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_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

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

HLA-I binding affinity of eluted peptides derived from the invariant chain.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-g002" target="_blank">Figure 2</a> for amino acid position in the invariant chain protein.</p>†<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>‡<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Surface display of the CLIP epitope on HLA-II-negative leukemic cells and influence of the invariant chain on HLA-I expression.

<p>(A) Surface expression of CLIP, HLA-DR, total HLA-II (‘HLA-DRPQ’) and invariant chain (‘CD74’) of myeloid cells from an acute promyelocytic leukemia (APL) patient, as determined by flow cytometry. Myeloid cells were defined as CD45^dim/SSC^low/int and expression thresholds were set according to unstained myeloid cells. (B) Quantitative analysis on frequencies of myeloid cells from APL patients that express CLIP (n = 9), HLA-DR (n = 9), total HLA-II (n = 6) and CD74 (n = 6). Frequencies indicate percentage tumor cells that reach threshold expression based on unstained leukemic cells. (C) The effect of invariant chain Ii down-modulation in KG-1 (CLIP^-) and THP-1 (CLIP⁺) leukemic cells on HLA-I expression at the cell surface. Intracellular staining (ICS) of Ii (PIN1.1) and surface staining of HLA-I (W6/32) were compared between Ii siRNA-transduced and non-transduced cells.</p

Promiscuous binding of CLIP-peptide to distinct HLA-I alleles, representing four supertypes.

<p>The CLIP peptide RMATPLLMQALPM (peptide 3) was tested for binding affinity in a competition-based cellular peptide binding assay. The four tested HLA-I alleles (HLA-A0201, -A0301, -B0702 and -B4002) harbor a completely distinct binding pocket and bind different peptide ligands. CLIP peptide shows intermediate to high binding affinity to all of these. Separate positive control peptides efficiently bind to their respective HLA allele: GILGFVFTL (A0201 peptide), QVPLRPMTYK (A0301 peptide), SPSVDKARAEL (B0702 peptide) and GEFGGFGSV (B4002 peptide) with IC50 values of 3.7, 0.2, 0.7 and 0.2, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>. The peptide concentration started at 100 µg/ml for HLA-A0201, -A0301 and -B4002 and 25 µg/ml for HLA-B0702, followed by a serial dilution of a factor two. Exact IC50 values of the CLIP peptides are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-t002" target="_blank">Table 2</a>.</p

<i>Salmonella</i> is actively excreted by B cells.

<p>(A) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy. Depicted is the GFP signal projected on the transmission image with images taken every 30 min. Scalebar = 10 µm. Arrows indicate the B cell, white arrow: B cells moves op top of the monolayer, black arrow: B cells moves below the monolayer. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s005" target="_blank">Video S4</a>. (B) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy in the presence of TexasRed labeled anti-LPS mAbs. Depicted are GFP and Texas-Red signals projected on the transmission image. Scalebar = 10µm. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s006" target="_blank">Video S5</a>. (C) Quantification of <i>Salmonella</i> secretion from B cells. Primary B cells were incubated with live uncoated GFP-<i>Salmonella</i>. Cells were stained with antibodies against LPS, fixed and analyzed using FACS. Left panel: increase in cell surface exposed LPS from bacteria exposed at the cell surface after initial uptake by B cells. Middle panel: percentage of B cells having excreted <i>Salmonella</i> as calculated from the percentage of B cells containing GFP-<i>Salmonella</i> followed in time. Right panel: left and middle panels are projected to illustrate that both processes show similar kinetics. Error bars represent SD from three independent experiments. (D) Primary B cells were incubated with live uncoated GFP-expressing <i>Salmonella</i> and followed for the time points indicated. The fraction of living B cells is plotted to demonstrate that loss of GFP-<i>Salmonella</i> positive B cells is not correlated with cell death.</p

HLA-I binding affinity of peptide length variants located in the CLIP region of the invariant chain.

*<p>Peptide found with HLA-I elutions.</p>†<p>Amino acid (AA) position in the invariant chain protein.</p>‡<p>Predicted HLA-I allele to which the peptide binds. Peptide binding prediction was done with netMHC (<a href="http://www.cbs.dtu.dk/services/NetMHC" target="_blank">http://www.cbs.dtu.dk/services/NetMHC</a>). Binding predictions can be made for peptide lengths between 8 and 11 for all alleles with a novel approximation algorithm using artificial neural networks trained on 9-mer peptides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard2" target="_blank">[27]</a>. Only peptides are shown with a predicted binding affinity of 500 nM or stronger.</p>§<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>¶<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Invariant chain-derived peptides identified in isolated HLA-I molecules of B-LCLs.

<p>Peptide elutions of purified HLA-I molecules from EBV-transformed B-LCLs resulted in the identification of five peptides originating from the invariant chain. HLA-I purification and subsequent mass spectrometry analysis are described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#s2" target="_blank">Methods</a></i>. Of note, peptide 3 and 4 are located in the CLIP region, known for universal binding to HLA-II molecules.</p