Characterization of glycoprotein IIb/IIIa-specific alloantibodies induced by cross-strain platelet immunization in mice

Abstract

Background:

We previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition, post-transfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies.

Study Design:

Hybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies (mAbs) were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by mAbs were identified by mutagenizing target glycoproteins expressed in CHO cells and observing the effects on antibody binding.

Results:

Three mAbs (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. MAbs 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs.

Conclusions:

Findings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen (HPA)-specific antibodies found in transfused and pregnant humans.

INTRODUCTION

In recent studies, we found that inter-strain platelet immunizations given intraperitoneally (IP) with adjuvant or intravenously (IV) between selected strains of inbred mice routinely lead to production of alloantibodies that recognize immunizing but not autologous platelets.¹ With certain strain combinations IP immunizations also induced platelet-reactive autoantibodies and severe thrombocytopenia. The mouse model resembles the human disorder, post-transfusion purpura (PTP) in which profound, often life-threatening thrombocytopenia develops in conjunction with the alloimmune response against a transfused human platelet alloantigen (HPA), usually HPA-1a, defined by a proline to leucine substitution at position 33 of platelet glycoprotein IIIa. ^2-4

A longstanding question that has puzzled clinicians and investigators ever since PTP was first described⁵ is how an alloantibody incapable of reacting with autologous platelets might, nonetheless, cause profound thrombocytopenia. Findings made in individual PTP patients^6-12 have hinted at the possibility that platelet destruction may actually be caused by a platelet-specific autoantibody that is usually overlooked in serologic studies because it is dominated by the much more potent alloantibody.

In this report, we describe studies to characterize the specificity of alloantibodies produced by mice that developed severe thrombocytopenia following cross-strain platelet immunization in these previous studies and provide evidence that, as in human patients with PTP, they recognize single amino acid (AA) polymorphisms in GPIIb/IIIa integrin that differ between the immunizing and immunized mouse strains. The findings demonstrate further similarity between the mouse model and the human disorder, PTP. Observations made concerning the immunogenicity of single AA differences in GPIIb/IIIa across mouse strains suggest it may be feasible to characterize GPIIb/IIIa-specific alloantigen systems in mice that are comparable to the human HPA antigens (HPAs) and could serve as models for study of human alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (FNAIT) and platelet transfusion refractoriness.

METHODS

Reagents:

Monoclonal antibody (mAb) 290. 5¹³ is specific for human GPIIb and was obtained from the Versiti-Blood Research Institute Hybridoma Core facility (Milwaukee, WI). Monoclonal antibody MWReg30 (rat anti-mouse GPIIb) was from BD Biosciences (San Jose, CA).

Mice:

C57Bl/6J (C57) 129S1/SvlmJ (129), SPRET/EiJ (SPRET), and PWK/PhJ (PWK) mouse strains were obtained from The Jackson Laboratory (Bar Harbor, ME) and were bred under pathogen-free conditions. Male and female mice, 8-15 weeks of age were included in this study

Immunization of mice and hybridoma preparation.

Mice were immunized as previously described.¹ For intraperitoneal (IP) immunizations, 10⁸ washed donor mouse platelets were suspended in Sigma Adjuvant System (Millipore Sigma, St. Louis, MO) and injected IP at weekly intervals. EDTA Blood samples (Microvette; Sarstedt, Numbrecht Germany) were collected from the submandibular vein, complete blood counts were performed using an automated animal blood counter as described previously¹. Selected mice were sacrificed and spleens collected. Splenocytes were isolated and fused with NP-3 cells as previously described.¹⁴ Culture supernatants from the resulting hybrids and subsequent clones were screened for reactivity against platelets from the donor and recipient mouse strains by flow cytometry using FITC labeled goat F(ab’)₂ (Jackson Immunoresearch) specific for mouse Ig (H+L) chains for detection of platelet-bound mouse antibody.

Expression of GPIIb/IIIa integrins in Chinese hamster ovary (CHO) cells.

Stably transfected CHO cell lines expressing various forms of GPIIb/IIIa integrins were created as previously described.¹⁴ Single AA mutants were generated using a site-directed mutagenesis kit (QuikChange II XL, Stratagene, La Jolla, CA) as previously described.¹⁵ Cells were selected for high expression of GPIIb/IIIa using MWReg30 (rat anti-mouse GPIIb) or (for mouse/human chimeras) mAb 290.5 (mouse anti-human GPIIb) on a Melody cell sorter (Becton Dickinson, Franklin Lakes, NJ).

Flow cytometric detection of antibodies.

Details have been described previously.¹ Washed platelets or CHO cells expressing recombinant proteins were incubated with 10 μL of test serum in a final volume of 50 μL. After one hour at room temperature, the cells were washed and bound IgG was detected with diluted fluorescein isothiocyanate (FITC)–labeled goat anti-mouse IgG (Fc-specific) F(ab’)2 (Jackson Immunoresearch, West Grove PA)

Statistics

Student t-Test, unpaired, 2 tailed, was utilized for comparison of 2 groups and was calculated with Excel.

