Detection of anti-eDithelia1 cell antibodies in association with pehiatric renal transplant failure using a novel microcytotoxicitv assav

d J

S.Martin, P. E. Brenchley, R. J. Postlethwaite, R. W. G. Johnson, P. A. Dyer. Detection of anti-epithelial cell antibodies in association with pediatric renal transplant failure using a novel microcytotoxicity assay. Tissue Antigens 1991: 37: 152-155.

Abstract: We have developed a microcytotoxicity assay allowing sera to be screened for anti-epithelial cell cytotoxic antibodies. Cells from the epithelial cell line A549 were cultured overnight in Terasaki trays prior to the addition of the sera to be screened. Using this assay, 63 pediatric recipients of 78 renal transplants have been studied retrospectively. Seven- teen transplants carried out in 13 patients were found to be associated with the production of antibodies reactive only against epithelial cells (AEC). Eleven of these transplants failed as compared with 19 failures out of 52 transplants not associated with AEC production (Fisher's p = 0.04). We conclude that transplantation in the face of pre-existing AEC should be approached with caution.

Introduction

The occurrence of IgM anti-epithelial cell anti- bodies (AEC) has recently been reported in associ- ation with pediatric renal allograft failure (1, 2). Production of AEC by renal dialysis and transplant patients with concomitant graft failure was first observed during 1989. The AEC were detected using the epithelial cell line A549 as the target cell in a flow cytometry assay. We have developed a simple microcytotoxicity assay in order to investi- gate the incidence of AEC in our renal transplant population and to assess the role of such antibodies in graft rejection.

Patients and methods

Patients: 63 pediatric recipients of 78 renal trans- plants were studied retrospectively. Between 1973 and May 1990, 113 pediatric renal transplants (17 regrafts) were performed at this center. We have studied all those recipients who are still under the care of the pediatrician (56), or who were so at the time of their transplant failure (7). Forty pediatric recipients now being followed-up as adults were not included in this study. Recipient selection for transplantation, transfusion protocols and im- munosuppressive therapy were as previously de-

Susan Martin', Paul E. Brenchlef, Robart J. Postlsthwaite', Robert W. 6. Johnson' and Philip A. Dyer' NW Regional Tissue Typing' and Immunology2 Laboratories, St Mary's Hospital, Renal Unit, Booth Hall Children's Hospital3 and Renal Trans- plant Unit, Manchester Royal Infirmary', Man-

Key words: anti-epithelial cell cytotoxic anti- bodies - pediatric renal transplant

Received 14 August 1990, revised, accepted for publication 23 January 1991

scribed (3). Of the 63 recipients, 46 received pre- transplant blood transfusions and none had had pregnancies.

Lymphocytotoxic antibody screening: serum samples had been collected monthly whilst patients were on the transplant waiting list and at regular intervals posttransplant. These were screened at the time of collection for lymphocytotoxic antibodies (PRA) against a selected panel of HLA-typed lymphocytes (4). Autoantibodies were identified by their reactivity with peripheral blood lymphocytes but not with lymphocytes from patients with chronic lymphocytic leukemia, their inhibition by treatment of the serum with 0.01 M dithiothreitol and their reactivity against autologous lympho- cytes both in cytotoxicity and absorption studies.

Epithelial cell culture: the human lung carcinoma epithelial cell line A549 (ATC no. CCL185) was maintained in culture in 10 ml Dulbecco's modifi- cation of Eagle's medium (DMEM) + 10% fetal calf serum (FCS) in a 50 ml flask at 37"C, 5% COz. Confluent cells were removed from the base of the flask by treatment with 2.5 ml 0.5% (w/ v) Trypsin+0.2% EDTA in phosphate buffered saline, for 2-3 min at 37°C. After the addition of 1 ml FCS, the cells were centrifuged for 5 min at

152

Cytotoxic AEC and renal transplant failure

Table 1 The effect of temperature on cytotoxicity in the anti-epithelial cell microcyte toxicity assay

300 g and resuspended in DMEM + 10% FCS for passaging or use in the microcytotoxicity assay. Epithelial cell microcytotoxicity assay: A549 cells at 105/ml in DMEM+ 10% FCS were plated out into Terasaki trays (10 pl/well). The trays were incubated overnight in a humid box at 37"C, 5% COz. Culture suspernatants were removed by "flicking" and 1 pl of serum was added to each well. After incubating for 1 h at 4" or 22°C in a humid box, 3 p1 of rabbit serum (as a source of complement) was added and the incubation con- tinued for a further hour at 22 "C. The assays were stained with a cocktail of ethidium bromide (0.2 mglml), acridine orange (0.1 mglml), bovine hemo- globin (15% w/v) and EDTA (2.5% w/v) and read under UV light on an inverted microscope. The percentage of cell killing/well was recorded and a cell death > 10% was taken as positive.

