Human Immunology xxx (2015) xxx–xxx

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Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation Rob Higgins a,⇑, David Lowe b, Sunil Daga a,c, Mark Hathaway b, C. Williams b, F.T. Lam a, Habib Kashi a, Lam Chin Tan a, Chris Imray a, Simon Fletcher a, Nithya Krishnan a, Pat Hart a, Daniel Zehnder a,c, David Briggs b a b c

Transplant Unit, University Hospitals Coventry and Warwickshire, Coventry, West Midlands, United Kingdom Histocompatibility Laboratory, National Blood and Transplant, Birmingham, West Midlands, United Kingdom Clinical Sciences Research Laboratory, Warwick Medical School, University of Warwick, Coventry, West Midlands, UK

a r t i c l e

i n f o

Article history: Received 16 April 2014 Revised 10 October 2014 Accepted 1 June 2015 Available online xxxx Keywords: HLA antibodies Antibody incompatible kidney transplantation Antibody mediated rejection Transfusion Pregnancy HLA Class 1 HLA Class 2

a b s t r a c t Acute antibody mediated rejection after HLA-specific antibody incompatible renal transplantation is related to donor specific HLA antibody (DSA) levels. DSA levels may rise sharply after transplant, and aim of this study was to examine changes in DSA levels, particularly according to the primary sensitising event. Changes in 220 HLA specificities in 64 patients over the first 30 days after transplantation were evaluated using microbead assays. The greatest increase from pre-treatment to peak DSA levels was seen in pregnancy-stimulated specificities, median (IQR) increase in MFI of 1981 (94-5870). The next highest increase was for those sensitised by transplant with repeat HLA epitope mismatch, at 546 (-308-2698) (p < 0.01). The difference was especially marked when the pre-treatment antibody level was low; with pre-treatment MFI 1000 in 19/26 (73%) of pregnancy stimulated specificities, compared with 9/29 (31%) for all others (p < 0.001). DSA production to specificities stimulated by previous pregnancy was marked, even from very low pre-transplant levels. By contrast, there was a lower rate of antibody resynthesis to specificities repeated from previous transplants, both at antigen and epitope levels. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction HLA specific antibodies are a well known obstacle to renal transplantation but which can be overcome. This is becoming increasingly successful using risk stratification and knowledge of how these antibodies behave [1,2]. In such cases early post-transplant antibody resynthesis can occur and this is typically associated with rejection [3]. Although all desensitisation recipients have, by definition, donor HLA-specific immune memory, the magnitude and timing of this response and the concomitant rejection can be hard to predict.

Abbreviations: ATG, anti-thymocyte globulin; CDC, complement dependent cytotoxicity; DSA, donor specific antibody; FC, flow cytometry; IQR, interquartile range; MFI, mean fluorescence intensity. ⇑ Corresponding author at: Renal Unit, University Hospital, Clifford Bridge road, Coventry CV2 2DX, UK. E-mail address: [email protected] (R. Higgins).

Changes in HLA antibody levels post transplantation have been described using Leucocyte crossmatching [4–6] and microbead technology [7–9], but these data do not inform on the drivers of early responses. There is an increased risk of early antibody mediated rejection, even down to the low DSA levels detectable by bead assays [10–15]. However the heterogeneity of post transplant DSA responses means that some patients may unnecessarily be denied transplants for fear that their DSA levels might rise sharply post-transplant, and others may receive high level immunosuppression yet subsequently do not show an antibody response post transplant, suggesting possible over-treatment and excessive exposure to the risk of treatment-related adverse events. We aimed to determine whether the original source of mismatched HLA protein exposure was important in the post-transplant antibody response, as we have previously found a high rate of rejection when pre-sensitised women received transplants from their spouses or children [9]. On immunological first principles, a repeat HLA mismatch in a second or subsequent

http://dx.doi.org/10.1016/j.humimm.2015.06.013 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Please cite this article in press as: Higgins R et al. Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation. Hum Immunol (2015), http://dx.doi.org/10.1016/j.humimm.2015.06.013

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R. Higgins et al. / Human Immunology xxx (2015) xxx–xxx

transplant might be associated with a high risk of antibody resynthesis. Some studies of clinical outcomes have shown no variation in graft function with repeat mismatches [17–20], though these studies are limited by either not have data on DSA levels, or measuring DSA by CDC assays.

