(!) SPR assay for detection of mucosal B cell responses. In addressing the question whether IgA and IgM responses appear in mucosal fluids during HIV infection, there is a need for the development of a sensitive assay using small quantities of mucosal fluids. We have developed a multiplex SPR assay that allows measurements of total Ig concentration, isotype and specificity for antibody binding to MPER peptide and oligomeric HIV Env gp140 (Figure 1). When equipped with high throughput capability the SPR binding assay is a highly sensitive assay that also provides quantitative data in terms of antibody binding avidity. This work was initiated using 2F5 IgG spiked into buffer, saliva and bovine skimmed milk. We have determined the limit of detection of total Ig to be between 121 pM to 380pM and the biologic limit of detection (LOD) for binding to HIV-1 Env gp140 to be about 1.1 nM. In general these limits are dictated by binding avidity and signal-to-noise responses, which was 3-5 fold higher when the mAb was spiked into complex mixtures (saliva, milk). To further improve signal-to-noise ratio we are currently testing additives (soluble carboxymethylated dextran to block non-specific binding to sensor surface), and flat surface sensor chips with self assembled monolayers and polymer brushes with shortened linkers (oligoethylene glycol). The CHAVI consortium has also provided us with 2F5 IgA, and 2F5 IgM and these 2F5 isotypes are being currently used to determine LOD in mucosal samples from healthy controls. The sensitivity of SPR assay in terms of LOD will be compared to conventional assays for antibody detection. (ii) Proteomic analysis of microparticles released by apoptotic cells during HIV infection. In acute HIV infection, autologous neutralizing antibody responses are delayed and appear much later after viral load ramp up. Recent work suggests that this delay could be due to massive immune cell apoptosis since all of the markers of apoptosis (Fas/FasL, TNFR2, TRAIL) are elevated during viral load ramp-up in the plasma of AHI patients (Smith &Haynes, unpublished data). It has been reported that such massive apopotosis is immunosuppressive;it promotes TGF-_ 1 secretion via phosphatidyleserine (PS) dependent ingestion of apoptotic cells (Huynh et al., 2002, J. Clin. Invest., 109:41) and could also inhibit T and B cell responses (Hoffman et al., 2005, J. Immunol., 174:1393;Fadok et al., 2005, J. Immunol., 174:1393). Furthermore, the released apoptotic microparticles themselves can bind to non-apoptotic cells and induce cell death (Distler et al., 2005, Apoptosis 10:731) and proinflammatory responses (Distler et al., 2005, Proc. Natl. Acad. Sci., 102:2892;Cerri etal., J. Immunol., 2006,177:1975). Thus in order to follow the apoptotic process during the early phases of AMI and to determine the fate of immune cell subpopulations we are using a proteomic approach in the analysis of microparticles. To address this issue we are initially comparing the proteomes of cell specific microparticles in order to distinguish parental cell protein expression profiles of T cell, B cell, macrophage, NK cells, monocytes. This will be followed by analysis of human plasma microparticles to identify the cellular origin of the released microparticles. We have shown that the proteomes of microparticles generated from T cells (Jurkat T cells) and human B cells from peripheral blood is vastly different (Figure 2). Several unique protein peaks were identified in these two groups of microparticles and validation of these results are currently on-going. Together with phenotypic functional analysis, the greater sensitivity of the proteomic approach has the potential to identify additional protein markers that could be useful in understanding the fate of immune cells during acute HIV infection. The differentially expressed proteins will be candidate host cell specific markers, and possibly targets for molecular intervention and/or therapy. (ii) Characterization of antigen specific T cell microparticles using pMHC tetramers. Microparticles released from immune cells upon activation or apoptosis differ quantitatively and qualitatively and vary depending upon the inducing stimulus (Jimenez et al., Thromb. Res., 2003;Distler et al., 2005;Proc.Natl. Acad. Set., 102:2892;Kolowas et al., 2005;Scand. J. Immunol., 61:226). Since there particles carry membrane derived from the parental cell (Figure 3A, 3B), characterization of surface antigens can be used to identify the cellular source of the microparticles. Microparticles have been reported to contain surface proteins of the immunoglobulin family (TCR, BCR), glycosyphosphatidylinositol- (GPI-) anchored molecules and members of the tetraspan family (Koopman et al., 1994, Blood, 84:1415;Denzer et al., 2000;J. Cell Sc/., 113:3336). For T cells, Src kinases, Lck and Fyn, and the adapter protein LAT are associated with lipid micrdomains and are the key components involved in T cell signaling (Zhang et al., 1998;Immunity, 9:239;Resh et al., 1997;Nature, 386:617;Schade &Levine, 2002;Biochem. Biophys. Res.Commun., 296:637). The SPR assay that we have developed is being used to detect antigen specific T cells using peptide-MHC (pMHC) tetramers or monomers. In our initial studies, we have used T cell microvesicles isolated from OT-1 T cells, and Jurkat T cells. We have demonstrated antigen specific binding of T cell microvesicles to MHC monomers and tetramers (Figure 3C). However, limitation of cell numbers is a potential hurdle to the application of this assay. The limit lies largely in the isolation technique and less in the sensitivity of tetramer binding. In our modified protocols for microvesicle isolation, we have determined that microvesicles isolated from 50,000 T cells can be used to detect antigen specific binding to MHC tetramers. This will be further refined using antibody or CT toxin conjugated magnetic beads to reduce the number of cells required for MHC tetramer binding. However the strength of the SPR assay lies in being able to provide quantitative data by measuring the avidity of TCR binding. Using the Ova specific OT-1 system, we have determined that off-rate measurements of monomeric pMHC binding to T cell microvesicles can distinguish between low and high avidity TCR. Furthermore, the methodologies described will allow us to study the correlation of protein phosphorylation status identified in microparticles with immune T cell activation or HIV infection. The strategy that we are employing include first the characterization of T cell microparticles in terms of antigen specificity using MHC tetramers, and then subjected to further characterization of the functional status (e.g. phosphorylation) of the identified antigen specific microparticles. The MHC tetramer captured microparticles will be eluted from the BIAcore sensor surface using the BIAcore 3000 recover capability. The eluted particles will then be lysed and then the phosphorylation status determined by: (a) immunoblotting with anti-signaling molecule antibodies (Src kinases, Lck, Fyn, LAT);anoVor b) 2D-LC followed by identification by mass spectrometry.

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