Search

BACKGROUND: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted. STUDY DESIGN AND METHODS: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated. RESULTS: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion. CONCLUSIONS: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/-or C1q-/-mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors.In-vitrostudies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/-than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first “hit” is recipient inflammation and the second “hit” is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided approximately 15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare post-translational modifications.

Naïve T cells proliferate independently of cognate antigen when introduced into lymphopenic hosts. Lymphopenia-induced proliferation depends on low-affinity MHC/self-peptide complexes and on IL-7. To elucidate the intracellular signals mediating this proliferation, we analyzed changes in gene expression in naïve CD8 + T cells at different times after their transfer into a lymphopenic environment. The genes induced in response to lymphopenia were largely an attenuated subset of those turned up by full antigenic stimulation, including genes related to cell cycling, whereas excluding genes specifically associated with effector activity. After the initial phase of proliferation in an empty compartment, the naïve T cells adopted a stable pattern of gene expression similar to that of antigen-experienced memory cells. Thus, T cells proliferating in lymphopenic hosts do not exhibit a unique gene-expression profile, instead relying on “traditional” signals for this antigen-independent proliferation; this process ultimately results in differentiation to “authentic” memory cells.

Advances in chemistry and massively parallel detection underlie DNA-sequencing platforms that are poised for application in personalized medicine. In stark contrast, systematic generation of protein-level data lags well behind genomics in virtually every aspect: depth of coverage, throughput, ease of sample preparation and experimental time. Here, to bridge this gap, we develop an approach based on simple detergent lysis and single-enzyme digest, extreme, orthogonal separation of peptides and true nanoflow liquid chromatography-tandem mass spectrometry that provides high peak capacity and ionization efficiency. This automated, deep efficient peptide sequencing and quantification mass spectrometry platform provides genome-scale proteome coverage equivalent to RNA-seq ribosomal profiling and accurate quantification for multiplexed isotope labels. In a model of the embryonic to epiblast transition in murine stem cells, we unambiguously quantify 11,352 gene products that span 70% of Swiss-Prot and capture protein regulation across the full detectable range of high-throughput gene expression and protein translation.

Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a two-dimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase (RP)-strong anion exchange (SAX)-RP configuration. Both techniques provide for automated, online data acquisition, with the 3-D platform providing the highest performance both in terms of separation peak capacity and the number of unique phosphopeptide sequences identified per μg of cell lysate consumed. Our integrated RP-SAX-RP platform provides several analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (2) high separation peak capacity (3) efficient retention of singly- and multiply-phosphorylated peptides; (4) compatibility with automated, online LC-MS analysis. We demonstrate the reproducibility of RP-SAX-RP and apply it to the analysis of phosphopeptides derived from multiple biological contexts, including an in vitro model of acute myeloid leukemia in addition to primary polyclonal CD8(+) T-cells activated in vivo through bacterial infection and then purified from a single mouse.

The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and in-frame tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia.

Previous results from this laboratory demonstrated that the dominant influenza A epitope recognized by HLA-A2.1-restricted cytotoxic T lymphocytes (CTL) from HLA-A2.1 transgenic mice was the matrix protein 1 (M1) peptide epitope that is immunodominant in human CTL responses. However, analysis of a large number of CTL lines revealed a subset of influenza A/PR/8/34-specific murine CTL that recognized an HLA-A2.1-restricted epitope distinct from M1. Using recombinant vaccinia viruses encoding different influenza gene segments, the epitope recognized by these CTL was shown to be derived from A/PR/8 non-structural protein 1 (NS1). Because these CTL did not recognize targets infected with the A/Alaska/6/77 strain of influenza, candidate peptide epitopes were synthesized based on sequences that included an HLA-A2.1-specific binding motif, and that differed between A/PR/8 and A/Alaska. All of these CTL recognized a nonamer and a decamer peptide which contained a common eight amino acid sequence and two distinct sets of binding motif residues. However, the nonamer peptide was able to sensitize CTL for half-maximal lysis at 80- to 2500-fold lower doses than either the octamer or decamer. The homologous peptide derived from A/Alaska NS1 contained conservative amino acid changes at positions 4 and 8, and was not recognized at any tested concentration, although it bound with higher affinity to HLA-A2.1 than the peptide from A/PR/8. The A/PR/8 NS1 nonamer epitope was also recognized by human influenza A-specific CTL derived from two individuals. These results substantiate the general utility of HLA class I transgenic mice for the identification of human CTL epitopes for other pathogens.