Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism

Abstract

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20–39 and 24–45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by ³H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.

Introduction

The human platelet antigen (HPA)-1a1b system (previously known as Pl^A1/A2 and Zw^a/b) results from a single amino acid polymorphism (Leu/Pro) at residue 33 of glycoprotein (GP) IIIa [1]. On platelets, GPIIIa (β3 integrin) is noncovalently associated with GPIIb (αIIb integrin) forming the platelet receptor for fibrinogen (CD41/CD61) which mediates platelet aggregation [2]. The β3 chain can also associate with αv integrin forming the vitronectin receptor (CD51/61) that is expressed on many tissues [3] and is also involved in platelet adhesion and aggregation at sites of vascular injury.

The incidence of HPA-1b1b is approximately 2·5% in Caucasians [4]. The immune response to HPA-1a is HLA class II restricted with a very strong association with HLA-DR52a (DRB3*0101) for antibody development [4,5]. HPA-1a alloantibodies recognize a conformational epitope that includes Leu33 on the platelet GPIIIa [6,7]. If present during pregnancy, maternal anti-HPA-1a IgG may cross the placenta causing destruction of fetal HPA-1a⁺ platelets. Fetomaternal incompatibility for HPA-1a is the most common target antigen in fetal and neonatal alloimmune thrombocytopenia (NAIT) [8]. Severe thrombocytopenia may result in purpura or, much more seriously, fetal or neonatal intracranial haemorrhage (ICH) that may be fatal or have severe neurological sequelae [9]. Most cases, especially in a first-born infant, are not diagnosed until after birth, when the recommended treatment is transfusion of HPA compatible platelets [10]. Approaches to the antenatal management of subsequent pregnancies include serial fetal blood sampling and intrauterine transfusion (IUT) of platelets [11] but the current preferred option, which is less invasive, is administration of intravenous immunoglobulin (IVIG) with or without steroids to the mother [12,13]. Severe thrombocytopenia is usually but not always associated with high maternal HPA-1a antibody levels during the third trimester [4] or at delivery [14]. Currently there is no noninvasive predictive test for the severity of NAIT [15] although the clinical history is important in deciding appropriate clinical management [16].

The HLA class II-restricted antibody response to HPA-1a is T helper cell dependent. For such responses, foreign antigens are endocytosed and processed by antigen presenting cells, most efficiently by dendritic cells (DC). Peptides resulting from proteolysis of the antigen are loaded onto the cleft of HLA class II molecules. The HLA class II-peptide complexes are then expressed on the DC cell surface and presented to CD4⁺ T cells for recognition through their T cell receptors (TCR) [17]. If the TCR interaction with a HLA class II-peptide complex is long lasting, the T cells become activated by the DC. These T cells then stimulate those B cells that also express the same peptide-HLA class II complex after they have recognized and endocytosed the conformational antigen through their B cell receptors. This results in B cell activation and antibody secretion.

For HPA-1a responses, the T cell epitope is likely to include Leu33 in the peptide that is processed and presented by DC. A peptide encompassing the Leu33 polymorphism (residues 24–45) was found to bind to soluble recombinant HLA-DRB3*0101 whereas the corresponding Pro33 peptide did not. The core peptide sequence and C-terminal anchor residue (Leu33 but not Pro33) were also identified [18].

We used the HPA-1(24–45) peptide and also a shorter peptide with an extended N-terminal sequence (20–39) in both the Leu33 (HPA-1a) and Pro33 (HPA-1b) forms in T cell proliferation assays to detect responding T cells from women who had been alloimmunized by pregnancy. Most samples were taken from patients during pregnancy or postpartum. Our aim was to determine the frequency, specificity and magnitude of responses of T cells from these patients to these HPA-1a and HPA-1b peptides and ascertain whether the responses correlated with the clinical severity of NAIT. Initial results indicated that proliferation of T cells from these patients was depressed and experiments were therefore performed to elucidate and overcome this problem. Most patients had HPA-1a reactive T cells and some of these also had T cells reactive with HPA-1b peptides. Proliferation of T cells postpartum was greatest in women with a severe history of NAIT.

Materials and methods

Blood samples

Blood was obtained by consent from 13 pregnant or postnatal women with HPA-1a alloantibodies with an HPA-1a⁺ infant. All except one had had one or more previous fetuses affected by thrombocytopenia. Two patients (nos. 1,2) each had two pregnancies during which samples were taken. Seven blood samples were received postnatally from five patients (nos. 2,3,4,5,11) and one from a patient (no. 13) prior to the index pregnancy. The six pregnancies of patient no. 2 have been reported [19] and the first fetus of patient no. 6 had hydrocephalus caused by anti-HPA-1a [20]. Blood was also obtained from a female blood donor (no. 14) who had been found to have HPA-1a alloantibodies presumably resulting from immunization by pregnancy over 20 years previously. Clinical data are provided in Table 1. Control samples were obtained from 11 non-HPA-1a alloimmunized adults (five male, six female) representing a random, nonpregnant population.

