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From the Department of Obstetrics and the Department of Immunohematology and Blood Bank, University Hospital Leiden, Leiden, The Netherlands.
Intrauterine transfusion (IUT) therapy is the treatment of choice in severe hemolytic disease of the fetus. This treatment automatically implies the introduction of alloantigens in the fetal circulation, which might potentially influence the unprimed fetal immune system. The present study provides evidence that the fetal immune system is indeed prone to modulations of the T-cell receptor BV (TCRBV) repertoire as a result of IUT treatment. Most notably, IUT therapy affects the composition of the CD4+ repertoire, whereas this effect may be obscured in the CD8+ subset. The CD8+ subset was found to be influenced by alterations of the TCRBV repertoire both in IUT patients and controls, suggesting that modulations in this subset could be the result of developmental influences. A more detailed analysis on the composition of the individual TCRBV families was performed by evaluating the distribution of the complementarity determining region 3 (CDR3) size lengths of [32P]-radiolabeled TCRBV transcripts. Using this technique, referred to as spectratyping, only marginal changes were observed in the CD4+ and CD8+ subset during the course of treatment and gestational development of both IUT-treated patients and controls. Therefore, the alterations in the overall TCRBV repertoire were of a quantitative rather than a qualitative nature. To evaluate whether the observed alterations in TCRBV usage-frequencies were a reflection of an allo-reactive response, a primed lymphocyte test (PLT) was performed in 3 IUT-treated patients. We observed that IUT, performed as early as 23 weeks of gestation, may induce the establishment of memory T cells against the IUT donor. However, there was no association between the observed changes in TCRBV repertoire and the magnitude of the secondary allo-reactive response.
DESPITE THE USE of Rhesus(D) immunoprophylaxis and matching for this antigen in blood transfusion therapy, approximately 1% of the Rhesus(D)-negative fertile women develop antibodies to erythrocyte antigens.1 Pregnancies complicated by severe hemolytic disease can be successfully treated with IUT therapy. From a technical point of view, IUT has become an established procedure, resulting in a survival of approximately 90% of affected fetuses.2,3
Under physiological circumstances, the development of the fetal immune system occurs in an almost sterile and allo-antigen-free environment. Throughout gestation, the fetus is mainly confronted with both self and also selected maternal antigens. Therefore, it would not be unreasonable to assume that the introduction of allo-antigens into the fetal circulation by intrauterine transfusion (IUT) might have an impact on the fetal immune system. However, the immunologic consequences of IUT treatment have, until now, not been investigated in great detail.
Blood transfusions are known to have potent immunomodulatory properties. For instance, administration of an HLA-(B),DR-matched blood transfusion before transplantation can result in an increased allograft survival.4-7 This toleration effect might be the result of induction of anergy, suppression, or deletion of allo-reactive T cells.8 It has been suggested that these transfusions result in cytotoxic T-lymphocyte nonresponsiveness to donor antigens9 and can result in the absence of several T-cell receptor BV (TCRBV) families.10 Because tolerance is easier to induce in neonates than in adults,11,12 the effect of allogeneic blood transfusion might be even more pronounced in the fetus.
IUT treatment offers a unique model to simultaneously investigate the effect of blood transfusion on the fetal immune repertoire and the allogeneic response of an unprimed immune system in vivo. Direct access to the umbilical cord under ultrasound guidance has made it possible to obtain peripheral blood of the fetus at different time points in gestation.2,3
To evaluate semiquantitative influences of IUT therapy on the fetal CD4+ and CD8+ TCRBV repertoire, the polymerase chain reaction (PCR) technique with 25 TCRBV family-specific oligonucleotide primers was used to determine the TCRBV gene element usage both before and after IUT treatment in 5 fetuses. Changes in the distribution of complementary determining region 3 (CDR3) length of the TCRs during the course of therapy were analyzed using spectratyping.13 The results were compared with 5 patients who underwent fetal blood sampling (FBS) but did not receive IUT therapy. In 3 IUT-treated patients, the magnitude of the alloresponse against several IUT donors was determined by a primed lymphocyte test (PLT) in cord blood (CB), obtained after delivery.
