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Immunological studies on women receiving leukocyte immunotherapy
Transfus. Sci. 1989; 10:257-264 Printed in Great Britain. All rights reserved
09553886/89 $3.00+0.00 Copyright 0 1989 Pergamon Press plc
Immunological Studies on Women Receiving Leukocyte Immunotherapy David C. Kilpatrick, BSc, PhD, MRCPath William A. Liston, BSc, MBChB, FRCOG*
n Leukocyte immunotherapy is increasingly being used in the prophylaxis of spontaneous abortion. We have measured immunoglobulins, complement components, T4/T8 ratios and alloantibody responses of 26 women before and after immunotherapy consisting of vaccination with their husbands’ mononuclear cells. There were no significant changes in IgG, IgA, IgM, C3, C4, CH,, or T4/T8 ratios in the 3 months after immunotherapy. No antibodies to red blood cell antigens were induced. Cytotoxic anti-lymphocyte antibodies were detected in 6 women (24% ) after a single course of immunotherapy, and one other woman seroconverted after a second course. There was no significant correlation between lymphocytotoxin production and subsequent pregnancy or pregnancy outcome. 4 (67%) of the women who produced lymphocytotoxins, but none of the non-responders, had HLA-Bw 22 (P
belief that lymphocytes and trophoblast share an epitope important for the induction of a protective maternal immune response to the conceptus. Encouraging results have been reported using erythrocyte-compatible buffy coats2 and leukocyte-rich red blood cell concentrates3 from multiple unrelated donors, and also using mononuclear cell preparations from husbands not matched for erythrocyte antigens4 Only one double-blind placebo-controlled trial has been carried out,4 however, and the validity of the therapy remains controversial.5 Administration of blood components always carries some risk to the recipient, and safety considerations are of particular importance when a blood component is given to healthy people for what could be considered an experimental procedure. We therefore measured immunological factors before and after injection of mononuclear cell concentrates. This study is of particular interest as it provides a rare opportunity to observe the effect of a well-defined blood product on the immune system of apparently healthy individuals.
INTRODUCTION Idiopathic spontaneous abortion is sometimes thought to have an immunological basis.’ Consequently, women with a history of repeated miscarriages have been treated by injection or infusions of leukocyte-rich blood products in the
PATIENTS
AND METHODS Patients
The patients were 26 healthy women (mean age 3 1, range 23-39) with a history of recurrent miscarriage. All were antinuclear factor, anti-cardiolipin antibody
From the Blood Transfusion Service and ‘Simpson Memorial Maternity Pavilion, The Royal Infirmary, Edinburgh EH3 9HB, Scotland. Received and Accepted 5489. 257
258
Transfus. Sci.
Vol. 10, No. 3
and VDRL
negative, and had normal activated partial thromboplastin times. Some had had diagnostic laparoscopy; most had been screened for possible metabolic, hormonal or infectious causes of abortion. Both the women and their partners had normal karyotypes. Husbands who were acceptable as blood donors were considered acceptable as leukocyte donors irrespective of blood group. Where a Rh-D negative wife had a Rh-D positive husband, anti-D was given 24 h after the mononuclear cell injection. Only one woman had cytotoxic antilymphocyte antibodies prior to immunization; she had had two children by a previous husband. Preparation and Administration of Mononuclear Cell Concentrates 1OOmL of blood was removed from the husband by venepuncture and immediately defibrinated. All subsequent procedures were performed under strictly aseptic conditions. The defibrinated blood was mixed with an equal volume of 0.9% saline, and Dextran-500 (10%) solution was added to a final concentration of 1% (v/v). After 15 min incubation at 37”C, the resulting leukocyte-rich upper phase was aspirated and layered into Ficoll solution (Lymphocyte Preparation Medium, Flow Laboratories, Irvine, U.K.) to prepare mononuclear cells.6 The mononuclear cells were collected from the interface, harvested and washed twice with 0.9% saline before finally resuspending in about 3.5 mL saline. The mean yield was lo* cells. The mononuclear cell concentrate was injected into the wife as follows: 3 x 0.1 mL intradermally into one arm; 3 x 0.1 mL subcutaneously into another arm; the remainder intravenously. The patients were observed for 1 h after the injections; blood pressure, body temperature and pulse were monitored regularly. The procedure was initially carried out while the patient was not pregnant. Where applicable, the procedure was repeated in early (within 6 weeks menstrual age) pregnancy.
Most patients were followed 24 h, 3-4 weeks and 3 months.
up at
Laboratory Methods Tissue-typing and cytotoxic anti-lymphocyte screening and cross-matching were performed by microcytotoxicity testing.’ Cross-matches were performed using serum from each woman against her husband’s unfractionated mononuclear cells (mainly T cells) and B cells prepared from the former by rosetting with sheep erythrocytes.* Each serum was also screened against a panel of lymphocytes from 30 unrelated individuals representing a wide variety of tissue types, and a panel of B lymphocytes from at least 10 patients with chronic lymphatic leukaemia. Immunoglobulin and complement components were measured by rate nephelometry; total haemolytic complement (CH,,) by lysis of antibody-sensitized sheep erythrocytes; blood grouping and erythrocyte antibody by standard screening methods; and T4:T8 ratios were measured using indirect immunofluorescence and flow cytometry, all as described elsewhere.9 IgE was determined by radioimmunoassay according to the manufacturer’s instructions (Tandem).
RESULTS The immunization procedure was well tolerated by the patients. There were no systemic adverse reactions, although some patients developed erythema, which usually subsided within an hour, at the injection sites. The delayed-type reaction at the intradermal sites was also mild: the area of erythema and induration was always small (mean 7 mm, range 1-15 mm, and observed 24 h after injection), never painful and only mildly, if at all, pruritic. 18 of the group became pregnant and were immunized again: local and systemic effects were similar to those experienced after the first immunization course. Measurements of immunoglobulin
Leukocyte Immunotherapy for Aborters
classes, complement components and haemolytic activity, and T4/T8 ratios, before and after immunotherapy, are summarized in Table 1. There were no significant changes in any of those immunological measurements in the group as a whole, nor were any consistent Table 1.
