Characterization of the T-lymphocyte response elicited by mouse immunization with rat platelets

Experimental Hematology 2011;39:676–685

Characterization of the T-lymphocyte response elicited by mouse immunization with rat platelets Laurent Detallea,*, Anubha Saxenaa, Nadia Ouled Haddoua, Catherine Uyttenhoveb, Jacques Van Snickb, and Jean-Paul Couteliera a

Unit of Experimental Medicine; bLudwig Institute for Cancer Research, Christian de Duve Institute, Universit
e Catholique de Louvain, Brussels, Belgium (Received 14 January 2010; revised 14 February 2011; accepted 2 March 2011)

Objective. Immunization of normal CBA mice with rat platelets leads to an autoantibody response directed against mouse platelets. The purpose of this work was to determine the involvement of T lymphocytes in this response. Materials and Methods. T-lymphocyte responses were analyzed in vivo by depletion and transfer experiments and ex vivo by proliferation assay and cytokine measurements. Results. Mouse immunization with rat platelets induced production of antibodies reacting with rat and mouse platelets. This response was found to depend on CD4+ T-helper lymphocytes reacting with rat, but not with mouse platelets. These anti-rat platelet T-helper cells were mainly of the Th1 phenotype. When transferred into na€ıve mice, they enhanced the anti-mouse platelet antibody response induced by subsequent immunization with rat platelets. In addition, depletion of CD25+ cells enhanced the thrombocytopenia induced by immunization with rat platelets, whereas adoptive transfer of CD4+CD25+ cells from immunized mice suppressed it. Conclusions. Our results suggest that activation of anti-rat platelet T-helper cells can bypass the mechanism of tolerance and result in the secretion of autoreactive antibodies, but this response is still controlled by regulatory T cells that develop progressively after immunization. Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

A transient and moderate thrombocytopenia develops in normal CBA mice repeatedly immunized with rat platelets [1]. This thrombocytopenia is correlated with production of antibodies binding mouse platelets. Analysis of these selfreacting antibodies, eluted from the platelets of immunized mice, shows that they recognize epitopes shared by both rat and mouse platelets. After injection into na€ıve animals, these autoreactive antibodies lead to platelet destruction [1]. Therefore, this model of immunization can mimic some aspects of diseases such as post-transfusion purpura or immune thrombocytopenic purpura. Little is known about the mechanisms leading to production of these self-reacting antiplatelet antibodies and to the consequent thrombocytopenia. Autoantibody-producing B

*Current address: Ablynx nv, 9052 Ghent/Zwijnaarde, Belgium. Offprint requests to: Jean-Paul Coutelier, M.D., Ph.D., Unit of Experimental Medicine, ICP, Universit
e Catholique de Louvain, Avenue Hippocrate 7430, B-1200 Brussels, Belgium; E-mail: [email protected]

lymphocytes are known to exist in the normal repertoire [2,3], and may recognize blood cells or proteins like immunoglobulin G (IgG). Antibodies produced by these autoreactive B cells may have important physiological functions in the clearance of aging blood cells or regulation of immune responses. On the other hand, most autoreactive T lymphocytes are expected to be suppressed by the mechanisms of tolerance. It is therefore of interest to better characterize the cellular mechanisms that regulate production of autoreactive antibodies. To further investigate the pathogenesis of the autoreactive immune response triggered by mouse immunization with rat platelets, we analyzed the role of T lymphocytes in both their generation and control. Our results indicate that T-helper lymphocytes are required for development of this antiplatelet antibody response, but that they react mostly with epitopes expressed on rat, but not on mouse, platelets. Moreover, the antimouse platelet antibody response is quickly controlled by generation of cells with the characteristics of regulatory T cells.

