Letters to the Editor
Predominant autoantibody response to GPIb/IX in a regulatory T-cell-deficient mouse model for immune thrombocytopenia
Article first published online: 7 FEB 2013
© 2012 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 11, Issue 2, pages 369–372, February 2013
How to Cite
Predominant autoantibody response to GPIb/IX in a regulatory T-cell-deficient mouse model for immune thrombocytopenia. J Thromb Haemost 2013; 11: 369–72. Doi:10.1111/jth.12079.
- Issue published online: 7 FEB 2013
- Article first published online: 7 FEB 2013
- Accepted manuscript online: 24 NOV 2012 09:15AM EST
- Manuscript Accepted: 7 NOV 2012
- Manuscript Received: 1 OCT 2012
Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder caused by increased platelet destruction and impaired platelet production, induced by IgG antiplatelet autoantibodies . Recently, we found that CD4+CD25+ regulatory T-cell (Treg) depletion in mice causes the spontaneous development of sustained thrombocytopenia with increases in the platelet-associated IgG and the proportion of reticulated platelets . Platelet eluates and the supernatants of splenocyte cultures prepared from these thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets derived from normal mice, indicating that the primary mechanism was autoantibody-mediated, analogous to the pathophysiology of ITP. To identify molecular targets recognized by antiplatelet autoantibodies in Treg-deficient ITP mice, we performed antiplatelet antibody detection assays using platelets from mice deficient in functional platelet membrane glycoprotein (GP) IIb/IIIa or the GPIb/IX complex, which are the major targets of antiplatelet autoantibodies in ITP patients .
Treg-deficient mice were generated by transferring Treg-depleted CD4+ T cells from BALB/c mice into syngeneic nude mice . Approximately one-third of the mice spontaneously developed thrombocytopenia 3 weeks after the transfer. As IgG bound to platelets from wild-type (WT) BALB/c mice were detected in platelet eluates and supernatants of splenocyte cultures, but not in plasma samples, from thrombocytopenic mice , splenocyte culture supernatants were used for evaluation of antigenic specificity of antiplatelet autoantibodies. Specifically, splenocytes were obtained from 10 Treg-deficient mice with thrombocytopenia (ITP mice; #60, #77, #105, #106, #108, #135, #139, #182, #199 and #200) 8 weeks after the transplantation and from 10 mice without thrombocytopenia (control mice), and cultured in RPMI1640 plus 10% fetal bovine serum for 4 days without antigenic or mitogenic stimulation. Platelets from WT mice were incubated with culture supernatants, and subsequently with AlexaFluor® 488-labeled anti-mouse IgG polyclonal antibodies (Life Technologies, Carlsbad, CA, USA). Platelets derived from GPIb-knockout (GPIb−/−) or GPIIIa-knockout (GPIIIa−/−) mice , which lack a functional GPIb/IX or GPIIb/IIIa complex on platelet surfaces, were also used instead of WT platelets. The IgG bound to platelets was quantitatively analyzed by a FACS® Calibur flow cytometer and CellQuest™ software (Becton Dickinson, Franklin Lakes, NJ, USA). The amount of bound IgG antibody was calculated as the ratio of the mean fluorescence intensity (MFI) of platelets treated with culture supernatant and fluorophore-conjugated anti-mouse IgG to the MFI of platelets treated with fluorophore-conjugated anti-mouse IgG alone. Relative IgG binding to the GPIb−/− or GPIIIa−/− platelets was obtained by comparison with the binding to WT platelets. All experimental protocols were approved by the Keio University Ethics Committee for Animal Experiments.
Flow cytometric analysis showed IgG antibodies capable of binding to WT platelets in the culture supernatant from representative ITP mouse #60, but not from control mouse #101 (Fig. 1A). Consistent results were obtained from 10 ITP and 10 control mice. When GPIb−/− platelets were used instead of WT platelets in the assay, IgG from a representative ITP mouse, #106, failed to bind (Fig. 1B). Concordant results were obtained from all ITP mice, although IgG from three ITP mice (#77, #105 and #199) bound to GPIb−/− platelets to a small degree. Binding of IgG from another representative ITP mouse, #77, to GPIIIa−/− platelets was reduced, compared with binding to WT platelets (0Fig. 1C), but binding to GPIIIa−/− platelets and WT platelets was nearly concordant in IgG from the remaining nine ITP mice. Relative binding of IgG from 10 ITP mice to GPIb−/− or GPIIIa−/− platelets compared with their binding to WT platelets is summarized in Fig. 1(D). IgG binding was diminished by the use of GPIb−/− platelets in all ITP culture supernatants, and was completely lost in half the samples. IgG binding to GPIIIa−/− platelets was reduced in six (60%) samples, but the reduction in binding was much less than with GPIb−/− platelets. IgG from ITP mouse #105 bound both GPIb−/− and GPIIIa−/− platelets, suggesting that in this mouse, antiplatelet antibodies also recognized other platelet surface molecules.