IgA IgM I@ c3 c4 Cf.&c
T4:T8
changes apparent in individuals (data not given). The erythrocytes of the patients and their partners were typed for ABO, Rhesus, MNS, Kell, Duffy (a) and Kidd (a) blood groups. All but two of the men had one or more alloantigens not shared by their wives (Table 2). Despite this, no red
Immune Profile Before and After Immunotherapy (n = 26)
24h (n=26)
Post: 3-4 weeks (n = 26)
3 months (n= 17)
11.452.4 1.98kO.89 1.65 + 0.64 36+41 1.12kO.33 0.27 + 0.08 110?29 2.1+0.7
11.2k2.3 1.97 + 0.86 1.61 kO.61 35f47 1.05 -t-0.23 0.27 + 0.08 114?28 2.0 f 0.5
ll.lk2.2 1.89 + 0.80 1.62 + 0.60 36+40 1.04 + 0.28 0.26 + 0.08 114*35 1.9f0.5
10.9 + 2.0 2.08 f 1.01 1.60 + 0.60 33+29 1.04 It 0.22 0.25 + 0.06 116528 1.8+0.5
Pre: IgC
259
All data are expressed as a mean + 1 SD Immunoglobulin G, A and M and complement components C3 and C4 are given in g/L; IgE is given in IU/mL; and total haemolytic complement (CH,,) is expressed as a percentage of the value obtained with a pool of normal blood donor sera. Table 2.
Red Blood Cell Incompatibilities ABO
Rhesus
Lewis
1
-
C
Le”
2’ 3’ 4’ 5 6’ 7’ 8’
B B -
Couple
9 10 11’ 12’ 13* 14* 15 16” 17’ 18’ 19 20* 21’ 22’ 23’ 24* 25 26
A B B A A A A A, B -
C, e : c E/c ; C, D -
Leb -
Leb
C
-
-
-
Fy” Fy” -
-
E M -
E, c
-
Kidd
-
Le” -
E, c -
Duffy
S
M -
Le” Le” -
Kell
S M,S -
-
Lea Le” -
CCD C: D C -
MNS
Cl Kl -
N N
Iz -
-
-
S NY s -
FY” Fy” -
G -. -
Fy” Fy”
-
-
-
Incompatability refers to blood groups possessed by the male leukocyte donor and not shared by his partner. All patients’ sera were negative for anti-red blood cell antibodies when tested 2-4 weeks before the commencement of immunotherapy. 18 patients became pregnant and were immunized a second time; these women are indicated by asterisks.
260
Transfus. Sci.
Vol. 10, No. 3
blood cell antibodies could be detected after immunization. This was also true after the second course of vaccination for the 18 women who became pregnant. Both spouses were fully tissue-typed for HLA-A, B and DR antigens (Table 3). All but one of the men were HLA-incompatible with their partners and 6 of 25 (24%) of the women produced cytotoxic anti-lymphocyte antibodies (Table 3 J. The production of lymphocytotoxins was unrelated to the number of cells injected, and only weakly associated with the degree of HLA-incompatibility (Fig. 1). Four of the six antibody producers possessed HLA-Bw22. This frequency of Bw22 is highly significant whether compared with its absence in the 19 nonantibody producers (P=O.O012) or with its presence in only 11 out of 264 in the general population (P = 0.00009) (Fisher’s exact test). Even after correcting for the number of antigens tested, the values were still significant: P
minimal erythrocyte contamination. These modifications eliminated the risk of alloimmune thrombocytopenia in a subsequent pregnancy and we hoped would reduce the risk of haemolytic disease of the newborn. Secondly, the dosage was considerably smaller: the equivalent of a quarter of a standard blood donation unit was used, but this amount was used again during pregnancy. It is noteworthy that more recent data from Mowbray and colleagues indicate that, while exact dosage is unimportant, the best pregnancy success is obtained after immunization in very early pregnancy.‘O This form of immunotherapy is of unique value to transfusion medicine as an instance where a well defined blood product is administered to healthy subjects. Interest in the effect of blood components on the immune system has recently been stimulated by several considerations. Kidney transplant recipients who received blood transfusions have better graft survival statistics, and this “transfusion effect” may be associated with the infusion of leukocytes.” Patients with haemophilias and congenital anaemias receiving blood components exhibit immunological abnormalities, including a decreased T4/T8 ratio, which have been suspected of contributing to the development of AIDS in individuals infected with the human immune deficiency virus. 12J3 Several retrospective surveys have suggested that blood transfusion increases susceptibility to various infections and malignant diseases.14 Such observations have usually been made on ill patients suffering from clinically-apparent diseases who had received whole blood or individual components in varying amounts and combinations. It is therefore an important finding that the intravenous administration of mononuclear cells to healthy, normal (apart from their reproductive problems) women produced no significant alteration in T4/T8 ratio either immediately (24 h) or during the course of the following 3 months. None of the women became sensitized to erythrocyte alloantigens,
Leukocyte Immunotherapy for Aborters 261 Table 3.
