Biomedical subjects

Periasamy Selvaraj

Publications and source records attributed to Periasamy Selvaraj.

13 recordsLinked to original sources

PubMed2007

Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

Animals↗

PubMed2006

Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles.

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.

Animals↗

PubMed2006

Probabilistic modeling of rosette formation.

Rosetting, or forming a cell aggregate between a single target nucleated cell and a number of red blood cells (RBCs), is a simple assay for cell adhesion mediated by specific receptor-ligand interaction. For example, rosette formation between sheep RBC and human lymphocytes has been used to differentiate T cells from B cells. Rosetting assay is commonly used to determine the interaction of Fc gamma-receptors (FcgammaR) expressed on inflammatory cells and IgG coated on RBCs. Despite its wide use in measuring cell adhesion, the biophysical parameters of rosette formation have not been well characterized. Here we developed a probabilistic model to describe the distribution of rosette sizes, which is Poissonian. The average rosette size is predicted to be proportional to the apparent two-dimensional binding affinity of the interacting receptor-ligand pair and their site densities. The model has been supported by experiments of rosettes mediated by four molecular interactions: FcgammaRIII interacting with IgG, T cell receptor and coreceptor CD8 interacting with antigen peptide presented by major histocompatibility molecule, P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1), and L-selectin interacting with PSGL-1. The latter two are structurally similar and are different from the former two. Fitting the model to data enabled us to evaluate the apparent effective two-dimensional binding affinity of the interacting molecular pairs: 7.19x10(-5) microm4 for FcgammaRIII-IgG interaction, 4.66x10(-3) microm4 for P-selectin-PSGL-1 interaction, and 0.94x10(-3) microm4 for L-selectin-PSGL-1 interaction. These results elucidate the biophysical mechanism of rosette formation and enable it to become a semiquantitative assay that relates the rosette size to the effective affinity for receptor-ligand binding.

Animals↗

PubMed2006

B7-1-HSA (CD80-CD24), a recombinant hybrid costimulatory molecule retains ligand binding and costimulatory functions.

Optimal activation of naïve T lymphocyte requires two signals; an antigen-specific signal initiated by engagement of TCR with the antigen-MHC complex and a costimulatory signal independent of the antigen receptor complex. Without the costimulatory signal, T cells become anergic. Various adhesion molecules, such as B7-1 (CD80) and heat stable antigen (HSA, CD24), expressed on antigen presenting cells have been demonstrated to provide costimulatory signals to T cells. It was reported that the combinations of different adhesion molecules could induce even stronger immune response. In this study, we made a hybrid costimulatory molecule, B7-1-HSA, and tested its T cell stimulatory function. Chinese hamster ovary (CHO) cells expressing this hybrid molecule bound both anti-CD80 and anti-CD24 monoclonal antibodies, and induced stronger T cell proliferation than CHO cells expressing B7-1 or HSA alone. These results suggest that the B7-1-HSA hybrid molecule can deliver two costimulatory signals simultaneously that can synergize in inducing T cell proliferation. The purified B7-1-HSA protein reacted with both anti-B7-1 and anti-HSA mAbs in Western blotting and specifically mediated adhesion of Jurkat cells. Furthermore, purified B7-1-HSA molecule spontaneously incorporated onto cell membrane through its glycolipid anchor suggesting that this hybrid costimulatory molecule can be used in protein transfer to develop effective cancer vaccines.

Animals↗

PubMed2005

Human tumor membrane vesicles modified to express glycolipid-anchored IL-12 by protein transfer induce T cell proliferation in vitro: a potential approach for local delivery of cytokines during vaccination.

