Gyan C. Mishra

B Cells Activated by Lipopolysaccharide, But Not By Anti-Ig and Anti-CD40 Antibody, Induce Anergy in CD8+ T Cells: Role of TGF-β1

B cells recognize Ag through their surface IgRs and present it in the context of MHC class II molecules to CD4+ T cells. Recent evidence indicates that B cells also present exogenous Ags in the context of MHC class I to CD8+ T cells and thus may play an important role in the modulation of CTL responses. However, in this regard, conflicting reports are available. One group of studies suggests that the interaction between B cells and CD8+ T cells leads to the activation of the T cells, whereas other studies propose that it induces T cell tolerance. For discerning this dichotomy, we used B cells that were activated with either LPS or anti-Ig plus anti-CD40 Ab, which mimic the T-independent and T-dependent modes of B cell activation, respectively, to provide accessory signals to resting CD8+ T cells. Our results show that, in comparison with anti-Ig plus anti-CD40 Ab-activated B cells, the LPS-activated B cells (LPS-B) failed to induce significant levels of proliferation, cytokine secretion, and cytotoxic ability of CD8+ T cells. This hyporesponsiveness of CD8+ T cells activated with LPS-B was significantly rescued by anti-TGF-β1 Ab. Moreover, it was found that such hyporesponsive CD8+ T cells activated with LPS-B had entered a state of anergy. Furthermore, LPS-B expresses a significantly higher level of TGF-β1 on the surface, which caused the observed hyporesponsiveness of CD8+ T cells. Therefore, this study, for the first time, provides a novel mechanism of B cell surface TGF-β1-mediated hyporesponsiveness leading to anergy of CD8+ T cells.

Evidence That Glycoprotein 96 (B2), a Stress Protein, Functions as a Th2-Specific Costimulatory Molecule

After the engagement of Ag receptor, most of the Th cells for their optimal activation require a second (costimulatory) signal provided by the APCs. We demonstrate the isolation and characterization of a 99- to 105-kDa protein (B2), from LPS-activated B cell surface, and its function as a Th2-specific costimulatory molecule. Appearance of B2 as a single entity on two-dimensional gel electrophoresis and as a distinct peak in reverse-phase HPLC ascertains the fact that B2 is homogeneous in preparation. Electron microscopy as well as competitive binding studies reveal that 125I-labeled B2 specifically binds anti-CD3-activated T cell surface and also competes with its unlabeled form. Internal amino acid sequences of B2 are found to be identical with stress protein gp96. The identity of B2 as gp96 is also revealed by immunological characterization and by confocal microscopic colocalization studies of B2 and gp96 on LPS-activated B cells. Confocal imaging studies also demonstrate that gp96 can be induced on B cell surface without association of MHC molecules. Furthermore, the novel role of gp96 in Th cell proliferation skewing its differentiation toward Th2 phenotype has also been established. Ab-mediated blocking of gp96-induced signaling not only abrogates in vitro proliferation of CD4+ T cells, but also diminishes the secretion of Th2-specific cytokines. Notably, the expression of CD91 (receptor of gp96/B2) is up-regulated on anti-CD3-activated Th cells and also found to be present on Th1 and Th2 subsets.

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0.1 mg/ml. The APTES modified, protein G coated crystal were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4 degrees C in a desiccator.

Differential CD40/CD40L Expression Results in Counteracting Antitumor Immune Responses

Establishment of host-protective memory T cells against tumors is the objective of an antitumor immunoprophylactic strategy such as reinforcing T cell costimulation via CD40-CD40L interaction. Previous CD40-targeted strategies assumed that T cell costimulation is an all-or-none phenomenon. It was unknown whether different levels of CD40L expression induce quantitatively and qualitatively different effector T cell responses. Using mice expressing different levels of CD40L, we demonstrated that the greater the T cell CD40L expression the less tumor growth occurred; the antitumor T cell response was host-protective. Lower levels of CD40L expression on T cells induced IL-10-mediated suppression of tumor-regressing effector CD8+ T cells and higher productions of IL-4 and IL-10. Using mice expressing different levels of CD40 or by administering different doses of anti-CD40 Ab, similar observations were recorded implying that the induction of protumor or antitumor T cell responses was a function of the extent of CD40 cross-linking. IL-10 neutralization during priming with tumor Ags resulted in a stronger tumor-regressing effector T cell response. Using IL-10−/− DC for priming of mice expressing different levels of CD40L and subsequent transfer of the T cells from the primed mice to nu/nu mice, we demonstrated the protumor role of IL-10 in the induction of tumor-promoting T cells. Our results demonstrate that a dose-dependent cross-linking of a costimulatory molecule dictates the functional phenotype of the elicited effector T cell response. The T cell costimulation is a continuum of a function that induces not only graded T cell responses but also two counteracting responses at two extremes.

