et al. (5). Frozen sections of biopsies from two cases were stained by using a Benchmark automated stainer (Ventana Medical Systems, Tucson, AZ) applying the same primary antibody at a 1:600 dilution and by using a horseradish peroxidase detection system. All biopsies were interpreted at the University of Wisconsin (A.F.), and biopsies with evidence of rejection were also reviewed independently by Dr. Robert Colvin (Massachusetts General Hospital) as an external study monitor.
The statistical significance of differences in proportion was analyzed with the chi-square test.
Human Leukocyte Antigen Antibody Production and Specificity
By using LAT ELISA and FlowPRA, we tested sera from 24 patients 1 to 24 months posttransplant for the presence of HLA class I and II antibody and the specificities of these antibodies. As shown in Table 1, 10 of the 24 patients treated with Campath-1H and sirolimus produced measurable HLA antibodies in serum. Six of the 10 (identification numbers: 3, 4, 5, 7, 8, and 10) developed both donor-specific antibodies (DSAs) and non–donor-specific antibodies (NDSAs), whereas only NDSAs were detected in the other four patients. Within 24 months posttransplant, 4 of the 10 patients who were HLA antibody positive (identification numbers: 3, 5, 8, and 10) experienced humoral rejection as evidenced by positive C4d staining of biopsies. At the time points when biopsy immunostaining was positive for C4d (six biopsies from four patients), NDSAs were detected in six of six sera and DSAs were detected in five of six samples. In these four patients demonstrating humoral rejection, as serum creatinine levels increased, there were a higher number of strong positive HLA antibody responses (both DSA and NDSA).
Correlation Between Human Leukocyte Antigen Antibodies and Serum Creatinine
Four patients demonstrating humoral rejection (identification numbers 3, 5, 8, and 10) were followed long enough posttransplantation to enable analysis of the numeric relationship between serum creatinine and HLA antibody and to observe their trends over time. As shown in Figure 1A for a particular patient (identification number 10, Table 1) who experienced reversible acute humoral rejection at 1-month posttransplant, levels of both DSAs (A1, 26) and NDSAs (A2, 29, 66 and 69) paralleled the patient’s serum creatinine level. The correlation coefficients between levels of serum creatinine and A1-, A26-, A2-, A29-, A66-, and A69-specific antibodies were 0.72, 0.966, 0.867, 0.973, 0.986, and 0.995, respectively. Most of the other positive DSAs and NDSAs showed similar correlation with serum creatinine (data not shown). The patient received rejection treatment (prednisone, intravenous immunoglobulin, Thymoglobulin, plasmapheresis, and rituximab) (6) because of an acute humoral rejection as evidenced by positive C4d staining and increased serum creatinine level at month 1. Production of both DSAs and NDSAs was significantly inhibited, and serum creatinine decreased to a normal level after rejection treatment (Fig. 1B).
Accumulating evidence has demonstrated that the presence of HLA antibody is associated with transplant rejection (7). It has been observed that preformed HLA antibodies can immediately destroy a transplanted organ. Therefore, crossmatch methods, which determine whether a recipient has preformed anti-DSA, were introduced into clinical use and proved to be helpful in improving graft survival (8–11). In a closely monitored renal transplant patient group, all graft failures caused by chronic rejection were preceded by the presence of HLA antibodies (12). Another recent renal transplantation study suggested that production of HLA antibodies is predictive of transplant failure (13).