Tissue Types and Anti-lymphocyte Antibody Responses of Immunotherapy Couples
Couple
Tissue Types
Cross-matches T B
Cell Panels T B
-
-
-
-
-
-
-
-
F: Al, 3; B7, 8; DR2, 5 M: A2; B14, 44; DRl, 4
-
-
-
-
F: A2, 28; B14, 44; DR2, w6 M: Al; B8; DR3
-
-
-
-
F: Al, 2; B5, 35; DR4 M: Al, 26; B5; DR2, 4 F: Al, 28; B35, 40; DR3, 5 M: A24, 25; B5, 7; DR5, 7
Cross-matches Positive after second immunization
-
-
-
-
comments
5
F: Al, 11; B7, 27; DR4, w6 M: A2,32; B14,35; DR7
6
F: A3, 30/31; B7, 18: DR2, 3 M: A2, 29; B7, 44; DR2, 7
-
-
-
-
7
F: A3, 11; B7, 22; DR2, 5 M: A3, 29; B7, 44; DR4, 7
+i-
++
56%
67%
8
F: Al, 21 B8, 441 DRl, 3 M: Al, llj B7, 35; DRl, 7
-
-
-
-
9
F: A3; B5, 73 DR2, 5 M: Al, 3j B7, 8; DR3, 7
-
-
-
-
10
F: A2, 24; B27, 35; DRw6, ~8 M: A2, 28; B21, 40; DR4, ~6
-
-
-
-
11
F: A3, 24; B7j DR2 M: All, 24; B7, 223 DR2, ~6
-
-
-
-
12
F: Al, 28; B22, 353 DR4, ~8 M: A2, 293 B44j DR2, 7
++
++
66%
70%
13
F: Al, 31; B15, 40; DR4, ~8 M: All; B5,35j DR5, ~9 F: A3, 263 B5, 8j DRw6 M: Al; B8, 153 DR3, 5
-
-
-
-
-
-
-
F: Al, 2; B8, 40; DRX M: A2, 29; B7, 44; DR7 F: Al, 24; B35j DR5 M: Al, 24; B35j DR5
-
-
-
-
-
-
F: Al, 23 B8, 44j DR3, 4 M: A2, 3; B14, 44; DR4, 7 F: Al, 2; B8, 40; DR4, 5 M: A2, 26; B7, 14; DR2, 7 F: Al, 3; B8, 181 DR3 M: A26, 30/31; B14, 18; DR3, 7
-
-
-
-
-
-
-
-
14 15 16 17 18 19
20
F: A2, 11; B22, 44; DR3 M: A2, 33 B7, 44; DR2, 3
21
F: A2, 29; B21, 443 DR4, ~6 M: A2j B16,44; DR2, ~6 F: Al, 3; B7, 8; DR2, 3 MI Al, 3; B8, 17; DR3, 7
22
Anti-Bw4
Anti-Bw4
(Reacted with 40% of cell panel and gave weakly positive cross-match before immunization). + + T(23%)and B (81%) cell panel positive after second immunization. -
-
-
continued overleaf
262
Tnn~sfus. Sci.
Vol. 10, No. 3
Table 3-continued Cross-matches T B
Cell Panels T B
Couple
Tissue Types
23
F: A23, 28; B22, 44; DR4, 7 M: Al, B8, 16; DR4, w6
+-I-
++
68%
24
F: Al; B35, 44; DR5, w9 M: A2, 3; B7, 8; DR2, 7
++
++
-
-
25
F: A3, 28; B35, 44; DR2, 7 MI All, 29; B35; 44, DRw6, ~8
-
-
-
-
F: A2, 33 B7, 44; DR2, ~6
-
+
-
-
26
M: All,
70%
Comments Anti-Al
+
Polyspecific (>90% ) towards cell panels after second immunization.
26; Bl6, 35; DR2, ~6
For anti-paternal cell cross-matches, half reactions (~60% cell death) are indicated by (+) and full reactions IS&JO%cell death1 bv I+ + 1.Reactivity against the cell panels is expressed as a percentage of strongly positive 1360% cell deat&) r&cti&s. ’ -
although most of the men had antigens not shared by their wives, and prophylactic action could only be taken against Rhesus-D sensitization. The couples included three cases of Kell positive men with Kell negative wives; Kell-mediated hameolytic disease is usually caused by blood transfusion and is often fatal to the fetus.r5 Although the extent of erythrocyte contamination in our mononuclear cell preparations was variable, it was always small relative to the cell number which might be expected to cause sensitization. Our finding that only six out of 25 patients (24%) produced lymphocytotoxins in response to a single injection of mononuclear cells was surprising in view of the higher rate of seroconversion (4476%) reported by others.4J6J7 This difference cannot be accounted for in terms of dosage, as only one of the other groups administered a significantly larger number of lymphocytes, and indeed within our own series sensitization was independent of cell number in the vaccine. Although only cells mismatched by at least 3 HLA specificities were im-
munogenic, there was no trend towards increased sensitization with increasing degree of HLA incompatability. Sensitization, therefore, may depend mainly on whether the subject is a high or low responder; responder status might show
2.5
2.0 : 8
CELLS (x10-81
‘.5
0 0
1.0
F 0 0
0.5
0 1
4
MISMATCHES
Figure 1. Anti-lymphocyte antibody production in relation to mononuclear cell dose and HLA-compatibility. Presence (W) or absence (0) of anti-paternal lymphocyte cytotoxic antibodies after a single course of injections is shown in relation to number of lymphocytes injected and the degree of HLA incompatibility. The latter is quantified as mismatches i.e. HLA specificities possessed by the man not shared by his partner.