Table 1. Clinical data of the patients and the donor.

Patient or donor and sample number	Week of sample (GA or PP)	Antibodies detected	Clinical history of NAIT in previous pregnancies	Administration of IVIG (weeks GA)	Fetal blood sampling (weeks GA)	Intrauterine transfusion (weeks GA)	Outcome of pregnancy included in this study GA at delivery; PC (if thrombocytopenic); status of neonate
1i	34 GA	HPA-1a (weak)	1)Thrombocytopenia at		24, 29, 35		36/40; healthy
		HPA-5b HLA class I	birth (PC 20) and in childhood
		HLA class I	childhood
1ii*	34 GA	HPA-1a (weak)	1)Thrombocytopenia at		34		36/40; healthy
		HPA-5b HLA class I	birth (PC 20) and in childhood
		HLA class I	childhood
			2) Healthy
2i	21 GA	HPA-1a	Splenectomised	24–29	22, 24, 25, 26, 28, 29	24, 25, 26, 28, 29	30/40; PC 127; healthy
2ii	2 PP		1)3) 4)IUD
			2)ICH < 34/40 (PC 15)
2iii*	28 GA	HPA-1a	Splenectomised	16–31	21, 23, 27, 31	21	34/40; healthy
2iv*	3 PP		1) 3) 4) IUD
			2) ICH<34/40 (PC 15)
			5) NAIT (treated: IVIG, IUT)
3i	24 GA	HPA-1a	1) TOP		28, 32	28, 32	36/40; healthy
3ii	28 GA		Blood donor, anti-HPA-
3iii	7 PP		la detected
4i	20 GA	HPA-1a	1) ICH (Pc 7)		20, 21, 22, 23, 24	20, 21, 22, 23, 24	33/40; healthy
4ii	1 PP		2) Purperic (PC 35)		25, 26, 27, 28,	25, 26, 27, 28,
					29, 30, 31	29, 30, 31
5i	3PP	HPA-1a (weak)					Term;
5ii	21 PP						thrombocytopenic
6i	20 GA	HPA-1a	1) TOP, 20/40 hydrocephalus	28–34			34/40; healthy
6ii	22 GA		2) IUD, 16/40
			3) neonatal death, 2/40
7i	15 GA	HPA-1a	10 TOP	15–21	21, 22, 23, 24, 25, 26	21, 22, 23, 24, 25, 26	26/40; PC 28;
7ii	21 GA		2) Healthy				required platelet
			3) Miscarriage, 8/40				transfusion;
			4) ICH & TOP, 21/40				premature; then
			5) Miscarriage, 11/40				healthy
			6) ICH & IUD, 30/40
8i	11 GA	HPA-1a (weak)	1) ICH and prolonged		21, 22, 23, 24, 25,	21, 22, 23, 24, 25,	33/40; healthy
			thrombocytopeni		26, 27, 28, 29,	26, 27, 28, 29,
8ii	20 GA				30, 31, 32, 33	30, 31, 32, 33
9i	13 GA	HPA-1a (weak)	1) Thrombocytopenia		20, 30, 35	35	35/40; healthy
10i	15 GA	HPA-1a	1) Thrombocytopenia	18–24	28, 31, 35	28, 31, 35	36/40; healthy
		HLA class I	(PC 3)
			2) Miscarriage, 15/40
11i	26 GA	HPA-1a	1) Thrombocytopenia	18–35	27, 29, 31, 35	27, 29, 31, 35	36/40; healthy
11ii	28 GA		(PC 9)
11iii	14 PP
12i	20 GA	HPA-1a	1) Miscarriage	22–35	29, 32, 35	29, 32, 35	35/40; healthy
		HLA class I	2) Petechiae (PC 9)
13i	36 PP	HPA-1a	1) Thrombocytopenia	n/a	n/a	n/a	n/a
			(PC 8)
13ii	28 GA	HPA-1a	1) Thrombocytopenia	16–36	31,36	36	37/40; healthy
			(PC 8)
14i	>20 years PP	HPA-1a	1) Healthy	n/a	n/a	n/a	n/a
14ii		HPA-1a	2) Healthy
14iii		HPA-1a
14iv		HPA-1a
14v		HPA-1a
14vi		HPA-1a

^*Second pregnancy included in this study GA = gestational age; PP, postpartum; PC, platelet count; thrombocytopenia PC < 150 × 10^9/l; ICH, intercranial haemorrhage; TOP, termination of pregnancy; IUD, intrauterine death; n/a, not applicable.