Transfusion sensitisation was assigned for antibodies in patients with a known transfusion history, the absence of a temporal relationship with other potential sensitising events containing the relevant HLA and the exclusion of specificities corresponding to any prior pregnancy or transplant.

2. Materials and methods

2.4. Treatment regimen

2.1. Patients

Plasmapheresis was administered with an HF-440 (Infomed, Geneva, Switzerland) machine with a Plasmacure plasma separator (Kuraray Medical Inc, Okayama, Japan) and Evaflux 2A plasma fractionator (Kawasumi Laboratories, Tokyo, Japan), to render the flow cytometric crossmatch negative, or to remove as much antibody as technically possible. Immunosuppression consisted of mycophenolate mofetil 500 or 1000 mg bd started 10 days before transplant, with the dose reduced if white cell count fell below 4.0  109/l. Tacrolimus was started 4 days before transplant at a dose of 0.15 mg/kg/day in divided doses, with a target trough level of 10–15 lg/l in the first month. Prednisolone 20 mg od was started at the time of surgery, and methylprednisolone 500 mg was given as a single intravenous dose during the transplant operation. Two doses of basiliximab 20 mg were given, at days 0 and 4. Rejection was diagnosed by renal biopsy if the renal function deteriorated, or clinically if there was rapid onset oliguria with a rise in both creatinine and in DSA levels. Rejection was treated with 3 days high dose methylprednisolone and daily plasmapheresis if DSA levels were raised, until patient 37 when post-transplant plasmapheresis was phased out, and with OKT3 (muromomabCD3) (Orthoclone) or rabbit (ATG) (Genzyme) if rejection was steroid resistant. Some patients felt to be at high risk of early rejection were started on ATG shortly after transplant if there was oliguria.

We studied 64 patients with preformed donor specific HLA antibodies transplanted in our academic transplant centre between June 2003 and June 2012. Serum samples were taken before and after each plasmapheresis treatment, typically daily for the first two post-transplant weeks and then three times a week for the next three to four weeks. A total of 104 consecutive HLA antibody incompatible transplants were performed during this period. Exclusions were 21 cases where single antigen bead analysis was not performed (assay not introduced at the time); 14 cases where the source of HLA sensitisation was uncertain; 3 early transplant losses and 2 cases who returned early to their referring centre and were unavailable for follow-up. The study was approved by the Coventry Research Ethics Committee. Informed and written consent was obtained. 2.2. Antibody detection and characterisation HLA specific antibodies were characterised using a single antigen microbead assay (One Lambda Inc, Canoga Park, CA, USA), analysed on the Luminex platform (XMap 200). Assay conditions followed the manufactures instructions. Antibody specificity was determined based on reaction pattern and relative antibody strength given as raw MFI (median fluorescence intensity). Thus, MFI was used as an arbitrary unit of antibody amount (u). We have previously shown that changes in MFI correspond to changes in antibody level [8]. For this analysis, the pre-treatment (i.e. immediately before the first plasmapheresis session, or pre-transplant if there was no plasmapheresis) antibody MFI and the highest antibody MFI during the first 30 days post-transplant was determined. For 20 HLA-specific antibodies, there was a progressive fall in MFI levels post-transplant, and the level at day 5 (or subsequent day if the level rose from this) was used for the analysis. All cases were subject to pre-treatment CDC and flow cytometry crossmatches as previously described [8]. 2.3. Assigning route of initial sensitisation Pre-treatment or historical HLA-specific antibodies were identified on the basis of specificity for HLA mismatches in the current transplant; thus at the time of desensitisation certain DSAs may have been below the manufacturers recommended cut-off level but were included if there was a higher level in the past. For all patients, the pregnancy, transplantation and transfusion history was obtained and each antibody specificity was assigned a primary sensitising source, as previously described [16]. Sensitisation by pregnancy was assigned if the antibody was seen to correspond with the partner’s HLA and the appearance of the antibody was consistent with the date of the pregnancy. Similarly, sensitisation by previous transplant was assigned when there was agreement between specificity of the donor HLA and antibody appearance. Repeat antigen mismatch was assigned if both donors carried the same HLA. Repeat epitope mismatch was assigned where antibodies, stimulated by a first transplant, reacted against the second donor in the absence of a repeat HLA type. Epitope reactivity was confirmed by reference to tables of known HLA epitopes [21].