Study approval.

Animal studies were approved by the institutional animal care and use committee of the Medical College of Wisconsin (Milwaukee, WI).

RESULTS

Production of allospecific monoclonal antibodies (mAbs) in immunized mice

In developing the mouse model of PTP,¹ cross-strain platelet immunizations were performed with mice of the C57Bl/6J (C57) 129S1/SvlmJ (129), SPRET/EiJ (SPRET), and PWK/PhJ (PWK) strains. A total of 48 cross-strain intraperitoneal immunizations given weekly for five weeks with adjuvant led in all cases to induction of IgG antibodies that recognized platelets from the immunizing strain. Using flow cytometry and immunoprecipitation, it was found that the main target for these alloantibodies was the glycoprotein IIb/IIIa complex (GPIIb/IIIa, α_IIbβ₃ integrin).¹ To further define alloantibody specificity, splenocytes were harvested from eight mice and hybridomas were prepared. Supernatants from the resulting clones were screened by flow cytometry for their ability to recognize platelets from the immunizing strain but not the strain of the spleen donor. In screening a total of about 6,000 clones, we identified three that produced potent IgG alloantibodies, two derived from a C57 to SPRET (mAbs 417.1 and 417.3) and one from a PWK to 129 (mAb 425.1) immunization (Figure 1). In each case, the spleen donors were mice that had experienced severe autoimmune thrombocytopenia during the course of immunization.¹ Sequence analysis and other descriptors of the mAbs are shown in Supplementary Table S1 and show that mAbs 417.1 and 417.3 encode nearly identical complementary determining regions (CDR). Mabs 417.3 and 425.1 were used for further studies.

Figure 1. Reactions of monoclonal antibodies induced by cross-strain platelet immunizations.

Left: MAbs 417.1 and 417.3 resulting from a C57 to SPRET immunization recognized C57 (dark gray) but not SPRET (light gray) platelets. Right: Mab 425.1 resulting from a PWK to 129 immunization recognized PWK (dark gray) but not 129 platelets (light gray). Values shown are representative of at least three experiments producing results that did not differ materially from one another.

Preliminary characterization of allo-specific mAbs

Immunoprecipitation studies using biotinylated platelets¹ showed that mAbs 417.3 and 425.1 recognized GPIIb/IIIa on platelets from the immunizing mouse strains C57 and PWK, respectively (data not shown). To identify AA differences that might have been responsible for alloimmunization, we consulted the Mouse Genome Database of the Wellcome Sanger Institute (https://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1303) and compared the AA compositions of the extracellular domains of GPIIb and GPIIIa of the donor and recipient mouse strains. As shown in Table 1, the combination C57 and SPRET, which produced mAb 417.3, differs by 7 AAs in these regions (5 in GPIIb and 2 in GPIIIa) and the combination PWK and 129 differs by three AAs (two in GPIIb and one in GPIIIa).

Table 1.

Extracellular amino acids of glycoproteins IIb and IIIa predicted to differ between four mouse strains used for platelet immunizations^*

	GPIIb (ITGA2B)							GPIIIa(ITGB3)
Mouse Strain	37	111	179	392	805	933	976	103	297	677
C57	E	G	A	P	R	V	R	A	H	A
SPRET	-	S	D	S	S	A	-	-	R	S
PWK	V	-	-	-	S	A	Q	S	-	-
129	-	-	-	-	S	A	-	-	-	-

^*Numbers denote amino acid positions at which extracellular domains of GPIIb and GPIIIa of the 4 mouse strains differ from one another. Amino acid 1 starts with the ATG of the translation start codon for each of the expressed proteins. C57 was chosen as the reference strain; a dash indicates identity with C57. Letters assigned to the other 3 strains indicate amino acids that differ from C57. Amino acids are designated by the conventional letter code.

When the two mAbs were tested against intact platelets from the remaining mouse strains employed in this study, it was found that mAb 417.3, made by a SPRET mouse, reacted with C57, 129 and PWK (Table 2), consistent with the possibility that it might be specific for AA G111, A179 or P392 in GPIIb or H297 or A677 in GPIIIa, the only AAs not present in SPRET that are shared by all three of the other strains. The target of this mAb was further narrowed to GPIIb by studying its reactions with CHO cells expressing chimeric GPIIb/IIIa in which either GPIIb or GPIIIa was switched to the human (non-reactive) version of the protein. Results showed that reactivity persisted when GPIIb of the immunizing mouse strain (C57) was present but was lost when this was switched to non-reactive human GPIIb (Figure 2). MAb 425.1, made by a 129 mouse, recognized only the PWK platelets used for immunization, suggesting it might be specific for V37 or Q976 of GPIIb or S103 of GPIIIa, the only AAs present in the immunizing PWK platelets that were absent from the other strains. Because only 3 AAs were implicated as targets for mAb 425.1, its specificity was defined directly by mutagenizing GPIIb and GPIIIa at these positions.