0301-472X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2011.03.002

L. Detalle et al./ Experimental Hematology 2011;39:676–685

Materials and methods Animals Specific pathogen-free CBA/Ht female mice were bred at the Ludwig Institute for Cancer Research by G. Warnier and CBA/ Ca mice were purchased from Harlan (Horst, The Netherlands). They were used when they were 8 to 12 weeks old. Wistar rats were bred in our local facility by J-P. Dehoux. The project was approved by the local commission for animal care. Immunization Rat platelets were prepared by successive centrifugations as described previously [1]. Mice were immunized first by intraperitoneal (IP) injection of 108 rat platelets in 0.5 mL saline, followed by weekly injections of 0.5
108 platelets. Antibodies Anti-CD4 monoclonal antibody (mAb) GK1.5 was made available by F. W. Fitch, and obtained through the courtesy of H. R. MacDonald [4]. It was used as ammonium sulfate-precipitated antibody and injected intraperitoneally at the dose of 1 mg/mouse. Rat anti-mouse CD25 (IL-2Ra) mAb PC61 [5] was administered at the dose of 300 mg/mouse in ascitic fluid. Platelet counts Blood was collected from the retro-orbital plexus of etheranesthetized mice using Unopette pipettes and kits (Unopette microcollection system, Becton Dickinson, Franklin Lakes, NJ, USA). Platelets were counted by microscopy with an improved Neubauer hemacytometer (Marienfeld, Germany). Enzyme-linked immunosorbent assay (ELISA) Reactivity of antibodies with rat platelets and with bovine serum albumin was analyzed by ELISA as described in [1]. Briefly, plates coated with rat platelets or bovine serum albumin were incubated with samples to be tested, followed by rat IgG2a anti-mouse k-chain mAb conjugated to horseradish peroxidase (LO-MK1, LO/IMEX, Universit
e Catholique de Louvain, Brussels, Belgium) and by 3,30 ,5,50 -tetramethylbenzidine (Thermo Fisher Scientific, Rockford, IL, USA). Total sera IgG were measured by sandwich ELISA [6] with plates coated with goat anti-mouse IgG polyclonal antibody and IgG binding revelation with peroxidase-conjugated goat anti-mouse antibody (BD Pharmingen, San Diego, CA, USA). Lymphocyte stimulation and cell proliferation assay After red cell lysis, T-helper lymphocytes were separated from spleen cells by magnetic cell sorting, using CD4 Microbeads and columns from Miltenyi Biotec (Bergisch Gladbach, Germany), following manufacturer’s recommendations. Purity of CD4þ cells obtained through this method was $85%. The 2
106 CD4þ cells were incubated for 6 days with 2
106 irradiated (2000 rads) CD4 cells as feeder cells and 50 to 200
106 platelets in 2 mL Iscove’s medium containing 10% decomplemented fetal calf serum and supplemented with 0.24 mM L-asparagine, 0.55 mM L-arginine, 1.5 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 100 mg/mL streptomycin, and 100 U/mL penicillin. After 6 days, 100 mL cell-containing media was transferred to 96-well plates. Bromodeoxyuridine was added in 100 mL medium

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and cell proliferation was analyzed after 16 hours incubation by measurement of bromodeoxyuridine incorporation, with a Cell Proliferation kit (Roche, Penzberg, Germany) following manufacturer’s recommendations. Cytokine assays Cytokine levels were measured by sandwich ELISAs. Interferon (IFN)-g assay was performed by using a Ready-SET-Go kit (eBioscience) according to manufacturer’s instructions. For interleukin (IL)-5 determination, plates were coated with TRFK5 antiIL-5 mAb (1 mg/mL; eBioscience) and captured IL-5 was revealed with biotinylated TRFK4 antimIL-5 mAb (1 mg/mL; eBioscience), followed by peroxidase-conjugated streptavidin. Standardization was performed with recombinant IL-5 (R&D Systems). For IL-4 assay, plates were coated with anti-IL4 BVD4-1D11 mAb (4 mg/mL; BD Biosciences, Erembodegem, Belgium) and revealed with biotinylated anti-IL4 BVD6-24G2 mAb (eBioscience). Recombinant IL-4 prepared in baculovirus was used as standard. IL-9 was titrated using sandwich ELISA with antimIL-9 RM9A3 mAb (5 mg/mL, obtained from a rat immunized with ovalbumine-conjugated IL-9) and a biotinylated polyclonal goat antimIL-9 antibody. Standard recombinant IL9 was prepared in baculovirus. IL-17A was measured on plates coated with MM17A.3G9 antimIL-17A mAb (5 mg/mL) and quantified with biotinylated MM17A.F3 mAb [7]. Standard IL-17A was from R&D Systems. Adoptive transfer experiments For adoptive transfer experiments, proliferating T lymphocytes (3
106 per recipient mouse) were injected intraperitoneally (IP) in 500 mL medium. For adoptive transfer of CD4þCD25þ cells, cell subpopulations were obtained from spleen cells of CBA mice immunized five times weekly with rat platelets. Cells were purified by magnetic cell sorting with a CD4þCD25þ Regulatory T Cell Isolation Kit (Miltenyi Biotech) first by negative selection of CD4þ cells followed by positive selection of CD4þCD25þ cells, according to manufacturer’s instructions. Flow cytometry analysis of the resulting enrichment showed 88% and 80% purity for the CD4þCD25þ and CD4þCD25 subpopulations, respectively. The 1
106 cells per mouse were administered by IP route to na€ıve animals in 500 mL medium. Flow cytometry Platelet-bound Ig were analyzed by flow cytometry after detection with fluoresceinated rat IgG2a anti-mouse k-chain mAb (LO-MK1) as described [1]. Positive platelets were determined with an arbitrary gate. Analysis of cell subpopulations in spleen cells was performed as described [8] using fluoresceinated anti-B220 (BD Biosciences) and anti-F4/80 (eBiosciences). Foxp3, CD4, and CD25 labeling was performed by using the Mouse Regulatory T Cell Staining Kit (eBioscience) following manufacturer’s instructions. Statistical analysis Statistical analysis was performed using Student’s t-test or, when appropriate, nonparametric unpaired two-tailed Mann-Whitney.