Leukocyte Immunotherapy for Aborters
HLA associations, and indeed several HLA specificities were over-represented in the antibody producing women, strongly and significantly so with HLABw22. The other workers referred to prepared their immunogen from anticoagulated blood which inevitably yields a mononuclear cell preparation contaminated by platelets. We speculate that the combination of lymphocytes and platelets may be more immunogenic than either alone. The observations of Fisher et ~1.‘~ that platelet transfusion stimulated lymphocytotoxin production only when contaminated with a small number of lymphocytes, indirectly supports that suggestion, although a different mechanism might be involved. While it has been claimed that the production of lymphocytotoxins is an indirect marker of successful immunotherapy, 4,17 MacLeod and coworkers have found that non-cytotoxic anti-lymphocyte antibodies correlate best with both pregnancy maintenance and successful renal graft survival.1y,20 On the other hand, sensitization to lymphocyte and platelet antigens may be undesirable and potentially harmful in the long term. Such sensitization is a well established problem with some patients requiring renal or cardiac transplants, or those requiring platelet transfusions for thrombocytopenia. There is also the rare but real possibility of combined immunodeficiency and maternal graft-vs-host disease.21 Although we have concentrated on safety aspects and do not have enough patients to evaluate the efficacy of the method, our preliminary results are encouraging. 10 women have delivered normal-sized babies, 2 are pregnant in the 3rd trimester, 1 aborted apparently from cervical incompetence at 20 weeks and 5 others had early abortions. (There was no correlation between anti-lymphocyte antibody production following immunization and pregnancy outcome: 3 (30%) of the women with live births and 1 (20%) of the early aborters were antibody producers.) The 6 children who were old enough were seen by a paediatri-
263
cian at 1 yr of age, and their development, assessed by the Denver screening system, was within the normal range. In conclusion, the results reported here are reassuring since they reveal no adverse, undesirable or unexpected responses to the administration of allogeneic leukocytes in these patients. Husband-specific preparations obviously carry less risk of infection than a pooled preparation from unrelated donors, and we have now shown that a low dose of a highly purified mononuclear cell preparation carries a relatively low risk of sensitization to alloantigens. Acknowledgements We thank the MLSO staffofthe Blood Transfusion Centre for technical assistance, the nursing and medical staff of Ward 36 for their co-operation, and Dr R. Hague for carrying out paediatric assessments.
REFERENCES 1. Mowbray JF, Underwood JL: Immunology of abortion. Clin Exp Immunol 1985; 60: l-7. 2. Taylor CC, Faulk WP, McIntyre JA: Prevention of recurrent spontaneous abortions by leukocyte transfusions. / R Sot Med 1985; 78:623-627. 3. Unander AM, Lindholm A: Transfusion of leukocyte-rich erythrocyte concentrates: a successful treatment in selected cases of habitual abortion. Am 1 Obstet Gynecol 1986; 154:516-520. 4. Mowbray JF, Gibbings C, Liddell H, Reginald PW, Underwood JL, Beard RW: Controlled trial of treatment of recurrent spontaneous abortion by immunisation with paternal cells. Lancet 19853 1:941943. habitual abor5. Adinolfi M: Recurrent tions, HLA sharing and deliberate immunisation with partner’s cells: a controversial topic. Human Reprod 1986; 1:45-58. 6. Boyum A: Isolation of lymphocytes, granulocytes and macrophages. Stand 1 Immunol 1976; 5(Suppl.5):9-15. 7. Tiwari JL, Terasaki PI: In HLA and Disease Associations. New York, Springer, 1985, pp. 14-17. 8. Kilpatrick DC, Darg C: Enzymatic modification of the lymphocyte surface: application to tissue-typing and rosetting with sheep erythrocytes Tissue Antigens 1983; 21:309-317.
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Transfus. Sci. Vol. 10, NO. 3
9. Kilpatrick DC, Liston WA, Neil1 G, Maxwell C: Immune profile of women experiencing recurrent spontaneous abortions of unknown aetiology. Disease Markers 1989; 7:87-94. 10. Mowbray JF: Immunology of early pregnancy. Human Reprod 1988; 3179-82. 11. Singal DP, Ludwin D, Blajchmon MA: Blood transfusion and renal transplantation. Br I Haematol 1985; 61595-602. 12. Carr R, Veitch SE, Edmund E, Peutherer JF, Prescott RJ, Steel CM, Ludlam C: Abnormalities of circulating lymphocyte subsets in haemophiliacs in an AIDS-free population. Lancet 1984; 1:1431-1434. 13. Kaplan J, Samaik S, Getlin J, Lusher J: Diminished helper/suppressor lymphocyte ratios and natural killer cell activity in recipients of repeated blood transfusions. Blood 1984; 64:308310. 14. Blumberg NG, Heal JM: Perioperative blood transfusion and solid tumour recurrence. Blood Rev 198 7; 1: 2 19-229. 15. Farr V, Gray E: Pregnancy outcome in mothers who develop Kell antibodies. Scott Med 11988; 33:300-303. 16. Beer AE, Semprini AE, Xiaoyu Z, Quibbeman JF: Pregnancy outcome in human couples with recurrent spontaneous abortion: HLA antigen profiles; HLA antigen sharing; female serum MLR
17.
18.
19.
20.
21.
blocking factors; and paternal leukocyte immunisation. Exp Clin Zmmunogenet 1985; 2: 137-153. Reznikoff-Etievant MF, Simonney N, Janaud A, Darbois Y, Netter A: Treatment of recurrent spontaneous abortions by immunisations with paternal leukocytes. Lancet 19853 1:1398. Fisher M, Chapman JR, Ting A, Morris P: Alloimmunisation to HLA antigens following transfusion with leukocyte-poor and purified platelet suspensions. VOX Sang 19853 49:331-335. MacLeod AM, Power DA, Mason RJ, Stewart KN, Skewan WG, Edward N, Catto GRD: Possible mechanism of action of transfusion effect in renal transplantation. Lancet 1982; 2:468470. Power DA, Catto GRD, Mason RJ, MacLeod AM, Stewart GM, Stewart KN, Shewan WG: The fetus as an allograft: evidence for protective antibodies to HLA-linked paternal antigens. Lancet 1983j 2:701-704. Bastain JF, Williams RA, Ornelas W, Tani P, Thompson LF: Maternal isoimmunisaresulting in imtion combined munodeficiency and fetal graft-versushost disease in an infant. Lancet 19843 1:1435-1437.