HPA genotyping and HPA-1a alloantibody detection

Genomic DNA isolated from mononuclear blood cells was typed for HPA-1a and -1b using a polymerase chain reaction with sequence-specific primers [21]. Platelet-specific antibodies were detected using an immunofluorescence test [22] and the monoclonal antibody immobilization of platelet antigens (MAIPA) assay [23], together with a panel of HPA-typed platelets.

Peripheral blood mononuclear cells (PBMC)

Blood was collected into EDTA anticoagulant and kept at room temperature for up to 24 h during transport before processing. Peripheral blood mononuclear cells (PBMC) from 15 to 30 ml blood were isolated by density gradient centrifugation over Histopaque-1077 (Sigma, Poole, UK), washed thrice in RPMI 1640 medium (PAA laboratories, Yeovil, UK) containing 10 iu/ml Sodium Heparin (CP Pharmaceuticals, Wrexham, UK), 100 U/ml penicillin (PAA), 100 µg/ml streptomycin (PAA) and 25 U/ml polymixin B sulphate (Gibco, Paisley, UK) and adjusted to a concentration of 1·4 × 10⁶/ml in Minimal Essential Medium (MEM) containing antibiotics (as above) and 1 mM glutamine (HyQ, Cramlington, UK).

Peptides and antigens

The 20- and 22-mer HPA-1 peptides were synthesized using F-moc chemistry on resin with 85–95% purity tested by HPLC and amino acid analysis. The lyophilized peptides were stored at −30°C. Aliquots (3–4 mg) of each peptide were solubilized and diluted to a concentration of 1–3 mg/ml in MEM and 0·2 µm filtered. The amino acid sequences were:

• HPA-1ai (20–39) SPMCAWCSDEALPLGSPRCD

• HPA-1aii (24–45) AWCSDEALPLGSPRCDLKENLI

• HPA-1bi (20–39) SPMCAWCSDEALPPGSPRCD

• HPA-1bii (24–45) AWCSDEALPPGSPRCDLKENLI

The control recall antigens were Mycobacterium tuberculosis purified protein derivative (PPD) obtained from the Central Veterinary Laboratory, Addlestone, Surrey, adsorbed tetanus toxoid (TT) supplied at 80 iu/ml (1 mg/ml) (Evans Medical Ltd, Leatherhead, UK) and tetanus toxin (TT) supplied at 6 mg/ml (Swiss Serum and Vaccine Institute, Berne, Switzerland).

T cell proliferation assay

This was based on the assay established by Plebanski & Burtles [24]. PBMC were aliquotted at 2 ml/well into 24-well tissue culture plates (Nunc International, Costar, New York, NY, USA) at 1·4 × 10⁶/ml. Autologous plasma was heat inactivated at 56 °C for 30 min followed by centrifugation at 2000 × g for 10 min to remove thrombin clots; 100 µl were added to the wells. In some assays, human AB serum was used in place of autologous serum (heat treated plasma). Stock solutions of the antigens were made in MEM and aliquots added to the wells to give final concentrations of 50 µg/ml of PPD, 0·1–1 iu/ml of adsorbed TT or 1–25 µg/ml of TT. HPA-1 peptides were added to the wells from stock solutions of 1 mg/ml in MEM to give final concentrations of 1, 3, 10 and 30 µg/ml. The pH of these stock solutions was 7·0 (7·01–7·04). Control wells received an equal volume of MEM. The plates were incubated at 37 °C in a humidified 5% v/v CO₂ incubator for 7 days and duplicate aliquots of 100 µl cell suspension transferred into a 96-well round bottomed plate on days 4–7. Then 16 µl ³H-thymidine (Amersham International, Amersham, UK) was added to each well (0·5 µCi/well) and the plates incubated as above for 16–24 h. The cells were harvested onto glass fibre filter mats (LKB-Wallac, Turku, Finland) using a Mach 111 harvester 96 (Tomtec, New Jersey, USA). Thymidine incorporation was determined using a Microbeta liquid scintillation counter (LKB-Wallac). The responses (counts per minute, cpm) of the test and control wells were expressed as the stimulation index (SI), calculated as SI = cpm (test)/cpm (control). Values of ≥3 SI were considered a positive response. Mean values of the duplicate wells were calculated. They were within 20% of each other in 94% of the positive samples. The cumulative SI was the total of the individual SI for the four days of sampling. The combined cumulative SI was the sum of the cumulative SI for all wells given HPA-1a or HPA-1b peptides. Samples were excluded from analysis if the cumulative SI to PPD plus TT was less than 20.