In our previous protocol with Campath-1H as an induction agent followed by sirolimus monotherapy, 20 mg Campath-1H was given intraoperatively on the day of transplantation (day 0), and a second dose of 20 mg was given the day after transplantation (day 1). Peripheral lymphocyte counts significantly decreased for both T and B cells, but this effect was more prolonged for T cells than for B cells (6). In the current study, we found that 42% of patients treated with Campath-1H and sirolimus developed either DSAs or NDSAs or both. This incidence is significantly higher than that found in a recent study by Terasaki and Ozawa (14). In Terasaki and Ozawa’s study, 4,763 patients from 36 centers were analyzed, and the overall frequency of HLA antibodies among kidney transplant recipients was 20.9% (19.3% in liver recipients, 22.8% in heart recipients, and 14.2% in lung recipients). Patients treated with cyclosporine A (CsA) and mycophenolate mofetil (MMF) demonstrated significantly lower antibodies (9.8%) than those treated with CsA and azathioprine (18.1%) (0.00008), which provided strong mechanistic evidence to explain why patients treated with MMF demonstrated significantly higher graft survival than those who received no MMF, as we reported previously (15). These data indicate that selective suppression of both T and B cells by MMF in combination with primary agents, tacrolimus, CsA, or Neoral, which are predominantly directed at T cells, more effectively prevent alloantibody formation than the combination of Campath-1H induction plus sirolimus monotherapy. Other data also indicate that B-cell–mediated antibody responses may play a role in chronic rejection, a major cause of late organ failure (12,16–18). The following possibilities may explain why HLA antibodies were detected in more patients from this Campath-1H and sirolimus clinical trial compared with those patients taking MMF: (1) Campath-1H was found to have an average half-life of 2 to 3 weeks in a pharmacokinetics study in bone marrow transplant recipients (19), which may not be sufficient to keep the B-cell counts low enough and inhibit antibody production. (2) There is no convincing evidence suggesting that sirolimus alone can inhibit antibody production despite its potent inhibitory effect on T cells (20). (3) MMF is a strong inhibitor for not only T cells but also B cells, which produce antibodies when activated (21).
Staining of C4d in graft capillaries is a useful method to detect antibody-mediated rejection in situ, and it is now incorporated in the “Banff classification” (22). Several studies have put much effort into attempting to correlate capillary C4d with the presence of circulating HLA antibodies. Unfortunately, this correlation was not perfect when a conventional microcytotoxicity panel test or the FlowPRA specific test with panel class I or II antigens was used to measure circulating alloantibodies (22–24). In this study, by using single-antigen ELISA trays and newly developed single-antigen flow beads, we successfully detected NDSAs in all six available sera from patients whose biopsies revealed C4d positive staining, whereas five of six sera were positive for DSAs (Table 1). Because C4d immunostaining was only performed in patients with graft dysfunction (elevation of baseline serum creatinine >20%) in this study, more biopsies with C4d staining would need to be performed to confirm the correlation between C4d and HLA antibody. Both serum and biopsy samples would also need to be collected at time points when serum creatinine levels are still within normal range. Single-antigen flow beads or single-antigen ELISA trays are more sensitive in detecting HLA antibody, which provide better tools to prove the correlation between these two humoral rejection indexes.
To prove the causal relationship between HLA antibody and transplant rejection, close correlation between those two factors has to be shown. Accumulated evidence has shown that antibody can cause hyperacute rejection; it is also associated with acute and chronic rejection (7). But in almost all of the studies reported so far on transplant rejection and HLA antibody, investigators provided sufficient evidence to show that transplant rejection is preceded by HLA antibodies in most cases of rejection. In this study, we demonstrated the numeric relationship between the hypothesized cause and effect of HLA antibody and serum creatinine. In this closely monitored small clinical trial, serum creatinine is significantly correlated with DSAs (Table 1 and Fig. 1). Notably, we also found that serum creatinine is closely correlated with NDSAs with correlation coefficients that are similar to those of DSAs. As shown in Table 1 and Figure 1, not all the mismatched donor HLA-specific antibodies were detected at all time points in peripheral blood. The levels of DSAs are often not high and are sometimes below the threshold of in vitro detection, and many of the NDSA levels in patient peripheral blood were higher than those of DSAs (Fig. 1). This may be because DSAs are produced, but they are absorbed in the graft during the process of rejection, whereas NDSAs, either produced by cross-activation or other mechanisms, remained in the periphery without being trapped within the graft. These data indicated that NDSA may be an accurate, although nonspecific, indicator of immune responsiveness to the donor. High levels of NDSAs, which might be produced by activated plasma cells by cross-reaction or other mechanisms, were also found in other studies (12,25). More important, compared with serum creatinine, which is an index of graft dysfunction, HLA antibody detection provides a more sensitive and specific index to monitor patients’ humoral responses against the allograft, especially for patients whose serum creatinine levels are still within normal range (Fig. 1).
Although HLA antibodies have been shown to be associated with transplant rejection by many studies, larger clinical trials that include monitoring of antibody are needed to further establish the causal relationship between HLA antibody and rejection. Meanwhile, it should also be realized that other polymorphic antigens may induce antibody responses that mediate organ injury (26–28).
The authors acknowledge Nadim El-Awar, Karen Ng, and Chris Macaraeg for their technical support; Shari Denham and Michiko Taniguchi for their helpful assistance in the preparation of this article.
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