09553886/89 $3.00+0.00 Copyright 0 1989 Pergamon Press plc
Immunological Studies on Women Receiving Leukocyte Immunotherapy David C. Kilpatrick, BSc, PhD, MRCPath William A. Liston, BSc, MBChB, FRCOG*
n Leukocyte immunotherapy is increasingly being used in the prophylaxis of spontaneous abortion. We have measured immunoglobulins, complement components, T4/T8 ratios and alloantibody responses of 26 women before and after immunotherapy consisting of vaccination with their husbands’ mononuclear cells. There were no significant changes in IgG, IgA, IgM, C3, C4, CH,, or T4/T8 ratios in the 3 months after immunotherapy. No antibodies to red blood cell antigens were induced. Cytotoxic anti-lymphocyte antibodies were detected in 6 women (24% ) after a single course of immunotherapy, and one other woman seroconverted after a second course. There was no significant correlation between lymphocytotoxin production and subsequent pregnancy or pregnancy outcome. 4 (67%) of the women who produced lymphocytotoxins, but none of the non-responders, had HLA-Bw 22 (P
belief that lymphocytes and trophoblast share an epitope important for the induction of a protective maternal immune response to the conceptus. Encouraging results have been reported using erythrocyte-compatible buffy coats2 and leukocyte-rich red blood cell concentrates3 from multiple unrelated donors, and also using mononuclear cell preparations from husbands not matched for erythrocyte antigens4 Only one double-blind placebo-controlled trial has been carried out,4 however, and the validity of the therapy remains controversial.5 Administration of blood components always carries some risk to the recipient, and safety considerations are of particular importance when a blood component is given to healthy people for what could be considered an experimental procedure. We therefore measured immunological factors before and after injection of mononuclear cell concentrates. This study is of particular interest as it provides a rare opportunity to observe the effect of a well-defined blood product on the immune system of apparently healthy individuals.
INTRODUCTION Idiopathic spontaneous abortion is sometimes thought to have an immunological basis.’ Consequently, women with a history of repeated miscarriages have been treated by injection or infusions of leukocyte-rich blood products in the
PATIENTS
AND METHODS Patients
The patients were 26 healthy women (mean age 3 1, range 23-39) with a history of recurrent miscarriage. All were antinuclear factor, anti-cardiolipin antibody
From the Blood Transfusion Service and ‘Simpson Memorial Maternity Pavilion, The Royal Infirmary, Edinburgh EH3 9HB, Scotland. Received and Accepted 5489. 257
258
Transfus. Sci.
Vol. 10, No. 3
and VDRL
negative, and had normal activated partial thromboplastin times. Some had had diagnostic laparoscopy; most had been screened for possible metabolic, hormonal or infectious causes of abortion. Both the women and their partners had normal karyotypes. Husbands who were acceptable as blood donors were considered acceptable as leukocyte donors irrespective of blood group. Where a Rh-D negative wife had a Rh-D positive husband, anti-D was given 24 h after the mononuclear cell injection. Only one woman had cytotoxic antilymphocyte antibodies prior to immunization; she had had two children by a previous husband. Preparation and Administration of Mononuclear Cell Concentrates 1OOmL of blood was removed from the husband by venepuncture and immediately defibrinated. All subsequent procedures were performed under strictly aseptic conditions. The defibrinated blood was mixed with an equal volume of 0.9% saline, and Dextran-500 (10%) solution was added to a final concentration of 1% (v/v). After 15 min incubation at 37”C, the resulting leukocyte-rich upper phase was aspirated and layered into Ficoll solution (Lymphocyte Preparation Medium, Flow Laboratories, Irvine, U.K.) to prepare mononuclear cells.6 The mononuclear cells were collected from the interface, harvested and washed twice with 0.9% saline before finally resuspending in about 3.5 mL saline. The mean yield was lo* cells. The mononuclear cell concentrate was injected into the wife as follows: 3 x 0.1 mL intradermally into one arm; 3 x 0.1 mL subcutaneously into another arm; the remainder intravenously. The patients were observed for 1 h after the injections; blood pressure, body temperature and pulse were monitored regularly. The procedure was initially carried out while the patient was not pregnant. Where applicable, the procedure was repeated in early (within 6 weeks menstrual age) pregnancy.
Most patients were followed 24 h, 3-4 weeks and 3 months.
up at
Laboratory Methods Tissue-typing and cytotoxic anti-lymphocyte screening and cross-matching were performed by microcytotoxicity testing.’ Cross-matches were performed using serum from each woman against her husband’s unfractionated mononuclear cells (mainly T cells) and B cells prepared from the former by rosetting with sheep erythrocytes.* Each serum was also screened against a panel of lymphocytes from 30 unrelated individuals representing a wide variety of tissue types, and a panel of B lymphocytes from at least 10 patients with chronic lymphatic leukaemia. Immunoglobulin and complement components were measured by rate nephelometry; total haemolytic complement (CH,,) by lysis of antibody-sensitized sheep erythrocytes; blood grouping and erythrocyte antibody by standard screening methods; and T4:T8 ratios were measured using indirect immunofluorescence and flow cytometry, all as described elsewhere.9 IgE was determined by radioimmunoassay according to the manufacturer’s instructions (Tandem).
RESULTS The immunization procedure was well tolerated by the patients. There were no systemic adverse reactions, although some patients developed erythema, which usually subsided within an hour, at the injection sites. The delayed-type reaction at the intradermal sites was also mild: the area of erythema and induration was always small (mean 7 mm, range 1-15 mm, and observed 24 h after injection), never painful and only mildly, if at all, pruritic. 18 of the group became pregnant and were immunized again: local and systemic effects were similar to those experienced after the first immunization course. Measurements of immunoglobulin
Leukocyte Immunotherapy for Aborters
classes, complement components and haemolytic activity, and T4/T8 ratios, before and after immunotherapy, are summarized in Table 1. There were no significant changes in any of those immunological measurements in the group as a whole, nor were any consistent Table 1.