All tests were carried out in triplicate. Human AB serum was used as a negative control and a pool of high titer anti-HLA alloantisera was used as the positive control. For all recipients, serum samples taken at the time of transplant were tested in this assay. For those with failed transplants, a sample taken at the time of nephrectomy was tested and for those with functioning transplants, the most recent sample was tested. Other samples were tested as necessary. Treatment of sera with dithothreitol: the use of di- thiothreitol (DTT) to remove IgM antibodies in serum is now an established clinical laboratory practice (5). Positive sera were retested with and without the addition of 0.01 M dithiothreitol (DTT). One microliter 0.1 M DTT was added to 9 pl of test serum which was then assayed as de- scribed above. As DTT and serum were always mixed at a ratio of 1 : 9, the possibility of a dilution effect was avoided and the abrogation of a positive reaction with DTT was taken to indicate that the reaction was due to an IgM antibody. Pretreatment of AS49 cells with a monomorphic anti-HLA class I antibody: patients known to have PRA were retested in dilution after pretreatment of the A549 cells in the Terasaki trays with the monomorphic anti-HLA class I antibody PA2.6 and F(ab') goat anti-mouse IgG (Northeast Bio- medical Laboratories) (6). Blocking of a reaction, or a reduction in titer by at least one doubling dilution, by treatment with PA2.6, indicated that reaction to be due to anti-HLA class I antibodies (6).

Results

The AEC microcytotoxicity assay gave consistent results, with complete concordance between repli-

Serum No. Percentage cytotoxicity'

4 "C 22 "C

1 2 3 4 5 6 7 8 9

10 11-18 19

100 100 100 100

80-1 00 40-80 20-50

20 20-50

20 0

50-1 00

80 1040 10-80 10-20 10-20 10-20

0 0 0 20 0

100

a: Range of results obtained in triplicates.

cates occurring in 98% of 770 assays. The viability of the A549 cells in the control wells at the end of the assay period was, with few exceptions, > 95%. The effect of temperature on the AEC microcyto- toxicity assay is shown in Table 1. All sera (no. 11-18) that were negative at 4 "C were also negative at 22°C. Serum no. 19, a pool of high titer anti- HLA alloantisera, was more reactive at 22°C. In contrast, sera no. 1-10 which contained IgM AEC were more reactive at 4°C which was therefore chosen as the assay temperature.

Of the 63 patients, 48 received only 1 transplant; 35 (73%) are still funcitoning and 13 (27%) have failed: 15 patients received 2 transplants; in 10 cases (67%) the 2nd transplant is funcitoning and in 5 cases (33%) both have failed. The outcome of the 78 transplants in relation to AEC production is depicted in Fig. 1.

78 transplants I

I 26 AEC

I 52 AEC

tive

1 19 fld

36% AEC: anti-epithelial cell antibody. Auto: autoantibody. fctn: functioning transplant. fld: failed transplant. Figure 1. The outcome of pediatric renal transplants performed between 1977 and 1990 in relation to anti-epithelial cell anti- body production.

153

Martin et al.

Table 2 The relationship between anti-epithelial cell antibody production and renal transplant outcome

years, the AEC antibody remained as IgM. The sole exception was a patient whose antibody was IgM for 9 months but subsequently became IgG.

Anti-epithelial - Of the 13 AEC-positive patfents, '3 (NA, SB,FP) cell antibody Failed transplant Functionina transplant were known to have PRA directed against HLA

Number of patients

class I antigens at the time of AEC detection. Pre- Positive 11 6 treatment of the A549 cells with PA2.6 failed to

block the cytotoxicity, showing the AEC to be Negative 1 9 33

independent of anti-HLA class I antibodies (Table 3).

Fischer's p = 0.04"

a: p value determined using Fisher's Exact test.

Twenty-six transplants (22 patients) were associ- ated with the production of non-PRA associated AEC. In 9 of these cases the patients with AEC were also known to have autoreactive lymphocyto- toxic IgM antibodies: 6 of the transplants are still functioning and 3 have failed. As autoreactive IgM lymphocytotoxic antibodies are known not to be graft damaging (7), these 9 cases were excluded from the analysis. Of the 17 non-auto AEC-posi- tive transplants (13 patients), 11 (65%) have failed. Three patients each lost 2 transplants. Fifty-two transplants (44 patients) were not associated with AEC production and, of these, 19 (36%) have failed. The association between AEC production and transplant failure was significant (Table 2). There was no association between AEC production and pretransplant blood transfusions which were receiced by 82% of recipients who produced AEC and by 78% of those who did not (Fisher's Exact Test p = ns).

In 10 cases, AEC were present at the time of transplantation. Five of these cases were 2nd trans- plants, for 3 of which AEC had also been present at the time of the 1st transplant. Eight of the trans- plants performed in the presence of AEC have failed, with the time of failure ranging from 1 day to 18 months (median 2 weeks) posttransplant.