Table 2.

Reactions of mAbs with platelets from four mouse strains or humans

Platelet source	mAb 417.3*	mAb 425.1**
C57	Yes	No
SPRET	No	No
PWK	Yes	Yes
129	Yes	No
Human	No	No

^*Mab 417.3 was produced in a SPRET mouse immunized with C57 platelets.

^**Mab 425.1 was produced in a 129 mouse immunized with PWK platelets.

Figure 2. Monoclonal antibody 417.3 recognizes GPIIb, but not GPIIIa from the immunizing C57 strain.

Left: A strong reaction was obtained with CHO cells expressing C57 GPIIb (mIIb) in a complex with human GPIIIa (hIIIa). Right: Weak reactions, comparable to those obtained with non-transfected CHO cells (not shown), were obtained with cells expressing C57 GPIIIa (mIIIa) in a complex with human GPIIb (hIIb). Values shown are representative of at least three experiments producing results that did not differ materially from one another.

Identification of single GPIIb AAs recognized by mAbs 417.3 and 425.1

These findings narrowed the likely specificities of mAb 417.3 to AAs G111, A179 or P392 of C57 GPIIb and those for mAb 425.1 to AAs V37 or Q796 of PWK GPIIb or S103 of PWK GPIIIa. To identify the target for mAb 417.3, three CHO cell lines were developed that expressed C57 GPIIIa in a complex with SPRET GPIIb mutated to change AAs at positions S111, D179 and S392 to the corresponding AAs found in C57 GPIIb (G, A and P, respectively). MAb 417.3 reacted strongly with the S111G mutant, but failed to react with D179A or S392P, indicating it was specific for G111 of the C57 GPIIb used for immunization (Figure 3A). Similarly, it was found that mAb 425.1 recognized the 129 strain GPIIb mutant E37V but not the GPIIIa mutant A103S, indicating specificity for V37 of PWK GPIIb (Figure 3B). The findings show that mAbs 417.3 and 425.1 are specific for G111 in C57 GPIIb and V37 in PWK GPIIb, respectively.

Figure 3. Monoclonals 417.3 and 425.1 are specific for single amino acid polymorphisms in GPIIb from the strain used for immunization.

A: MAb 417.3, made in a SPRET mouse, reacts with CHO cells expressing SPRET GPIIb mutant S111G (dark gray), but not mutants D179A or S392P (light gray), indicating specificity for AA residue G111 of GPIIb from the C57 strain used for immunization. B: MAb 425.1, made in a 129 mouse, reacts with CHO cells expressing 129 GPIIb mutant E37V (dark gray), but not 129 GPIIIa mutant A103S (light gray), indicating specificity for AA residue V37 of GPIIb from the PWK strain used for immunization. Values on the ordinate are expressed as a percent of the fluorescence signal obtained with control mAb MWReg30 (rat anti-mouse GPIIb). Values shown are representative of at least three experiments producing results that did not differ materially from one another.

Allospecific mAbs destroy platelets expressing GPIIb possessing the relevant target epitopes in mice.

To confirm the functional significance of mAbs 417.3 and 425.1, C57 mice were injected with 417.3 (3ug IP) or control antibody 290.5, (mouse anti-human GPIIb). As shown in Figure 4, platelet levels of C57 mice declined 72% on average following injection of mAb 417.3 but only about 20% in controls (p=0.003). Using a slightly different approach, mAb 425.1 was injected into PWK (antigen positive) and 129 (antigen negative) mice. The PWK mice all became profoundly thrombocytopenic (<10% of baseline) 24 hours later, whereas platelet levels in the 129 mice declined only slightly (p=0.00003) (Figure 4). There was a slight decrease (<10%) in RBC and Hgb and a slight increase in the white cell count in all groups, these were not statically different between the groups.

Figure 4. Mouse monoclonal allo-antibodies promote platelet clearance in antigen positive recipients:

Percent platelet count is calculated as the observed count/pre-challenge count *100. Left: Intraperitoneal injection of mAb 417.3 (3 ug IP) into C57 mice possessing GPIIb G111 induced thrombocytopenia at 24 hours in each of four mice (p=0.003). A non-reactive control mAb (290.5, anti-human GPIIb) was without effect. Right: Mab 425.1 (3 ug IP) induced profound thrombocytopenia at 24 hours in each of four PWK mice possessing GPIIb V37 but was without effect in four 129 mice possessing GPIIb E37 (p=0.00003).

