Increased cytotoxic T-lymphocyte-mediated cytotoxicity predominant in patients with idiopathic thrombocytopenic purpura without platelet autoantibodies

Idiopathic thrombocytopenic purpura (ITP) is a common hematologic disorder manifested by autoantibody-mediated platelet destruction. In the majority of ITP patients, autoantibodies were found to be specific to GPIIb/IIIa or GPIb/IX.1 It may not only damage platelets via the reticuloendothelial system,2 but may also inhibit platelet production.3,4 However, platelet autoantibodies can be detected in only 50–70% of ITP patients,5 indicating other mechanisms could be involved. Recently, in vitro studies suggested that CD8 cytotoxic T-lymphocyte (CTL) mediated lysis of autologous platelets may contribute to platelet destruction in ITP.6,7 In the present study, we prospectively measured CTL-mediated cytotoxicity toward autologous platelets between ITP patients with and without autoantibodies, and evaluated the effect of high dose dexamethasone (HD-DXM) on this action.

Forty-eight previously-untreated ITP patients were enrolled by diagnostic criteria for ITP.8 Blood sampling was performed before and two weeks after treatment with HD-DXM.9 The control group consisted of 17 healthy adult volunteers with no history of blood transfusion or pregnancies (Table 1). The study was approved by the Medical Ethical Committee of Qilu Hospital. Informed consent was obtained from all subjects.

All plasma samples for modified monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were obtained from ethylenediaminetetraacetic acid (EDTA) anticoagulated blood by centrifugation and stored at −80°C prior to use. Platelets were prepared by differential centrifugation from EDTA-anticoagulated blood and used as target cells. CD8 T lymphocytes were isolated by magnetic microbeads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) from peripheral blood mononuclear cells by density gradient centrifugation and used as effector cells. The purity of CD8 T cells was > 92%. The concentration of effector and target cells was adjusted to 10/mL and 10/mL respectively, and then diluted to achieve a final effector/target (E/T) ratio of 1:10. All cells were washed free from plasma.

Detection of specific anti-platelet GPIIb/IIIa and/or GPIb autoantibodies was performed as described by Hou et al.10 Measurement of CTL-mediated cytotoxicity was carried out according to the protocol developed by Goldberg et al.11 based on two-color flow cytometry to identify different populations of live targets (CD41a Annexin V-) and apoptotic targets (CD41a Annexin V) using antibodies from BD Biosciences (San Jose, CA, USA). Anti-CD3 antibody (BD Biosciences, San Jose, CA, USA) was added at a final concentration of 0.32 μg/mL to stimulate cytolytic effector T cells. Spontaneous lysis was determined in control tubes holding only culture media and platelets. A minimum of 10,000 events were acquired and data were analyzed using CellQuest Software on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). Percent lysis and specific lysis were calculated as follows: percent lysis=total (CD41a, Annexin V)=Total (CD41a); specific lysis=percent induced lysis=percent spontaneous lysis, and expressed as a percentage.

Table 1.Subjects’ characteristics.

In the plasma of 48 patients with ITP, antibodies against GPIIb/IIIa and/or GPIb/IX were detected in 22 samples (group I). Negative reactions to both glycoproteins were displayed in the remainders (group II). Before HD-DXM treatment, positive platelet lysis was seen in 11 of group I and 21 of group II (group I vs. group II, p<0.05) and in 4 of group I and 7 of group II after treatment (group I vs. group II, not significant (NS); pre-treatment vs. post-treatment in group I, p<0.05, and in group II, p<0.01) (Figure 1A–F). On the other hand, before treatment, both group I and group II had increased platelet lysis compared with controls (group I vs. controls, p<0.05; group II vs. controls, p<0.01; group I vs. group II, p<0.01), whereas platelet lysis was substantially decreased in both groups after treatment (pre-treatment vs. post-treatment in group I, p<0.01, and in group II, p<0.01) (Figure 1G). Interestingly, positive platelet lysis was also observed in one control subject (data not shown). Additionally, the platelet lysis was found to be negatively correlated with the platelet count in group II (r=−0.439, p<0.05) (Figure 2), but not in group I (r=−0.322, NS). Despite the use of the most sophisticated assays, platelet autoantibody cannot be detected in all patients with ITP.5 Thus, autoantibody-mediated platelet destruction cannot account for all the observations in ITP. Recently, Olsson et al.6 discovered increased expression of several cytotoxic genes in T cells from ITP patients and 6 out of 8 patients with active ITP showed platelet lysis by T cells, but none in remission. Subsequently, Zhang et al.7 found that platelet lysis was seen when using purified CD8 T cells as effector cells and that CTLs were abnormally activated in ITP. In the present study, we found a significant difference in CTL-mediated platelet lysis between MAIPA positive and negative ITP patients, as well as a negative correlation between platelet lysis and platelet count. This evidence strongly supports the view that CTL-mediated cytotoxicity toward autologous platelets is dominant in ITP patients without autoantibodies, and protection against this action might be an effective therapeutic approach for this subgroup of ITP patients. As a promising alternative to prednisone for the first- or second-line treatment in ITP patients,9 HD-DXM was demonstrated to correct the Th1 cytokine dominance, shown to promote cell-mediated cytotoxicity and IgG production by Guo et al.12 In this study, platelet lysis was found to be significantly lower after HD-DXM treatment, indicating that HD-DXM could be an effective protection against both autoantibody-mediated and CTL-mediated destruction of platelets in ITP patients. Finally, the limitation of all currently available platelet antibody tests, including MAIPA, must be noted. Furthermore, autoantibodies against other glycoproteins such as GPIa/IIa, GPIV, GPV, and GPVI do exist, although their incidence is scarce in ITP.

Figure 1.(A-D) One representative for CD41a-PEcy5 and Annexin V-FITC dual (A) and single (B-D) parameter analysis of CTL-mediated cytotoxity. In panels B-D, black lines designate the platelet incubated alone for estimation of spontaneous lysis, while the platelets and CD8⁺ cells mixed together for evaluation of induced lysis is indicated by red lines. The specific lysis is 10.03% (B), 4.33% (C), and 2.35% (D) respectively in one representative for group II and group I before treatment, and group II after treatment. (E, F) The number of patients with positive platelet lysis, defined as mean+2SD (dotted line) recorded for the controls, before (E) and after (F) treatment. Circles represent group I and triangles group II. (G) Analysis of cell-mediated cytotoxicity against autologous platelets in groups before and after treatment (n=48).

Figure 2.Negative correlation between platelet lysis and platelet count in group II.

Footnotes

• Funding: this work was supported by grants from National Natural Science Foundation of China (No. 30570779, No. 30470742, No. 30600259, No. 30770922, and No. 30300312), 973 Program (No. 2006 CB 503803), Foundation for the Author of National Excellent Doctoral Dissertation of PR China (No. 200561), Key Clinical Research Project of Public Health Ministry of China 2007–2009, the 1020 Program from Health Department of Shandong Province, and Commonweal Trade for Scientific Research (No. 200802031).