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Results and discussion T-helperdependence of thrombocytopenia after immunization with rat platelets To determine whether the thrombocytopenia elicited in normal mice by sequential immunization with rat platelets was a phenomenon dependent on T-helper lymphocytes, anti-CD4 GK1.5 mAb was administered during the immunization procedure. This treatment resulted in an almost complete suppression of CD4þ cells (!0.5% remaining CD4þ cells, Fig. 1D) and was previously shown to abrogate in vivo T-helperdependent responses [6]. As shown in Figure 1A, mice that received a mock treatment responded to the immunization protocol by developing a transient

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thrombocytopenia. In contrast, animals depleted of their T-helper lymphocytes by the anti-CD4 treatment conserved stable platelet counts during the whole procedure. The difference between T-helperdepleted and immunocompetent animals was significant at all time points analyzed (p 5 0.0072; p 5 0.0006; p ! 0.0001; p 5 0.0002; and p 5 0.0110 at 1, 2, 3, 4, and 5 weeks, respectively). Similar results were observed in three independent experiments. They indicate that the autoantibody-mediated thrombocytopenia induced by immunization of normal CBA mice with rat platelets requires the presence of functional T-helper lymphocytes. This is in agreement with previously reported observations demonstrating the role of T-helper lymphocytes in the pathogenesis of another mouse model of

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Figure 1. Involvement of T-helper lymphocytes in the response induced by mouse immunization with rat platelets. (A) T dependence of thrombocytopenia. Platelets were counted in groups of four to five CBA mice at different times after initiation of immunization with rat platelets. Anti-CD4 IP treatment was started 2 days before the first administration of rat platelets, at a weekly dose of 1 mg mAb, whereas control animals received saline. Results are expressed as mean 6 standard deviation (SD). (B) T-dependence of the anti-rat platelet antibody response. Anti-rat platelet antibody response was measured by ELISA in the serum of groups of three to four CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD4 IP treatment was started 2 days before the first administration of rat platelets, at a weekly dose of 1 mg mAb, whereas control animals received saline. Results of antibody binding to rat platelets (black columns) and to bovine serum albumin (open columns) are expressed as mean 6 SD. (C) T-independence of total IgG levels. Total IgG were measured by ELISA in the serum of groups of three CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD4 IP treatment was started 2 days before the first administration of rat platelets, at a weekly dose of 1 mg mAb, whereas control animals received saline. Results are expressed in mg/mL (mean 6 SD). (D) Effect of CD4þ cell depletion on spleen cell populations. The proportion of T-helper lymphocytes, B lymphocytes, and macrophages in spleen cells was analyzed by fluorescence-activated cells sorting in groups of three to four CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD4 IP treatment was started 2 days before the first administration of rat platelets, at a weekly dose of 1 mg mAb, whereas control animals received saline. Results are expressed as mean 6 SD of percent of cells positive for CD4, B220, and F4/80, respectively. BSA 5 bovine serum albumin.