IgA IgM I@ c3 c4 Cf.&c
T4:T8
changes apparent in individuals (data not given). The erythrocytes of the patients and their partners were typed for ABO, Rhesus, MNS, Kell, Duffy (a) and Kidd (a) blood groups. All but two of the men had one or more alloantigens not shared by their wives (Table 2). Despite this, no red
Immune Profile Before and After Immunotherapy (n = 26)
24h (n=26)
Post: 3-4 weeks (n = 26)
3 months (n= 17)
11.452.4 1.98kO.89 1.65 + 0.64 36+41 1.12kO.33 0.27 + 0.08 110?29 2.1+0.7
11.2k2.3 1.97 + 0.86 1.61 kO.61 35f47 1.05 -t-0.23 0.27 + 0.08 114?28 2.0 f 0.5
ll.lk2.2 1.89 + 0.80 1.62 + 0.60 36+40 1.04 + 0.28 0.26 + 0.08 114*35 1.9f0.5
10.9 + 2.0 2.08 f 1.01 1.60 + 0.60 33+29 1.04 It 0.22 0.25 + 0.06 116528 1.8+0.5
Pre: IgC
259
All data are expressed as a mean + 1 SD Immunoglobulin G, A and M and complement components C3 and C4 are given in g/L; IgE is given in IU/mL; and total haemolytic complement (CH,,) is expressed as a percentage of the value obtained with a pool of normal blood donor sera. Table 2.
Red Blood Cell Incompatibilities ABO
Rhesus
Lewis
1
-
C
Le”
2’ 3’ 4’ 5 6’ 7’ 8’
B B -
Couple
9 10 11’ 12’ 13* 14* 15 16” 17’ 18’ 19 20* 21’ 22’ 23’ 24* 25 26
A B B A A A A A, B -
C, e : c E/c ; C, D -
Leb -
Leb
C
-
-
-
Fy” Fy” -
-
E M -
E, c
-
Kidd
-
Le” -
E, c -
Duffy
S
M -
Le” Le” -
Kell
S M,S -
-
Lea Le” -
CCD C: D C -
MNS
Cl Kl -
N N
Iz -
-
-
S NY s -
FY” Fy” -
G -. -
Fy” Fy”
-
-
-
Incompatability refers to blood groups possessed by the male leukocyte donor and not shared by his partner. All patients’ sera were negative for anti-red blood cell antibodies when tested 2-4 weeks before the commencement of immunotherapy. 18 patients became pregnant and were immunized a second time; these women are indicated by asterisks.
260
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blood cell antibodies could be detected after immunization. This was also true after the second course of vaccination for the 18 women who became pregnant. Both spouses were fully tissue-typed for HLA-A, B and DR antigens (Table 3). All but one of the men were HLA-incompatible with their partners and 6 of 25 (24%) of the women produced cytotoxic anti-lymphocyte antibodies (Table 3 J. The production of lymphocytotoxins was unrelated to the number of cells injected, and only weakly associated with the degree of HLA-incompatibility (Fig. 1). Four of the six antibody producers possessed HLA-Bw22. This frequency of Bw22 is highly significant whether compared with its absence in the 19 nonantibody producers (P=O.O012) or with its presence in only 11 out of 264 in the general population (P = 0.00009) (Fisher’s exact test). Even after correcting for the number of antigens tested, the values were still significant: P
minimal erythrocyte contamination. These modifications eliminated the risk of alloimmune thrombocytopenia in a subsequent pregnancy and we hoped would reduce the risk of haemolytic disease of the newborn. Secondly, the dosage was considerably smaller: the equivalent of a quarter of a standard blood donation unit was used, but this amount was used again during pregnancy. It is noteworthy that more recent data from Mowbray and colleagues indicate that, while exact dosage is unimportant, the best pregnancy success is obtained after immunization in very early pregnancy.‘O This form of immunotherapy is of unique value to transfusion medicine as an instance where a well defined blood product is administered to healthy subjects. Interest in the effect of blood components on the immune system has recently been stimulated by several considerations. Kidney transplant recipients who received blood transfusions have better graft survival statistics, and this “transfusion effect” may be associated with the infusion of leukocytes.” Patients with haemophilias and congenital anaemias receiving blood components exhibit immunological abnormalities, including a decreased T4/T8 ratio, which have been suspected of contributing to the development of AIDS in individuals infected with the human immune deficiency virus. 12J3 Several retrospective surveys have suggested that blood transfusion increases susceptibility to various infections and malignant diseases.14 Such observations have usually been made on ill patients suffering from clinically-apparent diseases who had received whole blood or individual components in varying amounts and combinations. It is therefore an important finding that the intravenous administration of mononuclear cells to healthy, normal (apart from their reproductive problems) women produced no significant alteration in T4/T8 ratio either immediately (24 h) or during the course of the following 3 months. None of the women became sensitized to erythrocyte alloantigens,
Leukocyte Immunotherapy for Aborters 261 Table 3.