2.5. Statistical analysis Data are presented as median (inter quartile range) unless otherwise specified. Comparisons between groups were performed using Student’s T-test or Chi-squared testing (Microsoft Excel).

3. Results 3.1. HLA-specific antibody responses according to the sensitisation events The characteristics of the sixty-four patients are given in Table 1. These were transplanted against a total of 220 HLA mismatches. Prior antibodies against these mismatches were investigated and the primary source of sensitisation identified in 158. For each antibody, we compared the change in MFI between the pre-treatment and early peak sera. Testing was performed repeatedly on each patient, as the results were used for clinical care, and the outcomes were robust in terms of reproducibility and low and stable background fluorescence levels. The analysis by mode of primary sensitisation is given in Table 2 and displayed in Figs. 1 and 2. As a group, the pregnancy stimulated antibodies exhibited a significantly greater response from graft HLA restimulation compared to the other primary sensitisation groups for both HLA Class I and Class II specific antibodies (p = 0.007 and p = 0.0001, respectively). None of the other primary sensitisation groups showed significant differences in post-transplant antibody responses. Time to peak level was shorter in the pregnancy group, median 13 (9–19.75) days, compared to 15.5 (11–21) days in the transfusion group and 16 (12–23) days in the transplant group (p < 0.01).

Please cite this article in press as: Higgins R et al. Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation. Hum Immunol (2015), http://dx.doi.org/10.1016/j.humimm.2015.06.013

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R. Higgins et al. / Human Immunology xxx (2015) xxx–xxx Table 1 Baseline characteristics of patients (CDC – complement dependent cytotoxicity crossmatch; FC – flow cytometric crossmatch). No

64

Gender (Male/Female) Age (median, range)

27:37 44 (22–68)

Transplant number 1 2 3 4

31 (48%) 25 (39%) 5 (8%) 3 (5%)

Pre-treatment crossmatch status (number (%) patients) CDC positive FC positive (CDC negative) Microbead positive (CDC and FC negative)

13(20%) 35 (55%) 16 (25%)

Pre-transplant crossmatch, post-conditioning CDC positive FC positive (CDC negative) Microbead positive (CDC and FC negative) Living donor/deceased donor Rejection in first month HLA specificities, total Pregnancy-induced Transfusion-induced Transplantation induced Repeat epitope mismatch Repeat antigen mismatch No documented prior sensitisation Source sensitisation unknown

3 (5%) 31 (48%) 30 (47%) 61:3 35 (55%) 220 71 (32%) 42 (19%) 45 (21%) 20 (9%) 25 (12%) 42 (19%) 20 (9%)

Pregnancy induced sensitisation Number pregnancies per patient (median) Years since first pregnancy, median (IQR) Years since last pregnancy, median (IQR)

2 26 (17–36.5) 23 (14.5–32.5)

Transplantation induced sensitisation Number previous transplants per patient (median) Years since first transplant, median (IQR) Years since last transplant failed, median (IQR)

1 15 (10.75–19) 4 (1–6.5)