DISCUSSION

In previous work, we showed that cross-strain platelet immunizations between C57, 129, SPRET, and PWK mice consistently leads to production of platelet-reactive alloantibodies that predominantly recognize GPIIb/IIIa on immunizing platelets.¹ Findings described here show that two monoclonal antibodies (mAbs), one derived from a C57 to SPRET and the other from a PWK to 129 immunization, recognize single amino acid (AA) polymorphisms in glycoprotein IIb that differ between the immunizing and immunized strains, namely, G111 in C57 and V37 in PWK. The strain combinations that produced these mAbs each consist of one well-characterized and widely used strain (C57, 129) and one less well characterized, wild-derived strain (SPRET, PWK). The C57/SPRET and PWK/129 combinations differ by 7 and 3 AAs, respectively, in extracellular domains of the glycoprotein IIb/IIIa complex, (Table 1). The major histocompatibility complex (MHC) of the C57 and 129 strains is H-2^b but MHCs of SPRET and PWK are undefined. Greater genetic disparity between wild-derived strains and more widely used strains like C57 and 129¹⁶ could have contributed to the high incidence of alloimmunization against GPIIb/IIIa in immunizations involving at least one wild-derived mouse strain.¹

When appropriate serologic studies are performed in patients experiencing PTP, they are invariably found to have a potent IgG alloantibody that recognizes transfused but not autologous platelets. In 80-90% of cases, these antibodies are specific for HPA-1a (Pl^A1, Zw^a) encoded by leucine 33 in GPIIIa.⁴ Of the remainder, about two-thirds recognize other single AA polymorphisms in GPIIb/IIIa.¹⁷ Thus, 90-95% of the platelet-specific alloantibodies found in patients with PTP recognize GPIIb/IIIa. In previous studies involving cross-strain platelet immunizations in C57, 129, SPRET and PWK mice, we found that each of 48 mice immunized by intraperitoneal injection and all 15 immunized intravenously with platelets from one of the other three strains produced antibodies that recognized the platelets used for immunization.¹ Twelve of 15 alloantibodies tested were shown to recognize GPIIb/IIIa and, as described here, two mAbs produced from these mice were found to be specific for single AA differences found in GPIIb of the strain used for immunization. These observations further demonstrate the close similarity between the mouse model and the human condition, PTP. As noted, recent studies provide evidence that platelet-specific autoantibodies, rather than concomitantly produced alloantibodies, are the likely cause of profound thrombocytopenia in humans with PTP¹² and in the mouse model.¹ However, the alloantibodies may not simply be innocent bystanders. In previous reports, we have described a mechanism still under investigation suggesting that the autoantibodies evolve directly from alloantibodies in the course of random somatic hypermutation.^1,12

An unexpected finding made in our previous studies¹ was that nearly all mice immunized intraperitoneally or intravenously with platelets from one of the other three strains produced GPIIb/IIIa-specific alloantibodies, despite the relatively small numbers of AA differences in extracellular domains of GPIIb/IIIa in these strains (Table 1). This behavior contrasts markedly with experience in humans transfused with whole blood or platelets in whom alloantibodies against platelet-specific glycoproteins (HPA antibodies) are relatively uncommon^18-21 but who often become immunized against Class I HLA antigens and may become refractory to randomly selected platelet transfusions as a consequence.^18,22-24 Inter-strain platelet transfusions in mice have been used to model antibody-dependent platelet transfusion refractoriness that develops in multiply-transfused human patients, characterize its pathogenesis and examine ways that this complication can be circumvented.^25-30 However, many studies have failed to consider the possibility that the immunized mice may have produced both MHC-specific and GPIIb/IIIa-specific alloantibodies. Findings described here and in a previous report¹ show that inter-strain AA mismatches in extracellular domains of GPIIb/IIIa are common and highly immunogenic (at least in the mouse strains studied by us) and that antibodies recognizing them are capable of destroying platelets. It is important that the immunogenicity of GPIIb/IIIa and possibly other platelet-specific glycoproteins be considered in studies of platelet transfusion refractoriness conducted in mice.

To our knowledge, alloantibodies that recognize single AA differences in platelet-specific glycoproteins of mice have not previously been described. The ease with which such antibodies can be induced in selected mouse strains and the limited numbers of AA differences that occur naturally between strains (Table 1) suggest that it might not be particularly difficult to characterize immunogenic AA differences in mice that mimic the HPA antigen systems of humans. Fetal and neonatal alloimmune thrombocytopenia (FNAIT), a significant cause of morbidity and mortality in newborns^31-34 has been studied in mice under experimental conditions that deviate markedly from those found naturally in human patients with this condition.^35-38 Our findings raise the possibility that further studies of cross-strain immunogenicity of GPIIb/IIIa in mice, could identify strains in which pregnancy leads to “natural” maternal alloimmunization against epitopes on GPIIb/IIIa and recapitulates the human disorder, FNAIT.