IUT Patients
Controls
Preparation of IUT
Collection of the Samples Fetal blood was obtained by FBS in IUT patients before the onset of the transfusion. Immediately after birth, umbilical cord blood was collected before the delivery of the placenta. If this was not possible (patients no. 7 and 8), then peripheral blood was obtained after birth by venapuncture. All samples were collected in heparinized tubes, and 1 mL was used for TCRBV analysis. Mononuclear cells were isolated by Ficoll-Hypaque density gradient sedimentation within 12 hours after collection. The cells were then frozen in liquid nitrogen and stored at -170°C before fluorescence-activated cell sorting (FACS).FACS After thawing, mononuclear cells were washed twice with Hanks' balanced salt solution (GIBCO, Paisley, UK) and subsequently stained with fluorescein isothiocyanate-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8 (Becton Dickinson, San Jose, CA) monoclonal antibody for 30 minutes at 4°C. Cells were washed twice in Hanks' medium and resuspended in 50% RPMI 1640/50% fetal calf serum. CD4+ and CD8+ cells were separated using FACS (Becton Dickinson, Mountain View, CA) and collected in 50% RPMI1640/50% fetal calf serum. Cells were then washed twice in phosphate-buffered saline at 4°C.RNA Isolation, cDNA Synthesis, and PCR Amplification Analysis of the TCRBV was performed using a semiquantitative PCR as described previously.14,15 RNA was isolated from the sorted cells using the RNAzol method (Cinna/Biotecx Laboratories, Inc, Houston, TX), dissolved in 25 µL of distilled water, and stored at -80°C. Five micrograms of RNA was reversed-transcribed into first-strand cDNA using the Riboclone cDNA synthesis system (Promega, Madison, WI). This cDNA was first quantified with control primers and diluted with distilled H2O to an amount in which 1 µL per reaction resulted in PCR products in log phase for each primer used. The TCRBV repertoire was analyzed using 25 TCRBV-specific primers identifying 24 different TCRBV families. For each PCR, 1 µL of cDNA was used and added to a mixture of 20 pmol of 3' antisense TCRBC primer, 0.5 mmol/L of each dNTP, 10 mmol/L Tris HCl (pH 8.4), 50 mmol/L KCl, 4 mmol/L MgCl2 , 0.06 mg/mL bovine serum albumin, and 2.5 U AmpliTaq DNA polymerase (Perkin/Elmer, Roche Molecular Systems, Inc, Branchurg, NJ) and 20 pmol of each TCRBV-specific 5' sense primer in total volume of 100 µL. Twenty-five to 35 PCR cycles were performed in a Thermocycler 60 (Biomed Instruments, Fullerton, CA), depending on the amount of cDNA. The PCR cycles consisted of a 95°C denaturation, a 55°C primer annealing, and a 72°C extension step for 1 minute each. A 5' sense primer specific for the constant region of the TCR was used as an internal control. As a negative control, a PCR without template was performed to exclude contamination. Five microliters of the PCR-amplified products was size fractionated on a 1% agarose gel and subsequently transferred to a nylon filter (Hybond N+; Amersham International plc, Little Chalfont, Buckinghamshire, UK) for Southern analysis. The TCR chain-specific sequences were detected by hybridization with a [32P]-radiolabeled TCRBC-specific probe. The amount of each individual TCRBV PCR product was determined using either autoradiography on Kodak XAR films (Eastman Kodak, Rochester, NY) and laser densitometry (LKB 2220-020, Ultrascan XL; Pharmacia LUB Biotechnology, Uppsala, Sweden) or phosphor-imaging (PhosphorImager 445SI; Molecular Dynamics, Sunnyvale, CA). All PCR amplifications were performed in duplicate with correlation coefficients (R2 ) of >.95 between the two analyses.
Spectratyping Spectratyping of the different TCRBV families was determined according to the methodology described by Gorski et al.13 Briefly, 1 µL of cDNA was used for each PCR. PCR conditions were comparable to those described above, with the exception that 20 pmol of a 3' antisense primer was used for each reaction that was end labeled with -[32P]-ATP. PCR consisted of 30 to 40 amplification cycles, including a 94°C denaturation, a 58°C primer annealing, and a 72°C extension step for 1 minute each. Before transfer of the radioactive PCR products to a prewarmed 6% acrylamide-urea sequencing gel (Ultrapure Sequagel-6; National Diagnostics, Atlanta, GA), samples were boiled for 5 minutes. Gels were run for 2.5 to 3.5 hours and subsequently dried. The spectratypes were visualized by autoradiography and phosphor-imaging.