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autoantibody-mediated blood autoimmune disease [6]. Moreover, the anti-rat platelet antibody response was largely suppressed by the anti-CD4 treatment in animals immunized with rat platelets (Fig. 1B, difference between immunized mice treated with NaCl and anti-CD4 mAb; p ! 0.0001). However, although mouse platelet-associated immunoglobulin levels were also decreased in some experiments by the anti-CD4 treatment, this suppression was not reproducible (not shown). These data may indicate that production of the bulk of antiplatelet antibodies, including anti-rat platelet and pathogenic anti-mouse platelet antibodies, requires the presence of T-helper lymphocytes, whereas, some antibodies reacting with mouse platelets, but without clear pathogenic function are secreted even in the absence of T-helper cells. This might be related, for instance, to an IgM isotype of these platelet-associated immunoglobulins that was previously shown to be present in anti-mouse platelet antibody bulk [1]. Moreover, total IgG levels were not decreased by suppression of T-helper lymphocytes (Fig. 1C, no difference between immunized mice treated with saline or GK1.5

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Specificity of T-helper cells activated by immunization with rat platelets To further analyze T-helper lymphocytes that were stimulated by the immunization with rat platelets, the proliferation of spleen CD4þ cells was measured by bromodeoxyuridine incorporation in the presence of mouse or rat platelets and of antigen-presenting cells. As shown in Figure 2A, no proliferation was induced in CD4þ cells from control mice by stimulation with either rat or mouse platelets. In contrast, T-helper cells from immunized mice proliferated in response to rat platelets (p 5 0.0040 between control and rat platelet stimulation). However, mouse platelets did not induce any proliferation of these lymphocytes (p 5 0.0006 between stimulation with mouse and rat platelets). A similar observation was made in three independent experiments.

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mAb; p 5 0.7589). After anti-CD4 treatment, the proportion of B lymphocytes (B220þ) and macrophages (F4/80þ) in spleen cells showed a slight relative increase corresponding to the disappearance of CD4þ cells (Fig. 1D).

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Figure 2. Characterization of T-helper lymphocytes activated after mouse immunization with rat platelets. (A) Proliferation of pooled spleen CD4þ cells from groups of three control CBA mice or of mice immunized twice with rat platelets restimulated for 6 days ex vivo with medium, rat or mouse platelets, was measured by bromodeoxyuridine incorporation. Results are means 6 standard deviation (SD) of triplicate measurements. (BF) Cytokines were measured by ELISA in the supernatants of pooled CD4þ spleen cells from groups of three control CBA mice (open columns) or of mice immunized twice with rat platelets (closed columns) restimulated for 6 days ex vivo with medium, rat or mouse platelets. Results are shown as mean 6 SD of triplicate measurements.

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T lymphocytes are involved in a similar model where hemolytic anemia is induced in immunocompetent mice through immunization with rat red blood cells [9]. In this model, autoantibodies recognizing epitopes shared between rat and mouse target cells were also found [10]. However, in contrast with our observations in the antiplatelet response, T lymphocytes that recognize rat as well as mouse erythrocyte epitopes are activated after immunization with rat red blood cells [11]. These T lymphocytes proliferated equally well after presentation of either rat or mouse erythrocytes [12]. This suggested that, at the T-helper cell level, mechanisms of tolerance were more efficient in our model of mouse immunization with rat platelets, than in the corresponding model with erythrocyte immunization. The role of T-helper cells in the initiation of the autoreactive antibody response observed here would therefore be closer to what has been reported for rheumatoid factor production in secondary immune responses [13]. Indeed, in this case also, production of autoantibodies (anti-immunoglobulin rheumatoid factors) results from the interaction of autoreactive B lymphocytes with T-helper lymphocytes that recognize a foreign epitope expressed on the carrier antigen involved in the immune complex. Alternatively, the T-cell repertoire of CBA mice might be initially restricted to epitopes that are present in rat but not mouse platelets. The nature of the epitopes recognized by mouse T-helper lymphocytes after immunization with rat platelets remains undetermined. An involvement of T-helper lymphocytes in antiplatelet autoimmune diseases has long been recognized and, in human patients, autoreactive T cells have been reported [14]. Moreover, in patients, CD4þ T cells that react with GPIIb-IIIa have been described [15,16], an antigen also recognized by many autoantibodies. Interestingly, these T-helper cells responded to chemically modified, but not to native GPIIb-IIIa [17]. As in our model, autoantibodies have been found to react with both GPIIIa and GPIb ([1], unpublished results), it would be interesting to find out whether mouse anti-rat platelet T-helper cells display the same specificity. Cytokine production by antiplatelet T-helper cells Cytokine production was measured by ELISA in the supernatants of similarly stimulated CD4þ spleen cells (Fig. 2BF). Restimulation with mouse platelets did not induce any significant cytokine production. In contrast, a strong IFN-g response was observed after restimulation of CD4þ T cells from immunized mice with rat platelets (Fig. 2C). Such a large IFN-g production elicited specifically by rat but not mouse platelets was found in three independent experiments. Although production of other cytokines such as IL-4 (Fig. 2B), IL-5 (Fig. 2D), IL-9 (Fig. 2E), and IL-17A (Fig. 2F) by stimulated anti-rat platelet T lymphocytes could also increase, this was at a much lower and more variable level.