Administration of soluble human interleukin-12 (hIL-12) has been shown to induce a potent anti-tumor response. However, the use of soluble hIL-12 is hindered by its cytotoxicity when systemically administered and the difficulty of transferring multiple genes into primary tumor cells. In this study, we developed a membrane-anchored hIL-12 and expressed it on tumor membrane vesicles to deliver and confine IL-12 to the vaccination site. We constructed a glycolipid-anchored hIL-12 (GPI-hIL-12) by fusing the coding region of p35 and p40 subunits of hIL-12 with the GPI-signal sequence of CD59 at the C-terminal ends. The two subunits were processed correctly and expressed as a GPI-anchored disulfide-linked heterodimeric protein on the cell surface. GPI-hIL-12 cells induced proliferation of activated T cells and augmentation of allogeneic T cell generation in an MLR assay. Purified GPI-hIL-12 was efficiently intercalated onto isolated tumor cell membrane vesicles prepared from various human tumor cell lines. Further, the incorporation of GPI-hIL-12 onto tumor membrane vesicles induced proliferation of T cells and the release of IFN-gamma by activated T cells. Notably, GPI-hIL-12 enhanced the proliferative response initiated by CD80, a principal costimulatory molecule for T cell activation. These studies suggest that tumor membrane vesicles modified with GPI-anchored cytokines can be used to create potent immunogenic tumor vaccines for use in human immunotherapy. Since protein transfer can be used to modify tumor membrane vesicles obtained from surgical specimens, this approach offers a useful alternative to gene therapy as a means of developing cancer vaccines.

Animals↗

PubMed2005

Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain.

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.

Animals↗

PubMed2005

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification.

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).

Animals↗

PubMed2005

Signal-specific activation and regulation of human neutrophil Fc gamma receptors.

FcgammaRs with the ITIM domain have been shown to regulate the inflammatory signal delivered by the ITAM-containing FcgammaRs. In this study, we demonstrate that the function of human neutrophil FcgammaR type IIA (CD32A) is regulated in a distinct manner by different cell activation signals at the ligand-binding stage. Activation of neutrophils with fMLP up-regulated the ligand-binding function of CD32A, whereas PMA-mediated activation completely abolished ligand binding without altering CD32A expression. Furthermore, PMA treatment also abolished CD16B-dependent ligand binding irrespective of the level of expression. The effect of PMA was cell type specific, because the ligand-binding function of CD32A expressed on cultured cells such as K562 and CHO-CD32A transfectants was not affected by PMA. Interestingly, phorbol 12,13-dibutyrate, another phorbol ester, and IL-8 up-regulated CD32A-dependent ligand-binding function. These results demonstrate that regulation of CD32A-dependent ligand binding in human neutrophils is not only cell type specific but also activation signal specific. Moreover, these results suggest the possibility that signals delivered to neutrophils by various inflammatory stimuli can exert opposing effects on the function of human FcgammaRs, representing a novel inside-out regulatory mechanism of FcgammaR ligand binding.

Animals↗

PubMed2004

Functional regulation of human neutrophil Fc gamma receptors.

Interaction between Fc receptors expressed on phagocytic cells and antibodies play a critical role in innate immune response. Interestingly, immune cells such as neutrophils, monocytes, and dendritic cells (DCs) express multiple Fc receptors for IgG (FcgammaR) with overlapping ligand specificity. These receptors compete for the same ligand on the target and are known to transduce positive and negative signals to the same cell, depending on presence of type of signaling motif in their cytoplasmic domain. Neutrophils, the first line of defense against bacterial infection and the major phagocytic cell in the blood, express two types of FcgammaRs depending on the species. In humans, the neutrophils co-express immunoreceptor tyrosine-based activation motif (ITAM) containing CD32A and glycosyl-phosphatidyl inositol (GPI)-anchored CD16B, which is in contrast to co-expression of ITAM containing CD16A and ITIM containing CD32B in mouse neutrophils. Recent studies in gene knockout mice have demonstrated that the negative signaling by CD32B plays a critical role in preventing immune complex (IC)-mediated autoimmune diseases by regulating the activation signal delivered by CD16A. However, it is not known how the function of ITAM signaling CD32A is regulated in human neutrophils. Recent observations from our laboratory suggest that in human neutrophils, the CD32A receptor is regulated at the ligand-binding stage. Using a CD16B-deficient donor, we found that the CD32A expressed on resting neutrophils is unable to bind ligand; however, once neutrophils are activated with fMLP, a bacterial chemotactic peptide, the CD32A is functionally active in binding ligand. We also observed that this regulation is neutrophil-specific phenomenon. These observations suggest that FcgammaR can be regulated by distinct mechanisms and factors such as membrane-anchoring, cell-specific signaling, and avidity modulation that may be coordinately involved in regulating the function of human FcgammaR. Because neutrophils may be activated during infectious and inflammatory diseases, the knowledge of functional regulation of FcgammaR will be useful in designing therapies for many autoimmune diseases.

Animals↗

PubMed2004

Cancer vaccine development: protein transfer of membrane-anchored cytokines and immunostimulatory molecules.