Validation of the Procedure PCR analysis with HLA-A- and HLA-B-specific probes, followed by allele-specific DNA-typing, showed that cord blood of the IUT treated patients used for analysis was not contaminated with donor leukocytes.PLT The proliferative alloresponses of 3 IUT-treated patients were determined against 8 original IUT donors using PLT. Briefly, 5 × 104 responder cells were cultured with 5 × 104 irradiated stimulators (30 Gy) in 96-well round-bottomed microtiter plates (Costar, Cambridge, MA). Cultures were incubated for 3 days at 37°C in humidified air containing 5% CO2 . Cells were subsequently pulsed overnight with [3H]-thymidine and then harvested with an automated cell harvester. Simultaneously, the responses of the IUT patients were tested against individuals who were either HLA-DR-matched or mismatched with the IUT donors. The responses of the IUT patients against the different stimulators were compared with responses of donors that were HLA-DR matched with the IUT patients and tested against the same set of stimulators. In the same test, responder cells of both IUT patients and HLA-DR-matched donors were incubated with autologous irradiated stimulators. The proliferative response was assessed by measuring [3H]-thymidine incorporation using scintillation counting. The stimulation index (SI) is calculated by dividing the counts per minute measured when stimulator and responder cells were cultured together by the sum of the counts per minute when the stimulator and responder cells were cultured separately.Statistical Analysis Overall changes in the TCRBV gene usage frequencies between the two time points of analysis were determined by correlation coefficients calculation (R2 ). An R2 value of 1.0 resembles a perfect correlation between the first time point of analysis and the second time point, whereas an R2 value of .0 resembles no correlation between the two time points. Differences in the usage frequencies of individual TCRBV genes in CD4+ as well as CD8+ T-cell subsets between controls and IUT-treated patients were determined by the two-tailed unpaired Student's t-test. When the standard deviations in the IUT-treated patients and controls were not equal, the nonparametric Mann-Whitney U test was used. Results were also corrected for the number of parameters analyzed by the method of Edwards.16 PLT responses were compared with the two-tailed paired or unpaired Student's t-test. For all statistical analyses, a P value of <.05 was considered significant.
To determine whether IUT therapy had any influence on the TCRB repertoire, we compared the composition of the TCRBV repertoire, both before and after IUT treatment, in 5 IUT patients using a semiquantitative PCR and spectratyping. To exclude developmental influences, similar experiments were performed in 5 control patients. In all patients, the first FBS was used as a reference. In IUT-treated patients, the first FBS was performed before the onset of transfusion therapy to determine the fetal hematocrit or platelet count. On average, the first FBS was performed at 26.6 weeks in the IUT group compared with 27.4 weeks in the control group (Table 1). The composition of the TCRBV repertoire of this CB sample was compared with a sample obtained at a second time point. For 3 IUT patients (nos. 1, 2, and 5), we compared the first FBS with a CB sample before the last IUT. This was performed to exclude possible contamination with donor leukocytes, because the interval between the last IUT and delivery was less than 2 weeks. The second time point of measurement was at 35 weeks of gestation, on average in the IUT group, compared with 38.8 weeks in the control group (Table 1). The median interval between the two measurements was 7 weeks for the IUT group and 10 weeks for the control group. Semiquantitative Analysis of the TCRBV Gene Usage Frequencies Using PCR The TCRBV repertoires of CD4+ and CD8+ T-cell subsets were analyzed separately because they display different usage profiles.14,17,18 The individual TCRBV repertoires in IUT-treated patients and controls are shown in Fig 1A and B. Each individual patient showed a distinct usage pattern of the various TCRBV gene elements in the CD4+ and CD8+ subsets. All TCRBV gene elements were used for each individual patient both in IUT-treated patients and controls, except for patient no. 4 (Fig 1A). In this patient, several TCRBV gene segments remained undetectable in the CD4+ subset at 32 weeks of gestation (TCRBV 17 and 19 through 24). However, most of these genes were detected in cord blood after delivery, although the usage frequencies of TCRBV 20 through 22 remained low in this patient.