HLA antibody production where there was no record of previous exposure was limited to two specificities (Fig. 2A). One de novo DSA was against HLA-B61 (MFI 252-1526), and in another patient, HLA-DR8 (MFI 154-2054). Fig. 3 shows the change in each antibody level plotted against its pre-treatment level. Thus for pre-treatment MFI 10,000 occurred in 10/36 (28%) of the pregnancy specificities, 2/16 (13%) of the transfusion group, and 0/12 in the transplant groups (p < 0.01). Examination of the range of Class I and Class II specificities in each group did not show differences in potential immune responsiveness that might explain the results (for example, each group contained similar proportions of patients with potentially immunodominant HLA-A2 and HLA-B7 CREG groups). 3.2. Time since sensitisation The times from pregnancy and previous transplantation to the current transplant differed significantly (Table 1). Fig. 4A shows a negative association between time since first sensitising event and the pre-treatment antibody levels (proportions of antibodies with MFI >5000 at more and less than 25 years since first sensitising event differ significantly p < 0.01). Fig. 4B shows the change in each antibody level post-transplant and the time since first sensitising event. Some patients seem to retain a full immune potential for post-transplant increases in antibody levels after even nearly 50 years, even though the pre-treatment antibody levels had declined. However, excluding data from >25 years ago, there continues to be a significant increase in the antibody levels after pregnancy sensitisation (median (IQR) 843 (395-5894) pre-treatment to 4671 (1042-9508 peak, p < 0.05)), not seen after transplantation

Fig. 1. Percentage changes in HLA-specific antibody levels (from pre-treatment levels) according to mode of original sensitisation event. A = no known sensitising event; B = pregnancy; C = transfusion; D = transplant, repeat antigen mismatch; E = transplant, repeat epitope mismatch.

sensitisation p = ns).

4337

(919-7903-3719

(996-9721)

respectively,

3.3. Changes in DSA levels and plasmapheresis (PP) Pre-transplant plasmapheresis was administered to 48 patients (161 specificities), while 16 patients (59 specificities) did not receive pre-transplant plasmapheresis. Nine patients (27 specificities) had post-transplant PP, and 55 patients (193 specificities) did not have post-transplant PP. The use of post-transplant PP was phased out during the programme because it had little effect on rapid resynthesis of DSA, and the combination of PP and ATG markedly increased the infection rate [22]. It is important to consider whether the limited use of post-transplant PP could have affected the results of this study by preventing a rise in DSA. In the pregnancy group, where post transplant PP was given (two patients and six antibodies), DSA levels median (IQR) rose from MFI 4084 (1344-7706) to 9,062 (5325-11405), compared to 828 (258-3104) pre-treatment to 3957 (541-9652) at peak in all others. In the transplant induced DSA group who had PP (5 patients and 11 antibodies), DSA levels median (IQR) rose from MFI 9181 (3363-12001) to 11,865 (7560-15556) compared to a fall in DSA from MFI 3618 (627-6000) to 2023 (858-7140) in those who did not have PP. Thus, there was treatment bias to giving post-transplant PP to patients with high DSA levels, and this treatment did not prevent rises in DSA. In relation to other therapies that might have had an affect on DSA levels, no patient received bortezomib or therapeutic splenectomy. One patient received pre-transplant intravenous immunoglobulins (IVIg and rituximab; another received post-transplant IVIg. These patients had two DSA induced by transplantation, the respective levels were 11,568 pre and 5787 the peak, and 7481 pre and 9634 peak, i.e. one rose and one fell from relatively high pre-transplant levels. Exclusion of these data did not change the conclusions of the analysis. 3.4. Changes in DSA levels and infections Because infection may result in a change in HLA antibody levels, we examined the incidence of infection in these patients. During

Please cite this article in press as: Higgins R et al. Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation. Hum Immunol (2015), http://dx.doi.org/10.1016/j.humimm.2015.06.013

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R. Higgins et al. / Human Immunology xxx (2015) xxx–xxx

Fig. 2. Changes in MFI levels by type of sensitisation. Pre-treatment MFI level versus change to peak MFI level post-transplant; for each group data for the full range of pretreatment MFI are shown, together with a lower starting range of MFI (