Statistical analysis

Differences between proliferative responses of T cells cultured in various sera were analysed for significance using the Mann–Whitney U-test using GraphPad Instat version 4·0 for Windows 95 (GraphPad Software, San Diego, USA).

Results

Effect of pregnancy and postpartum sera on T cell proliferative responses

Initial experiments using PBMC from pregnant women with autologous sera indicated that the cells proliferated less than would be expected from normal adults [25]. To investigate this, the ability of PBMC in three samples from two patients (nos. 2,6) to respond to the HPA-1a peptides in the presence of autologous or AB serum was examined. Cells proliferated in 65% of wells (11/17) containing AB serum but in none of the wells containing autologous serum. This result suggested that the serum from the patients depressed T cell proliferation. To determine if this effect was due to pregnancy, the ability of PBMC from pregnant and postnatal patients to proliferate against the recall antigens, PPD and TT, were tested in the presence of autologous or AB serum. The results are illustrated in Fig. 1. It can be seen that the proliferative responses to PPD and TT were profoundly suppressed in the presence of autologous serum both from a patient at 20 weeks of pregnancy (Fig. 1a) and another patient seven weeks postpartum (Fig. 1b). In contrast, however, in one PBMC sample obtained 21 weeks postpartum, suppression by autologous serum was not observed when compared to AB serum (Fig. 1c). The experiments were repeated with a number of control donors and the results are summarized in Table 2. The suppressive serum from the patient taken seven weeks postpartum was then tested with PBMC of nine normal control donors. It markedly suppressed the proliferation of T cells from these subjects to PPD and TT when compared with the donors’ autologous sera (Table 2, Fig. 2). Together these experiments demonstrated that there was a heat stable non-MHC restricted suppressive factor(s) present in serum during and after pregnancy. To avoid this inhibitory effect, AB serum was used in the T cell proliferation assay rather than autologous serum for samples taken from patients during pregnancy and the early postnatal period.

Fig. 1.

Proliferative responses to PPD (♦,◊) and TT (▴,Δ) of T cells from three patients, (a) no. 6i, 20 weeks GA; (b) no. 3iii, 6 weeks PP; (c) no. 5ii, 21 weeks, PP cultured in the presence of autologous serum (♦,▴) or AB serum (◊,Δ). Mean ± SEM.

Table 2. Effect of pregnancy serum and AB serum on proliferative responses of T cells from pregnant and postnatal patients and control donors. T cells were stimulated with PPD or TT antigens.

	Cumulative SI, mean ± SEM of group
	PPD		TT
Source of PBMC	Autologous serum	Other serum	Autologous serum	Other serum
Pregnant patients	Pregnancy serum	AB serum	Pregnancy serum	AB serum
(n = 7)	8 ± 4	86 ± 25 *	14 ± 7	156 ± 23 ***
Early postnatal patients	Early postnatal serum	AB serum	Early postnatal serum	AB serum
(n = 3)	36 ± 18	183 ± 21	15 ± 8	213 ± 70
Late postnatal patient	Late postnatal serum	AB serum	Late postnatal serum	AB serum
(n = 1)	191	167	166	175
Control donors	Control serum	Early postnatal serum	Control serum	Early postnatal serum
(n = 9)	351 ± 82	42 ± 20 ***	351 ± 100	10 ± 4·6 ***
Control donors	Control serum	AB serum	Control serum	AB serum
(n = 7)	332 ± 105	200 ± 82	365 ± 115	300 ± 205

^*P < 0·05;

^***P < 0·001. Early postnatal 1, 3 and 7 weeks postpartum, patient sample numbers 3iii, 4ii, 5i. Late postnatal 21 weeks postpartum, patient sample number 5ii.

Fig. 2.

Proliferative responses to PPD of T cells from two nonpregnant donors (a) male (squares) and (b) female (circles) cultured in the presence of autologous serum (grey symbols), AB serum (open symbols) or seven weeks postpartum serum (filled symbols). Mean ± SEM.

Comparison of autologous and AB serum on T cell proliferative responses

In contrast to the enhanced proliferation of T cells from pregnant and postnatal women when using AB serum compared to autologous serum, there was no significant difference in similar experiments using T cells from control donors (Table 2; Fig. 2). However, the use of autologous serum generally gave the highest proliferation of T cells from control and nonperinatal subjects, confirming earlier work [24].