Many tumor cells escape host-immune recognition by the downregulation or lack of immunostimulatory molecules. Expression of immunostimulatory molecules on tumor cells by gene transfer can be used to induce an antitumor immune response. However, we have previously shown that protein transfer of glycosyl-phosphatidylinositol (GPI)-linked costimulatory molecules is a successful alternative to traditional gene transfer in preparing such a tumor vaccine. Vaccination with membranes modified by protein transfer to express GPI-linked B7.1 (CD80), a costimulatory adhesion molecule, induces protective immunity in mice and allogeneic antitumor T-cell proliferation in humans in vitro. Our goal is to develop an optimal tumor vaccine using tumor membranes modified by protein transfer to target and stimulate antigen-presenting cells (APCs) and T cells. We have investigated the efficacy of expressing GPI-anchored cytokine molecules on the surface of tumor cells. Expression of interleukin-12 (IL-12) on tumor-cell membranes in a GPI-anchored form induces a strong antitumor immune response that is comparable to the effects of secretory IL-12. Because many cytokines act synergistically, we are testing the membrane expression and immunostimulatory effects of cytokines individually as well as in combination to determine potential complementary effects of coexpression on the antitumor immune response. Ultimately, the protein-transfer vaccination may be used in humans alone or in multimodal combination therapies to induce tumor regression and to serve as a protective measure to prevent postsurgical secondary metastases.

Adjuvants, Immunologic↗

PubMed2002

Glycolipid-anchored IL-12 expressed on tumor cell surface induces antitumor immune response.

Systemic or local administration of cytokine has been used as a mode to enhance the antitumor immune response induced by many cancer vaccines. We have investigated whether the expression of cytokines on the tumor cell surface as a glycolipid (GPI)-anchored form will be effective in inducing antitumor immune response using a GPI-anchored interleukin (IL)-12 (GPI-IL-12) as a model. GPI-IL-12-induced the proliferation of concanavalin A-activated T cells and induced IFN-gamma secretion by activated and allogeneic T cells, indicating that the membrane-expressed IL-12 can stimulate T cells. GPI-IL-12 expressed on the tumor cell surface prevented tumor growth in mice in a highly tumorigenic murine mastocytoma model. These results suggest that the cell surface-expressed GPI-IL-12 can be effective in inducing antitumor immune response, and GPI-anchored cytokines expressed on the tumor cell surface may be a novel approach to deliver cytokines at the immunization site during vaccination against cancer. Furthermore, purified GPI-anchored cytokines can be used to quickly modify tumor membranes by the protein transfer method to express the desired cytokines for vaccine development.

Animals↗

PubMed2002

Recombinant CD16A-Ig forms a homodimer and cross-blocks the ligand binding functions of neutrophil and monocyte Fcgamma receptors.

Receptors for the Fc region of IgG (FcgammaR) are expressed as membrane-bound and soluble forms in inflammatory cells. These receptors mediate a variety of immunoregulatory functions. FcgammaR-bearing neutrophils and macrophages play a major role in mediating immune complex induced inflammatory diseases and antibody-mediated tissue injury in autoimmune diseases. To delineate the biological role of the soluble FcgammaR, a recombinant soluble CD16A (CD16A-Ig) was expressed and characterized. CD16A-Ig is secreted as a disulfide-linked homodimer of 70kDa. The purified CD16A-Ig bound to human IgG1 and IgG3 immobilized onto ELISA plates and on IgG1- and IgG3-coated erythrocytes. Saturation binding studies and Scatchard plot analysis showed that immune complex bound to the purified CD16A-Ig with high avidity. Moreover, CD16A-Ig competitively blocked the binding of cell surface-expressed CD16A-CHO cells to IgG-coated plates. The dimeric CD16A-Ig also efficiently cross-blocked the binding of IgG-coated target cells to other FcgammaR expressed on neutrophils and monocytes. These results demonstrate that CD16A-Ig forms a high avidity dimer and is capable of blocking cell-cell interactions mediated by inflammatory cells expressing FcgammaR and IgG-coated target cells. These findings suggest that the dimeric FcgammaR molecules could be used therapeutically for the intervention of immune complex-mediated inflammatory disease.

Animals↗

PubMed2002

GPI-anchoring of GM-CSF results in active membrane-bound and partially shed cytokine.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10-15% of all membrane-bound GM-CSF after 72h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.

Animals↗