To confirm the autoantibody binding of IgG from ITP mice to GPIb/IX and GPIIb/IIIa, we conducted antibody depletion assays, in which splenocyte culture supernatants were pre-incubated with platelets derived from WT, GPIb−/− or GPIIIa−/− mice and then subjected to the antiplatelet antibody assay using WT platelets as the source of antigen. Pre-incubation of the supernatant from representative ITP mouse #106 with WT platelets completely abolished IgG binding to platelets, but pre-incubation with GPIb−/− platelets had no effect (Fig. 1E). A concordant result was obtained from additional ITP mice (#135 and #200). In contrast, IgG binding of supernatant from ITP mouse #77 to platelets was partially suppressed by pre-incubation with GPIIIa−/− platelets (Fig. 1F).
Our results clearly indicate that both GPIb/IX complex and GPIIb/IIIa complex are the major autoantigens recognized by IgG antiplatelet autoantibodies in an ITP mouse model generated by Treg deficiency. This heterogeneous autoantigen recognition profile is analogous to the antiplatelet autoantibody response seen in ITP patients . Antiplatelet autoantibodies in our ITP mouse model preferentially targeted GPIb/IX over GPIIb/IIIa. In contrast, the most common autoantigen in ITP patients is GPIIb/IIIa, which is recognized by approximately 30–93% of them, depending on the assay [5–11], and anti-GPIb/IX is the second-most prevalent antibody, found in 12–83% of the patients [5–10]. However, some reports have shown that anti-GPIb/IX antibodies are more frequent than anti-GPIIb/IIIa antibodies in ITP patients [9,10]. Interestingly, ITP patients with anti-GPIb/IX antibodies are refractory to treatment with intravenous IgG (IVIG), and those without these antibodies are responsive . In addition, IVIG treatment is effective in a mouse model of experimental thrombocytopenia induced by an anti-GPIIb/IIIa monoclonal antibody, but not in a model induced by an anti-GPIbα monoclonal antibody . We have yet to examine the efficacy of IVIG treatment in Treg-deficient ITP mice.
The antiplatelet autoantibody response in individual mice was heterogeneous, including recognition of GPIb/IX alone and of GPIb/IX and GPIIb/IIIa together, even though all the Treg-deficient mice had an identical BALB/c genetic background and were grown in the same colony. This heterogeneous nature of the autoantibody response is in sharp contrast to another ITP mouse model, which is characterized by induction of antibody- and CD8+ T cell-mediated responses specific to GPIIIa . It has been recently reported that peripheral Treg deficiency is observed in association with thymic retention of Tregs in this ITP model , but the immune target of this model is restricted to GPIIIa. This is not surprising because this model is generated by immunization of a GPIIIa−/− mouse with wild-type platelets, and subsequent transfer of splenocytes from the immunized mouse into a severe combined immunodeficiency mouse. Alternatively, this finding strongly suggests that peripheral Treg deficiency alone is not sufficient to induce autoreactive responses to multiple GPs on platelets.
In contrast, in our ITP model, a small number of Treg-depleted CD4+ T cells derived from WT mice are transferred into syngeneic nude mice that lack functional T cells. The transferred CD4+ T cells proliferate in the lymphopenic condition by a process termed ‘homeostatic proliferation’ . The clonal expansion of an autoreactive CD4+ T-cell repertoire during this process is reported to be responsible for the development of autoimmunity . In our ITP model, the absence of Tregs during homeostatic proliferation is likely to promote expansion of a variety of autoreactive T-cell reportoires, resulting in elicitation of autoantibody responses to multiple platelet GPs. The mechanisms that control the expansion of autoreactive T cells have not been fully elucidated, but contributing factors may include precursor frequency in the transferred T cells and the presentation of a variety of agonistic peptides required for the survival and expansion of autoreactive donor T cells in the recipient nude mice . Differences in these factors among individual Treg-deficient ITP mice may contribute to their heterogeneous antiplatelet autoimmune responses.
In conclusion, IgG autoantibody responses to multiple platelet surface GPs, including GPIb/IX and GPIIb/IIIa, observed in Treg-deficient ITP mice are analogous to the antiplatelet autoantibody response in ITP patients. Our new mouse ITP model should be useful for analyzing the mechanisms behind the antiplatelet autoantibody response and for developing novel therapeutic strategies to suppress harmful autoimmune responses in ITP patients.
T. Nishimoto performed research, collected data, analyzed and interpreted data and wrote the manuscript; T. Satoh and E. K. Simpson performed research and collected data; H. Ni interpreted data and supervised the study; M. Kuwana designed the research, analyzed and interpreted data, supervised and organized the study and wrote the manuscript.
This work was supported by a research grant on intractable diseases from the Japanese Ministry of Health, Labour, and Welfare, and Canadian Institutes of Health Research (MOP-97918). E.K. Simpson was a recipient of the studentship award of LKSKI of St. Michael's Hospital and the Heart & Stroke Foundation of Canada/Ontario Graduate Scholarship in Science & Technology.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.