These results indicate that a differentiation toward the Th1 phenotype is preferentially triggered after mouse immunization with rat platelets, while less Th2 or Th17 cytokines are produced. This observation fits well with the predominance of IgG2a and IgG2b autoantibody detected in these animals [1]. Such a preponderance of Th1 cells among lymphocytes involved in antiplatelet autoimmune response has also been reported in patients with immune thrombocytopenic purpura [18–22]. Activation of antiplatelet Th1 lymphocytes might trigger a pathogenic response through distinct mechanisms; by providing the required help to B lymphocytes to produce antibodies that would be autoreactive to mouse platelets through antigenic mimicry, and by enhancing the phagocytic capacity of macrophages through IFN-g secretion. This cytokine has been shown to dramatically exacerbate antiplatelet antibody pathogenicity by increasing phagocytosis of opsonized platelets after viral infection, although in this case it was not produced by T lymphocytes [23]. Role of antiplatelet T-helper lymphocytes in autoreactive antibody response The ability of stimulated anti-rat platelet T-helper lymphocytes from immunized mice to effectively promote an in vivo autoreactive antibody response was analyzed in adoptive transfer experiments (Fig. 3A). Animals immunized twice with rat platelets, 3 and 10 days after transfer of 3
106 proliferating anti-rat platelet-specificT cells displayed an anti-mouse platelet antibody response, measured by flow cytometry, that was significantly higher than the response of mice that received either T lymphocytes or rat platelets only (p 5 0.0411 and p 5 0.0303, respectively). An increased anti-mouse platelet antibody response in mice receiving both anti-rat platelet T cells and rat platelets was observed in four independent experiments. This indicates that anti-rat platelet T-helper cells stimulated in mice immunized with rat platelets provide the required help to B lymphocytes producing antibodies that recognize epitopes shared by mouse and rat platelets. However, although the anti-mouse platelet antibody response continued to increase in mice transferred with activated anti-platelet T-helper cells (not shown), the total anti-rat platelet antibody response quickly reached a plateau and by day 17 post T-cell transfer, no difference was observed (Fig. 3B). This suggests that the natural anti-rat platelet T-helper cell response was sufficient to quickly induce the bulk of the anti-rat platelet antibody response, but that the production of the antiplatelet antibody fraction that was cross-reactive between rat and mouse epitopes may be enhanced by an addition of activated anti-rat platelet helper T cells. Transfer of activated anti-platelet T-helper cells followed by immunization with rat platelets also induced thrombocytopenia, when compared to immunized animals that did not receive the activated antiplatelet lymphocytes (Fig. 3C; p 5 0.0357), although the differences between groups did not always reach statistical significance (not shown).

L. Detalle et al./ Experimental Hematology 2011;39:676–685

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Figure 3. Pathogenic effect of anti-rat platelet T lymphocytes. (A) Groups of five to six CBA mice received by IP administration 3
106 anti-rat platelet T lymphocytes restimulated in vitro, or medium alone, followed after 3 and 10 days by immunization with rat platelets or mock immunization. Seven days after the second immunization, platelet-bound immunoglobulin was measured by flow cytometry. Results are shown as mean 6 standard deviation (SD) of positive platelets. (B) Groups of four to five CBA mice received by IP administration 3
106 anti-rat platelet T lymphocytes restimulated in vitro, or medium alone, followed after 3, 10, and 17 days by immunization with rat platelets or mock immunization. Seven days after the third immunization, anti-rat platelet antibodies were measured by ELISA with plates coated with rat platelets. Results in OD, after subtraction of values obtained with plates coated with bovine serum albumin, are shown as mean 6 SD. (C) Platelets were counted 7 days after the second immunization with rat platelets of groups of three to five CBA mice treated by IP administration 3
106 anti-rat platelet T lymphocytes restimulated in vitro, or medium alone. Results are expressed as mean 6 SD.