Proliferation of T cells from serial samples

Six blood donations were received at 3–17 monthly intervals over a 44-month period from the nonpregnant HPA-1a alloimmunized woman (no. 14) who had been immunized over 20 years previously. These donations were obtained when the units were not required for clinical use and were taken at the minimum statutory intervals of three months. T cell proliferative responses were always strong towards PPD but weak or negative to TT, whereas responses to the HPA-1 peptides only occurred in three samples (Table 3). The magnitude of the responses to these antigens varied independently of each other. For instance, the second sample had the highest PPD and TT responses but no activity towards the HPA-1 peptides, in contrast to the third sample that had very weak anti-TT activity but showed the greatest anti-HPA-1 responses.

Table 3. Proliferation of T cells in repeat samples from donor no. 14 who was immunized to HPA-1a over 20 years previously.

		Cumulative SI
Antigen (µg/ml)		24 Oct 2000	13 Aug 2001	27 Nov 2001	8 May 2002	13 Jan 2003	24 Jun 2004
PPD	(50)	182	1352	717	526	906	339
TT	(25)	19	62	9	nd	0	10
HPA-1ai	(30)	7	0	10	0	0	0
HPA-1ai	(10)	12	0	30	0	0	0
HPA-1ai	(3)	0	0	15	0	0	0
HPA-1ai	(1)	5	0	0	nd	0	0
HPA-1aii	(30)	0	0	0	0	0	0
HPA-1aii	(10)	0	0	0	0	0	0
HPA-1aii	(3)	4	0	0	0	0	0
HPA-1aii	(1)	9	0	59	nd	0	0
HPA-1 bi	(30)	0	0	16	0	0	0
HPA-1 bi	(10)	0	nd	0	0	0	14
HPA-1 bi	(3)	0	nd	0	0	0	4
HPA-1 bi	(1)	4	0	0	nd	0	0
HPA-1bii	(30)	5	0	0	0	0	0
HPA-1bii	(10)	0	nd	40	0	0	0
HPA-1bii	(3)	0	nd	nd	0	0	0
HPA-1bii	(1)	0	0	0	nd	0	0
Total HPA-1a		37	0	114	0	0	0
Total HPA-1b		9	0	56	0	0	18

nd, not done.

T cells in sequential blood samples from five patients during pregnancy and two of these patients postpartum also showed some fluctuations in responses to PPD, TT and the HPA-1 peptides; two examples are shown in Table 4. The variability in T cell responses of serial samples from the patients was, however, less than that of the donor (no. 14, Table 3). In addition, two samples from patient no. 5 taken at 3 and 21 weeks postpartum exhibited differential activity to the T cell antigens. Low proliferative responses to PPD, TT and the HPA-1a and HPA-1b peptides (cumulative SI of 9, 18, 8 and 3, respectively) were apparent 3 weeks after delivery but at 21 weeks the T cell proliferative activity to PPD and TT increased greatly (to 191 and 166, respectively) while activity towards the HPA-1 peptides was lost.

Table 4. Proliferation of T cells from two patients with NAIT during and after pregnancy.

	Cumulative SI
Sample number	3i	3ii	3iii	11i	11ii	11iii
Sample time	24 GA	28 GA	7 PP	26 GA	29 GA	14 PP
Antigen:
PPD	262	506	69	53	222	113
TT	329	250	27	41	164	50
HPA-1a	18	0	0	11	9	3
HPA-1b	7	0	0	0	6	0

GA, weeks gestational age; PP, weeks postpartum.

T cell proliferative responses of alloimmunized women to the HPA-1 peptides

The combined cumulative SI values for responses to HPA-1a and HPA-1b peptides for the 33 samples from the 14 women are shown in Fig. 3. Most women (86%) (but fewer samples, 70%) had T cells that responded to either or both of the HPA-1a and HPA-1b peptides (Table 5). T cells from more women (86%) and more samples (64%) responded to the HPA-1a peptides (both 1ai and 1aii) than to the HPA-1b peptides (64% of women and 42% of samples) (Table 5). Some women (21%) and samples (27%) had T cell reactivity solely to the HPA-1a peptides. Responses to the HPA-1b peptides nearly always occurred when T cell proliferation to the HPA-1a peptides was also present; only two samples (1ii, 14vi) had a T cell response solely to HPA-1b (Fig. 3). In all samples tested, the extent of proliferation was greater towards the HPA-1a than the HPA-1b peptides when T cells reactive to both allo-peptides were present in the same sample (Fig. 3); the total combined cumulative SI for all the samples was 612 and 214, respectively.

