image_1_Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion.PDF

<p>Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.</p

image_3_Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion.PDF

<p>Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.</p

image_5_Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion.PDF

<p>Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.</p

image_2_Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion.PDF

<p>Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.</p

Characteristics of the study population.

*<p>Two-tailed Mann-Whitney test; † Mean±SEM (range); ND: Not Documented; ^#Fisher's exact test.</p>‡<p>All patients received Steroids, Cyclosporin, Tacrolimus and/or Mycophenolate Mofetil; one patient received both Basiliximab and anti-thymocyte globulins.</p

Treatment with IVIg is associated with a transient decrease in levels of PFR-MCA hydrolyzing IgG.

<p>IgG was purified from the plasma of patients who received IVIg therapy prior to transplantation (full circles) and from patients who did not received IVIg (empty circles). Plasma had been collected prior to renal transplant (D0) and 3 (M3), 12 (M12) and 24 (M24) months after renal transplant. IgG (66.67 nM) was incubated with PFR-MCA (100 µM), a peptide chromogenic substrate, for 24 hr at 37°C. The amount of hydrolysis was quantified by measuring the fluorescence of the leaving MCA moiety, and is expressed in femtomoles of substrate hydrolyzed per minute per picomoles of IgG. Pooled normal human IgG was used as a control source of IgG. Panel A depicts the raw results as scatter dot plots. Panel B depicts the evolution of the mean ± SEM levels of PFR-MCA-hydrolyzing IgG in the two groups of patients with time (*: P = 0.004). The dotted line represents the hydrolysis of PFR-MCA by normal pooled human IgG (mean of 29 measurements; Coefficient of variation: 0.29). Panel C depicts the levels of PFR-MCA-hydrolyzing IgG in patients treated with anti-thymocyte globulins (ATG, full squares) or not (empty squares), as measured in plasma collected 3 months post-transplantation.</p