Therefore, the correlation between thrombocytopenia and detection of platelet-associated antibodies is not absolute. This might be explained by the production of various antiplatelet autoreactive antibodies, with distinct pathogenic potency.

Control of thrombocytopenia by CD25þ cells The transiency of the thrombocytopenia induced by CBA immunization with rat platelets suggests that this pathogenic response is controlled by regulatory mechanisms.

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As for other autoimmune models, these mechanisms could involve the activation of CD25þ regulatory T cells. To test this hypothesis, mice were treated with an antiCD25 mAb that depletes CD25þ regulatory T cells in vivo [24] before immunization with rat platelets. Analysis of spleen cells from mice untreated with anti-CD25 antibody showed a proportion of 2.7% 6 0.08% of CD4þCD25þ cells; 88.4% 6 3.5% of these CD4þCD25þ cells were positive for Foxp3 staining, indicating that these cells were mostly T-regulatory cells. In animals immunized with rat platelets and treated with anti-CD25 mAb, only 0.3% 6 0.06% CD4þCD25þ cells remained, showing the efficiency of the treatment (p ! 0.0001). As shown in Figure 4A, this treatment resulted in a deeper and more

prolonged thrombocytopenia, when compared with control animals (difference between treated and control groups; p 5 0.0216 at 3 weeks; p ! 0.0001 at 4 weeks; and p 5 0.0010 at 5 weeks). This result indicated that CD25 regulatory T cells control the development of this autoreactive antibodymediated thrombocytopenia as it has been proposed in humans [25,26]. This enhanced thrombocytopenia was not correlated with an increase in the binding of antibodies to circulating platelets as measured by flow cytometry (Fig. 4B). This might be explained by a lack of pathogenicity of these bound antibodies detected by flow cytometry, and by an absence of control of their secretion by regulatory T cells. This was

Figure 4. Control of thrombocytopenia by CD25þ cells. (A) Platelets were counted in groups of five CBA mice at different times after initiation of immunization with rat platelets. Anti-CD25 treatment was administered IP once, 2 days before the first administration of rat platelets, at a dose of 300 mg mAb, whereas control animals received saline. Results are expressed as mean 6 standard deviation (SD). (B) Effect of anti-CD25 treatment on platelet-bound Ig. Platelet-associated Ig was measured by flow cytometry in groups of four CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD25 treatment was administered IP once, 3 days before the first administration of rat platelets, at a dose of 250 mg mAb, whereas control animals received saline. Results are expressed as mean 6 SD of fluorescence intensity (log scale). (C) Effect of anti-CD25 treatment on the anti-rat platelet antibody response. Antirat platelet antibody response was measured by ELISA in the serum of groups of three to four CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD25 treatment was administered IP once, 4 days before the first administration of rat platelets, at a dose of 300 mg mAb, whereas control animals received saline. Results of antibody binding to rat platelets (black columns) and to bovine serum albumin (BSA; open columns) are expressed as mean 6 SD. (D) Effect of CD25þ cell depletion on spleen cell populations. The proportion of T-helper lymphocytes, B lymphocytes, and macrophages in spleen cells was analyzed by fluorescence-activated cell sorting in groups of three to four CBA mice at 4 weeks after initiation of immunization with rat platelets. Anti-CD25 treatment was administered IP once, 4 days before the first administration of rat platelets, at a dose of 300 mg mAb, whereas control animals received saline. Results are expressed as mean 6 SD of percent of cells positive for CD4, B220, and F4/80, respectively.

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also supported by the observation that the anti-CD25 treatment did not modify the anti-rat platelet antibody response (Fig. 4C, no significant difference between immunized mice with and without anti-CD25 treatment; p 5 0.6834). Finally, T-helper lymphocyte, B lymphocyte, and macrophage spleen cell proportions were not modified by the anti-CD25 treatment (Fig. 4D, no significant difference between immunized mice with and without anti-CD25 treatment for CD4þ, B220þ, and F4/80þ cell subpopulations; p 5 0.3212; p 5 0.2974; and p 5 0.3547, respectively), whereas total sera Ig levels were increased in mice depleted from their CD25þ cells, but not to a significant level (9.3 6 2.0 mg/mL in control animals vs. 24.0 6 11.7 mg/mL in antiCD25treated animals; p 5 0.0571). Transfer of CD4þCD25þ cells To further analyze a possible role of CD25þ regulatory T cells in the control of thrombocytopenia, both CD4þCD25 and