Fig. 3.

T cell proliferative responses of 33 samples from 14 HPA-1a alloimmunized patients to HPA-1a and HPA-1b peptides. The combined cumulative SI of values were obtained by summation of the individual daily responses at days 4–7 to all concentrations of the HPA-1ai plus HPA-1aii peptides (□) or the HPA-1bi plus HPA-1bii peptides (▪).

Table 5. Percentage of HPA-1a alloimmunized women and samples responding to HPA-1 peptides.

Peptide	Either or both 1a or 1b	1a and 1b together in a sample	1a ± 1b	Solely 1a	1b ± 1a	Solely 1b	None
% women	86	57	86	21	64	0	14
% samples	70	36	64	27	42	6	30

Samples with high proliferative activity generally exhibited T cell responses to both the short and long peptides (both HPA-1a and HPA-1b). However, more women (79%) responded to the shorter 1ai peptide (20–39) than to the longer 1aii peptide (24–45) (50%). This difference was more marked comparing women who had responses only to either the 1ai (36%) or the 1aii (7%) peptide. Interestingly, the opposite effect was observed for the HPA-1b peptides, when the 1bi peptide stimulated less reactivity than the 1bii peptide (36% and 57% of women responded, respectively, with 7% and 29% of women responding solely to the 1bi or 1bii peptide). There did not appear to be an optimum concentration of peptide. Although proliferation to the peptides was observed on any day of the assay, slightly more responses were early (days 4–5) rather than late (days 6–7).

T cell responses of non-HPA-1a alloimmunized donors to the HPA-1 peptides

None of six subjects exhibited T cell proliferation to the HPA-1a peptides, but on one occasion donor B (HPA-1a1a) reacted to the HPA-1bi and HPA-1bii peptides (Table 6). This donor had not been transfused. T cells from all these donors responded to PPD and most of them to TT.

Table 6. T cell proliferative responses of non-HPA-1a alloimmunized donors to control antigens and to HPA-1a peptides.

	Cumulative SI
Donor:	A	B(i)	B(ii)	C	D	E	F
Antigen:
PPD	75	160	303	158	38	112	83
TT	0	0	202	143	0	47	28
Total HPA-1a	0	0	0	0	0	0	0
Total HPA-1b	0	23	0	0	0	0	0

Comparison of clinical data and T cell proliferative responses of patients

All the patients and the donor were genotyped HPA-1b1b and all were found to contain HPA-1a antibodies as indicated in Table 1. Five of 13 patients had a previous fetus with an ICH, indicative of a severe history of NAIT (Table 1). The magnitude of the T cell proliferative responses to the HPA-1a peptides was compared between the patients who had had a mild or severe history. There was no significant difference in results from the antenatal samples (mean cumulative SI = 11·9 (mild history) and 23·4 (severe history); P = 0·22). In contrast, T cell reactivity to the HPA-1a peptides was significantly greater (P = 0·024) in three postnatal samples from two women with a severe history (samples 2ii, 2iv, 4ii; mean cumulative SI = 37·0) compared to the five postnatal samples from four women with a mild history (samples 3iii, 5i, 5ii, 11iii, 13i; mean cumulative SI = 4·0).

Most patients were treated with IUT's and/or IVIG during the current pregnancies and all had a good outcome with delivery of healthy babies unaffected by severe thrombocytopenia. Therefore it was not possible to determine any relationship between in vitro T cell reactivity to HPA-1a peptides and clinical severity.

The effect of IUT's on T cell responses could also not be determined as treatment was performed after samples were taken for the T cell study.

Patient no. 6 was treated early with prednisolone as she had a very severe history of NAIT. Over two weeks, her T cell reactivity to the HPA-1 peptides increased (Fig. 3) whereas that to PPD and TT decreased slightly (from 146 to 92 and 192 to 70, respectively). Prednisolone did not appear to suppress the alloimmune response.

IVIG was administered to seven patients (eight pregnancies), although only five pregnancy samples (2iii, 7ii, 11i, 11ii and 13ii) were taken for the study after the start of this therapy. The magnitude of the T cell responses to the HPA-1a peptides were lower during and after the last pregnancy of patient no. 2 (samples 2iii and 2iv) after she had had IVIG therapy than in her previous pregnancy (sample 2i) before IVIG was started. Patient no. 7 had two samples taken, one before and one after the start of IVIG administration. T cells in the second sample had lower HPA-1a reactivity (Fig. 3). Interestingly, in contrast to the reduced reactivity to HPA-1a, the T cell reactivity to PPD and TT was increased after IVIG was started (the cumulative SI to PPD, TT and HPA-1a were 79, 56 and 26 before IVIG and 322, 180 and 9 after IVIG). A similar phenomenon was observed for patient no. 13. Eighteen weeks before the index pregnancy (sample 13i), the cumulative SI's to PPD, TT and HPA-1a were 7, 19 and 9, respectively, but during the pregnancy (13ii) these values were 118, 533 and 0, respectively. Taken together, these data suggest IVIG may have ameliorated the T cell reactivity to HPA-1a but not to the control antigens.