Tissue Types and Anti-lymphocyte Antibody Responses of Immunotherapy Couples
Couple
Tissue Types
Cross-matches T B
Cell Panels T B
-
-
-
-
-
-
-
-
F: Al, 3; B7, 8; DR2, 5 M: A2; B14, 44; DRl, 4
-
-
-
-
F: A2, 28; B14, 44; DR2, w6 M: Al; B8; DR3
-
-
-
-
F: Al, 2; B5, 35; DR4 M: Al, 26; B5; DR2, 4 F: Al, 28; B35, 40; DR3, 5 M: A24, 25; B5, 7; DR5, 7
Cross-matches Positive after second immunization
-
-
-
-
comments
5
F: Al, 11; B7, 27; DR4, w6 M: A2,32; B14,35; DR7
6
F: A3, 30/31; B7, 18: DR2, 3 M: A2, 29; B7, 44; DR2, 7
-
-
-
-
7
F: A3, 11; B7, 22; DR2, 5 M: A3, 29; B7, 44; DR4, 7
+i-
++
56%
67%
8
F: Al, 21 B8, 441 DRl, 3 M: Al, llj B7, 35; DRl, 7
-
-
-
-
9
F: A3; B5, 73 DR2, 5 M: Al, 3j B7, 8; DR3, 7
-
-
-
-
10
F: A2, 24; B27, 35; DRw6, ~8 M: A2, 28; B21, 40; DR4, ~6
-
-
-
-
11
F: A3, 24; B7j DR2 M: All, 24; B7, 223 DR2, ~6
-
-
-
-
12
F: Al, 28; B22, 353 DR4, ~8 M: A2, 293 B44j DR2, 7
++
++
66%
70%
13
F: Al, 31; B15, 40; DR4, ~8 M: All; B5,35j DR5, ~9 F: A3, 263 B5, 8j DRw6 M: Al; B8, 153 DR3, 5
-
-
-
-
-
-
-
F: Al, 2; B8, 40; DRX M: A2, 29; B7, 44; DR7 F: Al, 24; B35j DR5 M: Al, 24; B35j DR5
-
-
-
-
-
-
F: Al, 23 B8, 44j DR3, 4 M: A2, 3; B14, 44; DR4, 7 F: Al, 2; B8, 40; DR4, 5 M: A2, 26; B7, 14; DR2, 7 F: Al, 3; B8, 181 DR3 M: A26, 30/31; B14, 18; DR3, 7
-
-
-
-
-
-
-
-
14 15 16 17 18 19
20
F: A2, 11; B22, 44; DR3 M: A2, 33 B7, 44; DR2, 3
21
F: A2, 29; B21, 443 DR4, ~6 M: A2j B16,44; DR2, ~6 F: Al, 3; B7, 8; DR2, 3 MI Al, 3; B8, 17; DR3, 7
22
Anti-Bw4
Anti-Bw4
(Reacted with 40% of cell panel and gave weakly positive cross-match before immunization). + + T(23%)and B (81%) cell panel positive after second immunization. -
-
-
continued overleaf
262
Tnn~sfus. Sci.
Vol. 10, No. 3
Table 3-continued Cross-matches T B
Cell Panels T B
Couple
Tissue Types
23
F: A23, 28; B22, 44; DR4, 7 M: Al, B8, 16; DR4, w6
+-I-
++
68%
24
F: Al; B35, 44; DR5, w9 M: A2, 3; B7, 8; DR2, 7
++
++
-
-
25
F: A3, 28; B35, 44; DR2, 7 MI All, 29; B35; 44, DRw6, ~8
-
-
-
-
F: A2, 33 B7, 44; DR2, ~6
-
+
-
-
26
M: All,
70%
Comments Anti-Al
+
Polyspecific (>90% ) towards cell panels after second immunization.
26; Bl6, 35; DR2, ~6
For anti-paternal cell cross-matches, half reactions (~60% cell death) are indicated by (+) and full reactions IS&JO%cell death1 bv I+ + 1.Reactivity against the cell panels is expressed as a percentage of strongly positive 1360% cell deat&) r&cti&s. ’ -
although most of the men had antigens not shared by their wives, and prophylactic action could only be taken against Rhesus-D sensitization. The couples included three cases of Kell positive men with Kell negative wives; Kell-mediated hameolytic disease is usually caused by blood transfusion and is often fatal to the fetus.r5 Although the extent of erythrocyte contamination in our mononuclear cell preparations was variable, it was always small relative to the cell number which might be expected to cause sensitization. Our finding that only six out of 25 patients (24%) produced lymphocytotoxins in response to a single injection of mononuclear cells was surprising in view of the higher rate of seroconversion (4476%) reported by others.4J6J7 This difference cannot be accounted for in terms of dosage, as only one of the other groups administered a significantly larger number of lymphocytes, and indeed within our own series sensitization was independent of cell number in the vaccine. Although only cells mismatched by at least 3 HLA specificities were im-
munogenic, there was no trend towards increased sensitization with increasing degree of HLA incompatability. Sensitization, therefore, may depend mainly on whether the subject is a high or low responder; responder status might show
2.5
2.0 : 8
CELLS (x10-81
‘.5
0 0
1.0
F 0 0
0.5
0 1
4
MISMATCHES
Figure 1. Anti-lymphocyte antibody production in relation to mononuclear cell dose and HLA-compatibility. Presence (W) or absence (0) of anti-paternal lymphocyte cytotoxic antibodies after a single course of injections is shown in relation to number of lymphocytes injected and the degree of HLA incompatibility. The latter is quantified as mismatches i.e. HLA specificities possessed by the man not shared by his partner.