Spectratyping of the TCRBV Repertoire We have used the technique of spectratyping to investigate whether the observed semiquantitative changes of the TCRBV repertoire in the CD4+ subset of the IUT-treated patients also resulted in qualitative alterations in the distribution of CDR3 size lengths of the [32P] -radiolabeled TCRBV transcripts.13 In Fig 2, the distribution of the CDR3 size lengths of the TCRBV families that showed the most pronounced modulations of the TCRBV gene usage frequencies are displayed in conjunction with the least affected families. These results show that, in general, the observed expansions or contractions of TCRBV gene usage, as determined by semiquantitative PCR analysis (Fig 1A and B), in the CD4+ subset did not result in alterations of the respective spectratypes. One exception was patient no. 4, in whom major changes in the distribution of the CDR3 lengths were noted during the course of IUT treatment. Before IUT treatment, this patient showed a very restricted TCRBV repertoire that differed from all other patients in that they showed a more or less binomial distribution of all TCRBV products. These results were comparable with the semiquantitative PCR, showing a restoration to a more or less normal distribution pattern after the course of IUT treatment. Both in the CD4+ and CD8+ subset of the other IUT-treated patients and controls, the individual TCRBV families remained relatively unchanged and showed only marginal changes during gestation (data not shown).
Proliferative Responses to the Original IUT Donors Finally, we determined if these molecular changes in the CD4 subset of IUT-treated patients were a reflection of an allo-reactive response against the IUT donor. Of 3 IUT-treated patients (patients no. 1, 2, and 3), sufficient numbers of CB mononuclear cells were available to perform such extended studies. Because no pretransfusion samples of these fetuses were available, proliferative responses against the IUT donors were compared with the responses of healthy nontransfused adult donors that were HLA-DR-matched with the IUT-treated patients (Fig 3). Both IUT patients and HLA-DR-matched responders were also tested against autologous mononuclear cells and third-party stimulators that were either HLA-DR matched or mismatched with the IUT donor. Clear proliferative responses of all IUT patients were observed against their original IUT donors (mean SI = 16.0). Responses of the IUT patients against stimulators mismatched with the IUT donors were much lower (mean SI = 5.4). Establishment of a statistical significance was not possible due to the limited number of individuals (mismatched with the IUT donor) tested. In all but 2 cases (fetus no. 1 against donors no. 2 and 4), the responses were higher than those of the HLA-DR-matched responders against the same set of IUT donors (P = .045). In 3 cases, the SI of IUT patients was 75% higher when compared with the HLA-DR-matched controls (patient no. 2 against the second IUT donor and patient no. 3 against both IUT donors). Patients no. 2 and 3 showed higher responses to all IUT donors when compared with HLA-DR-matched control responders. Patient no. 2 showed a memory response against the second IUT donor administered at 23 weeks of gestation. To determine if these responses were HLA class II specific, the allo-reactive responses against third-party individuals (that were HLA-DR matched with the original IUT donor) were determined (SI = 12.85). These responses were comparable to the allo-reactive capacity against the original IUT donor (P = .356), although in individual cases the responses were clearly lower. The spontaneous responses of IUT patients (average, 1,048 counts per minute; range, 425 to 2,168 counts per minute) were higher then those of the HLA-DR-matched controls (average, 349 counts per minute; range, 250 to 426 counts per minute) but was corrected for in the calculation of the SI.
In vivo exposure to allo-antigens can profoundly change the composition of the TCRBV repertoire.10,19,20 For example, heart transplantation patients showed quantitative changes of several TCRBV families in peripheral blood, just before a rejection crisis.20 Munson et al10 reported that transfusions with HLA-B,DR-shared donor blood can result in complete deletions of one or more TCRBV families. In vitro experiments have shown a restricted but heterogeneous TCRBV gene usage of the allo-reactive T cells.21-23
Submitted July 29, 1996;
accepted May 21, 1997.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hearly marked ``advertisment'' in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
The authors thank Dr E. Goulmy, Dr Frans Claas, Dr David Sherr, and Dr G.C. Beverstock for critically reading the manuscript; Jenny Verdoes and Annemiek van Rooden for collecting the cord blood samples; Maarten van der Keur and Arie van der Marel for FACsorting and analysis; and Jacqueline Anholts for her expert technical assistance.
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Abstract
Introduction
Abstract
) and least (
) pronounced alterations during the course of IUT treatment. PCR products were analyzed on a phosphor-imager before (B) and after (A) the start of treatment.
. DR 1, 2 
. DR 11(5), 3 
. DR 6, 8; 3 dp matched, 2 
. DR 6, 7, 2 
. DR 2, 4; 3dp matched, 1