Discussion

The pathogenesis of NAIT is due in most cases to maternal HPA-1a antibodies destroying fetal HPA-1a⁺ platelets. Alloantibody responses are driven by T helper cells. This study aimed to identify HPA-1a specific T cells in maternal blood, to quantify T cell proliferation in response to HPA-1a peptides and to determine whether these T cell responses were associated with clinical sequelae.

There were some limitations of our study as discussed below. Only small volumes of blood could be taken from pregnant women, limiting the number of peptides that could be tested. The suppressive effect of autologous pregnancy sera had to be overcome by using AB serum in the T cell proliferation assay. Natural fluctuations in antigen specific T cell responses were apparent. There also remained the possibility that the patients’ alloimmune T cell responses had been suppressed in vivo by IVIG therapy. Recently it was shown that administration of IVIG to patients with immune thrombocytopenic purpura (ITP) induced a transient increase of interleukin-10 (a regulatory cytokine) but not pro-inflammatory cytokines in vivo[26].

The 24–45 HPA-1aii and -1bii peptides were chosen based on results of a study of the binding of peptides to insect cell-derived HLA-DRB3*0101 using a sensitive cold target inhibition assay [18]. This data indicated that Leu33 was an anchor residue since the corresponding 24–45 Pro33 peptide did not bind to the purified recombinant HLA class II. Binding of HPA-1a peptides of residues 24–45 and 24–37 occurred but not of residues 32–45 or 26–38. Alignment of sequences of other published DRB3*0101-binding peptides suggested that acidic amino acids at relative position 4 and hydrophobic residues at positions 1 and 9 were important. Together this information led the authors to propose that the core anchor residues for HPA-1a were WxxDxxxxL at sequence numbering 25xx28xxxx33, with Leu33 (position 9) being the critical C-terminal anchor residue [18].

Sera from pregnant and postnatal women reduced T cell proliferative responses to whole antigens (PPD and TT) and to HPA-1 peptides. The suppressive effect on autologous and allogeneic T cells indicated the presence of non-MHC-restricted inhibitory factor(s) in the heat-inactivated pregnancy sera. This is in accordance with other reports showing pregnancy sera may contain suppressive factors to immune responses. First, human pregnancy serum suppressed the proliferative response of lymphocytes to autologous PHA-activated T cells but it did not affect IL-2 dependent proliferation, suggesting a non-MHC-restricted inhibitory factor in human pregnancy sera [27]. Second, murine pregnancy sera suppressed specific T cell proliferation and IL-2 production induced by PLP p139–151 (a peptide from myelin proteolipid protein) in mice with experimental autoimmune encephalomyelitis (EAE) [28]. Third, some systemic T cell mediated autoimmune diseases such as pristane-induced arthritis [29], multiple sclerosis (MS) [30] and psoriasis [31] are ameliorated during pregnancy [32]. Fourth, the pregnancy-specific hormones oestriol [33] and human chorionic gonadotrophin (hCG) [34] may contribute to reduction of multiple sclerosis (MS) [33] or experimental arthritis [34] through suppression of pro-inflammatory mediators. It is unlikely, however, that hCG or other substances with short half lives were responsible for the reduction in antigen-specific T cell proliferation observed, since suppression by serum was still apparent seven weeks after parturition when relapse of rheumatoid arthritis, psoriasis and MS would have occurred by this time in patients with these autoimmune diseases.

The presence of antigen-specific T cells to whole antigens (PPD or TT) and HPA-1 peptides fluctuated with time in serial samples from the blood donor and to a lesser extent from the patients. This phenomenon was previously reported with T cell proliferative responses to TT [25,35] from healthy donors and to PLP peptides [35] or myelin basic protein peptides [36] from patients with MS. It may reflect periodic reactivation of antigen-specific T cells and their appearance in the peripheral circulation.

Most of the women had T cell reactivity to one or both of the HPA-1a peptides (20–39 and 24–45), but this was not observed in the samples from control donors. The responses observed in the alloimmunized women were likely to be due to the presence of specific T cells preactivated towards the allelic peptides. Not all samples exhibited HPA-1a proliferative responses and they were generally of lower magnitude than those towards the control recall antigens, PPD and TT. The sporadic nature of responses to the HPA-1 peptides (at different concentrations of peptides) in individual culture wells of each sample may have reflected the infrequency of antigen-specific T cells.