Leukocyte Immunotherapy for Aborters
HLA associations, and indeed several HLA specificities were over-represented in the antibody producing women, strongly and significantly so with HLABw22. The other workers referred to prepared their immunogen from anticoagulated blood which inevitably yields a mononuclear cell preparation contaminated by platelets. We speculate that the combination of lymphocytes and platelets may be more immunogenic than either alone. The observations of Fisher et ~1.‘~ that platelet transfusion stimulated lymphocytotoxin production only when contaminated with a small number of lymphocytes, indirectly supports that suggestion, although a different mechanism might be involved. While it has been claimed that the production of lymphocytotoxins is an indirect marker of successful immunotherapy, 4,17 MacLeod and coworkers have found that non-cytotoxic anti-lymphocyte antibodies correlate best with both pregnancy maintenance and successful renal graft survival.1y,20 On the other hand, sensitization to lymphocyte and platelet antigens may be undesirable and potentially harmful in the long term. Such sensitization is a well established problem with some patients requiring renal or cardiac transplants, or those requiring platelet transfusions for thrombocytopenia. There is also the rare but real possibility of combined immunodeficiency and maternal graft-vs-host disease.21 Although we have concentrated on safety aspects and do not have enough patients to evaluate the efficacy of the method, our preliminary results are encouraging. 10 women have delivered normal-sized babies, 2 are pregnant in the 3rd trimester, 1 aborted apparently from cervical incompetence at 20 weeks and 5 others had early abortions. (There was no correlation between anti-lymphocyte antibody production following immunization and pregnancy outcome: 3 (30%) of the women with live births and 1 (20%) of the early aborters were antibody producers.) The 6 children who were old enough were seen by a paediatri-
263
cian at 1 yr of age, and their development, assessed by the Denver screening system, was within the normal range. In conclusion, the results reported here are reassuring since they reveal no adverse, undesirable or unexpected responses to the administration of allogeneic leukocytes in these patients. Husband-specific preparations obviously carry less risk of infection than a pooled preparation from unrelated donors, and we have now shown that a low dose of a highly purified mononuclear cell preparation carries a relatively low risk of sensitization to alloantigens. Acknowledgements We thank the MLSO staffofthe Blood Transfusion Centre for technical assistance, the nursing and medical staff of Ward 36 for their co-operation, and Dr R. Hague for carrying out paediatric assessments.
REFERENCES 1. Mowbray JF, Underwood JL: Immunology of abortion. Clin Exp Immunol 1985; 60: l-7. 2. Taylor CC, Faulk WP, McIntyre JA: Prevention of recurrent spontaneous abortions by leukocyte transfusions. / R Sot Med 1985; 78:623-627. 3. Unander AM, Lindholm A: Transfusion of leukocyte-rich erythrocyte concentrates: a successful treatment in selected cases of habitual abortion. Am 1 Obstet Gynecol 1986; 154:516-520. 4. Mowbray JF, Gibbings C, Liddell H, Reginald PW, Underwood JL, Beard RW: Controlled trial of treatment of recurrent spontaneous abortion by immunisation with paternal cells. Lancet 19853 1:941943. habitual abor5. Adinolfi M: Recurrent tions, HLA sharing and deliberate immunisation with partner’s cells: a controversial topic. Human Reprod 1986; 1:45-58. 6. Boyum A: Isolation of lymphocytes, granulocytes and macrophages. Stand 1 Immunol 1976; 5(Suppl.5):9-15. 7. Tiwari JL, Terasaki PI: In HLA and Disease Associations. New York, Springer, 1985, pp. 14-17. 8. Kilpatrick DC, Darg C: Enzymatic modification of the lymphocyte surface: application to tissue-typing and rosetting with sheep erythrocytes Tissue Antigens 1983; 21:309-317.
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9. Kilpatrick DC, Liston WA, Neil1 G, Maxwell C: Immune profile of women experiencing recurrent spontaneous abortions of unknown aetiology. Disease Markers 1989; 7:87-94. 10. Mowbray JF: Immunology of early pregnancy. Human Reprod 1988; 3179-82. 11. Singal DP, Ludwin D, Blajchmon MA: Blood transfusion and renal transplantation. Br I Haematol 1985; 61595-602. 12. Carr R, Veitch SE, Edmund E, Peutherer JF, Prescott RJ, Steel CM, Ludlam C: Abnormalities of circulating lymphocyte subsets in haemophiliacs in an AIDS-free population. Lancet 1984; 1:1431-1434. 13. Kaplan J, Samaik S, Getlin J, Lusher J: Diminished helper/suppressor lymphocyte ratios and natural killer cell activity in recipients of repeated blood transfusions. Blood 1984; 64:308310. 14. Blumberg NG, Heal JM: Perioperative blood transfusion and solid tumour recurrence. Blood Rev 198 7; 1: 2 19-229. 15. Farr V, Gray E: Pregnancy outcome in mothers who develop Kell antibodies. Scott Med 11988; 33:300-303. 16. Beer AE, Semprini AE, Xiaoyu Z, Quibbeman JF: Pregnancy outcome in human couples with recurrent spontaneous abortion: HLA antigen profiles; HLA antigen sharing; female serum MLR
17.
18.
19.
20.
21.
blocking factors; and paternal leukocyte immunisation. Exp Clin Zmmunogenet 1985; 2: 137-153. Reznikoff-Etievant MF, Simonney N, Janaud A, Darbois Y, Netter A: Treatment of recurrent spontaneous abortions by immunisations with paternal leukocytes. Lancet 19853 1:1398. Fisher M, Chapman JR, Ting A, Morris P: Alloimmunisation to HLA antigens following transfusion with leukocyte-poor and purified platelet suspensions. VOX Sang 19853 49:331-335. MacLeod AM, Power DA, Mason RJ, Stewart KN, Skewan WG, Edward N, Catto GRD: Possible mechanism of action of transfusion effect in renal transplantation. Lancet 1982; 2:468470. Power DA, Catto GRD, Mason RJ, MacLeod AM, Stewart GM, Stewart KN, Shewan WG: The fetus as an allograft: evidence for protective antibodies to HLA-linked paternal antigens. Lancet 1983j 2:701-704. Bastain JF, Williams RA, Ornelas W, Tani P, Thompson LF: Maternal isoimmunisaresulting in imtion combined munodeficiency and fetal graft-versushost disease in an infant. Lancet 19843 1:1435-1437.