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CD4þCD25þ cells were enriched from spleen cells of animals repeatedly immunized with rat platelets. These cells were transferred to na€ıve animals that then received a similar immunization protocol. As shown in Figure 5A, mice that received no cells or CD4þCD25 cells developed a significant thrombocytopenia when compared to control nonimmunized animals (p 5 0.0015 and p 5 0.0072, respectively). In contrast, mice that had received CD4þCD25þ cells from previously immunized animals were protected from development of thrombocytopenia (very significant difference between mice receiving CD4þCD25 and CD4þCD25þ cells; p 5 0.0030). A protection was also observed in a second independent experiment, whereas in a third experiment, no protection by CD4þCD25þ cells was observed (not shown). These differences may be due to variations in the number of actual regulatory cells that are present in the CD4þCD25þ subpopulation obtained from spleens of immunized mice, or to a moderate efficiency of these transferred regulatory

Figure 5. Regulation of the response elicited by rat platelets by CD4þCD25þ cells. (A) Adoptive transfer of CD4þCD25þ cells. Groups of four to five CBA mice received PBS or 106 CD4þCD25 or CD4þCD25þ cells, followed by weekly immunization with rat platelets, or NaCl. Platelets were counted after 3 weeks and results are shown as mean 6 standard deviation. (B) Regulation of CD4þ cell proliferation by CD4þCD25þ cells. The 2
106 pooled CD4þ cells isolated from the spleens of a group of 10 CBA mice immunized twice with rat platelets were restimulated with feeder cells and rat platelets with or without approximately 0.8
105 purified CD4þCD25þ cells obtained from pooled spleen cells of a group of 10 CBA mice immunized five times with rat platelets. Results of bromodeoxyuridine incorporation measured in triplicates are shown for two to four distinct cultures dishes (mean 6 standard deviation).

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cells. Similarly, immunosuppressive therapy in humans with isolated or expanded regulatory T cells is difficult [27]. Interestingly, in different mouse models of autoimmune and alloimmune responses against erythrocytes, similar results to those reported here for anti-platelet responses have been found by depletion and transfer of CD25þ cells [28,29]. This indicates that common effector and regulatory mechanisms control the immune responses against platelets and erythrocytes. In our anti-platelet model, the anti-mouse, but not the anti-rat antibody response is downregulated with time, although they are both initiated by anti-rat T-helper lymphocytes. Therefore, it may be postulated that this suppression is exerted at the level of B lymphocytes, as it has been reported in other cases [30], rather than on T cells. However, addition of activated CD4þCD25þ cells to CD4þ T-helper cells restimulated ex vivo with rat platelets, largely suppressed proliferation of these activated T lymphocytes (Fig. 5B, significant difference between CD4þ cells without and with CD4þCD25þ cells; p 5 0.0115). Thus, at least part of the regulation of the response elicited by rat platelet immunization might be directed toward CD4þ cells. Further work will be required to determine the precise mechanisms by which these regulatory cells may control development of thrombocytopenia in mice immunized with rat platelets.

Conclusions In conclusion, our results indicate that immunization of immunocompetent CBA mice with rat platelets elicits a normal T-helper lymphocyte response that recognize rat, but not mouse, platelet epitopes. Those anti-rat T lymphocytes provide adequate help to B cells that produce antibodies reacting with epitopes shared by rat and mouse platelets. Therefore, this xenoimmune response, without self-reactivity at the antiplatelet T-helper lymphocyte level leads to an autoreactive antibody production. Similar mechanisms might be involved in an antiplatelet response induced in the marmoset [31] and in human posttransfusion purpura or immune thrombocytopenic purpura [32]. As in these human diseases, and in other experimental models of blood autoimmune diseases, regulatory T cells play an important role in the control of the autoimmune response and of the progression of the disease. At least part of this control seems to be directed toward T-helper cells responding to rat platelets. Therefore, our model of antiplatelet autoreactive response seems appropriate to study the mechanisms at play in corresponding human diseases.

Acknowledgments The authors would like to thank Isabelle Bar for expert technical assistance. This work was supported by the Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique

M
edicale (FRSM), Loterie Nationale, Fonds de D
eveloppement Scientifique and the State-Prime Minister’s Office - S.S.T.C. (Interuniversity Attraction Poles) and the French Community (Concerted Actions, 04/09-318 and 09/14-021), Belgium. L.D. is a FRIA fellow and J.P.C. is a research director with the FNRS.

Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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