In contrast to the lack of binding of the HPA-1b peptide to recombinant DRB3*0101 in the study described by Wu et al. [18], T cell proliferation in response to the HPA-1b peptides was occasionally observed. This may have been because they were presented on non-DRB3*0101 HLA class II molecules. Unless homozygous for this allele, the antigen presenting cells would have other HLA class II molecules that may be able to present the HPA-1b peptides in addition to DRB3*0101 that would efficiently present the HPA-1a peptides. Alternatively, since the HPA-1b peptides contain two (of three) anchor residues, enough peptides may have bound sufficiently well to the DRB3*0101 expressed on human antigen presenting cells in the cultured PBMC to signal to T cells. Presentation of exogenous peptides on an antigen presenting cell may be by passive binding of the peptide to empty HLA class II molecules [37] or after processing of endocytosed peptides. Passively bound peptide complexed to HLA class II may interact with T cell receptors but may not induce activation of the APC and production of the relevant cytokines and costimulatory molecules required for full T cell activation. Since the patients had no evidence of (auto) anti-HPA-1b antibodies the T cell proliferation observed in the in vitro assays may have been nonfunctional in stimulating B cells. T cell proliferative responses of a similar group of HPA-1b1b donors to HPA-1b peptides has been reported by others (see below). In the current study, the only control donor who responded was homozygous HPA-1a1a. Taken together it is unlikely that reactivity to HPA-1b peptides was a nonspecific assay artefact.

Alloimmunization to HPA-1b is a rare event [8] and the antibody response is not HLA restricted [38]. It is not known what the ‘natural’ immunizing peptides are and if they are similar for HPA-1a and HPA-1b. Previous studies aimed at identifying the DRw52a (DRB3*0101) restricted HPA-1a peptide(s) have given conflicting results, probably due to different assay techniques. First, there was no binding of 25–42 HPA-1a peptides to HLA-DRw52a molecules that had been purified from an EBV-transformed B cell line. The class II molecules may have been occupied by naturally processed endogenous peptides that could not be displaced by the added HPA-1 peptides [39]. Next, binding of HPA-1a peptides to baculovirus expressed soluble DRB3*0101 led to identification of the putative core sequence that contained the anchor residues Trp25, Asp28 and Leu33 as described above [18]. Recently, 15-mer peptides spanning position 33 with either Leu or Pro 33 were tested in T cell proliferation assays with PBMC from HPA-1b1b women who had delivered a HPA-1a⁺ baby; 64% responded to peptides with Leu33 and 22% to Pro33 peptides, with peptides 19–33, 20–34 and 21–35 being immunodominant [40]. In the present study, peptide 20–39 (1ai) with the extended N-terminal sequence (not tested by Wu et al. [18]) was more active than peptide 24–45 (1aii) in stimulating proliferation of T cells from HPA-1a alloimmunized women.

The incidence and magnitude of T cell proliferative responses to HPA-1a were not predictive of fetal disease in the index pregnancies but the pregnancies had been managed with IVIG, fetal blood sampling, IUT's or steroids and all resulted in good fetal outcomes.

However, T cells in postnatal samples from women who had had a severe history of NAIT gave greater reactivity towards HPA-1a peptides than T cells from women who had not had a fetus with an ICH. Prolonged or enhanced T cell activation to HPA-1a may have some relationship with pathogenicity. Confirmation of this finding using samples taken prior to or early in pregnancy in a larger cohort of HPA-1a alloimmunized women is required.

Two potential applications of HPA-1a peptides in the diagnosis and treatment of NAIT are suggested by this work. First, a noninvasive predictive diagnostic test such as the T cell proliferation assay might determine the severity of NAIT and would aid the management of patients. Second, there is evidence that antigen-specific T cells can be suppressed and the corresponding antibody response can be decreased by immunomodulatory peptides. Antigen-specific soluble peptides from autoantigens [41–43] and allergens [44] can lower specific immune responses in animals. We have reduced and curtailed human antibody responses to a foreign antigen, TT, using soluble immunodominant TT peptides in a preclinical model, whereby mice with severe combined immunodeficiency were transplanted with human PBMC, given TT peptides and TT antigen, and the serological anti-TT response monitored over three months [25]. If HPA-1a immunodominant peptides are also effective in this system they could be considered for immunotherapy to specifically reduce antibody responses to HPA-1a.