For 21 of the 22 AEC-positive patients, treat- ment of their sera with DTT abrogated the cytotox- ic reactions, indicating the antibody to be IgM. Even when patients were followed over several

Table 3 The effect of blocking with PA2.6 on the reactivity of sera with AEC and anti-HLA activity in the AEC rnicrocytotoxicity assay

Percentage cytotoxicitf Serum concentration

Serum Neat + PA2.6 1 :2 +PA2.6 1 : 4 +PA2.6

Anti-HLA 90 40 80 40 70 1 0 NA 70 80 20 30 1 0 15 SB 80 80 6 0 6 0 40 40 FP 60 60 30 30 0 0

a: Mean of results from triplicate assays.

Conclusions

We have developed a simple and reliable cytotox- icity assay, using A549 as the target cell, which has enabled us to investigate the incidence of AEC in our pediatric transplant recipients. A limited number of sera have been exchanged with Har- mer's group. Preliminary results show concordance between results obtained using the cytotoxicity as compared with the flow cytometry assay (personal communication). The advantages of this cytotox- icity assay over the previously described method using flow cytometry are its simplicity and low cost. It is thus readily applicable for use in the majority of tissue typing laboratories.

Our results support the findings of Harmer et al. (l), that AEC may be produced by pediatric recipients in association with transplant failure. Of the 17 transplants associated with AEC produc- tion, 65% failed as compared with 36% of the 52 transplants where there was no AEC production: this difference achieved statistical significance. We confirm that this antibody is unusual in that it remains as IgM and does not switch to IgG: the single patient who was an exception to this is under further investigation.

In addition, we have found autoreactive lymph- ocytotoxic antibodies to be associated with cyto- toxicity against A549 cells; this has not been pre- viously reported. Absorption studies involving A549 and autologous lymhocytes are clearly indi- cated to confirm that the same antibody is respon- sible for cytotoxicity against both A549 and auto- logous lymphocytes. However, as the antigenic stimulus which triggers autoantibody production has never been clearly defined, further studies with A549 could prove helpful in its characterization. Meanwhile, the apparent reactivity of autoanti- bodies, which are not graft damaging, with A549 must be taken into account when interpreting re- sults obtained using the assay which we have de- scribed.

The causative agent involved in AEC production has not been identified. The pattern of antibody production in the patients described by Harmer et al. (l), with its sudden appearance and then

154

sequential development in other members of the patient group, was suggestive of an infective agent which as yet remains undefined. Our experience differs in that this retrospective study has identified AEC in patients as early as 1977 and as recently as 1989 but provides no evidence for the spread of an infection through the pediatric dialysis and transplant population.

In this study, 10 transplants were performed when the patient had pre-existing AEC: 8 failed. Although 1 of these failures was at 18 months posttransplant and might therefore not be related to the AEC, the others all failed in less than 8 wk with a median of 2 wk. We would therefore suggest that the AEC could have contributed to the graft failure and that pediatric transplantation, when the recipient has pre-existing AEC, should be ap- proaced with caution. AEC have also been iden- tified in a few adult transplant recipients (1). Further studies are clearly necessary to fully assess the relevance of AEC to adult as well as pediatric transplantation. In conclusion, by developing a simple microcytotoxicity assay we have been able to monitor the production of AEC in our pediatric transplant population. We have shown AEC pro- duction to be associated with transplant failure and propose that AEC status should be taken into consideration when selecting children for renal transplantation.

Cytotoxic AEC and renal transplant failure References

1. Harmer AW, Rigden SPA, Koffman CG, Welsh KI. Pre- liminary report: dramatic rise in renal allograft failure rate. Lancet 1990: 335: 1184-5.

2. Harmer AW, Haskard D, Koffman CG, Welsh KI. Novel antibodies associated with unexplained loss of renal allo- grafts. Transplant Znt (in press).

3. Dyer PA, Johnson RWG, Martin S, et al. Evidence that matching for HLA antigens significantly increases trans- plant survival in 1001 renal transplants performed in the North West Region of England. Transplantation 1989: 48:

4. Sinnott PJ, Kippax RL, Sheldon S, Dyer PA. A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates. Tissue Antigens 1985: 26: 318-22.

5. Roy R, Belles-Isles M, Pare M, Lachange J, Noel R. The importance of dithiothreitol treatment in crossmatching selection of presensitized kidney transplant recipients. Transplantation 1990: SO: 532-4.

6. Taylor CJ, Chapman JR, Ting A, Morris PJ. Characteriza- tion of lymphocytotoxic antibodies causing a positive cross- match in renal transplantation: relationship to primary and regraft outcome. Transplantation 1989: 48: 953-8.

7. Jeannet M, Benzonana G, Arni I. Donor-specific B and T lymphocyte antibodies and kidney graft survival. Transplan- tation 1981: 31: 160-3.

131-5.

Address: Dr Susan Martin PhD NW Regional Tissue Typing Laboratory St Mary’s Hospital Hathersage Rd Manchester M13 OJH UK

155