Development of piezoelectric crystal based microgravimetric immunoassay for determination of insulin concentration.

A microgravimetric, piezoelectric crystal based immunoassay for the quantification of insulin concentration is described. The method utilizes a modified piezoelectric crystal device having an antibody specific to insulin bound to its surface. The antibody to insulin was immobilized on the surface of crystal electrode by using either 3-aminopropyltriethoxy silane (3-APTES), polyethyleneimine (PEI) or covalently coupled protein A-gold immobilization method. Coating an electrode with a cross linked protein A-antibody complex gave better results in terms of sensitivity and stability. Using the system described, the insulin concentration up to 1 ng ml-1 could be detected. The stability and reusability of the system was further improved by using a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized insulin antibody. Scanning tunneling microscopic (STM) study was also done to confirm the surface coverage and orientation of insulin and antibody molecules on the modified piezoelectric crystal electrode surface. A comparison between the present study and the well-established radioimmunoassay technique (RIA) revealed that the described microgravimetric immunoassay technique (MIA) could successfully be developed as an alternative of RIA.

Determination of immunoglobulin M concentration by piezoelectric crystal immunobiosensor coated with protamine

In the present study, the specific binding between protamine and immunoglobulin M (IgM) has been exploited to construct a piezoelectric crystal based immunobiosensor for the determination of concentration of IgM. The system consisted of highly stable IC based oscillator, 8-digit frequency counter and modified piezoelectric crystal device. The crystal surface was physically modified and chemically treated (refluxed) with strong acid to produce stable hydroxylic groups of silicon oxide. This modified surface reacted strongly with coupling reagents for binding of protein molecules. The protamine was immobilized by using either gamma-aminopropyltriethoxy silane (gamma-APTES) or 2.2.2-trifluoroethanesulfonyl chloride (tresyl chloride). Scanning electron microscope images of piezo crystal revealed that tresyl activated surface presented more surface area for binding than gamma-APTES modified surface and showed better sensitivity. This immobilization technique also improved the reproducibility and long term stability of the detection system. Using the system described, the IgM concentration up to the level of 10 ng/ml could be detected without interference of IgG.

Functional Dichotomy in CD40 Reciprocally Regulates Effector T Cell Functions

Activation of T cells requires signals through Ag-specific TCR and costimulatory molecules such as CD40L. Although the use of defined tumor Ags for the induction of protective T cells met with limited success, the CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely. Using a model for prostate tumor, a leading cause of tumor-induced mortality in men, we show that the failure is due to a novel functional dichotomy of CD40 whereby it self-limits its antitumor functions by inducing IL-10. IL-10 prevents the CD40-induced CTL and TNF-α and IL-12 production, Th1 skewing, and tumor regression. Priming mice with tumor lysate-pulsed IL-10-deficient dendritic cells (DCs) or wild-type DC plus anti-IL-10 Ab establishes antitumor memory T cells that can transfer the protection into syngenic nude mice. Infusion of Ag-pulsed IL-10-deficient but not wild-type DCs back into syngenic mice results in successful therapeutic autovaccination. Thus, we demonstrate the IL-10-sensitive antitumor T cell memory formulating a novel prophylactic and therapeutic principle.

Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Bone Loss in Collagen-Induced Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis leading to joint destruction and systemic bone loss. The inflammation-induced bone loss is mediated by increased osteoclast formation and function. Current antirheumatic therapies primarily target suppression of inflammatory cascade with limited or no success in controlling progression of bone destruction. Mesenchymal stem cells (MSCs) by virtue of their tissue repair and immunomodulatory properties have shown promising results in various autoimmune and degenerative diseases. However, the role of MSCs in prevention of bone destruction in RA is not yet understood. In this study, we investigated the effect of adipose-derived MSCs (ASCs) on in vitro formation of bone-resorbing osteoclasts and pathological bone loss in the mouse collagen-induced arthritis (CIA) model of RA. We observed that ASCs significantly inhibited receptor activator of NF-κB ligand (RANKL)–induced osteoclastogenesis in both a contact-dependent and -independent manner. Additionally, ASCs inhibited RANKL-induced osteoclastogenesis in the presence of proinflammatory cytokines such as TNF-α, IL-17, and IL-1β. Furthermore, treatment with ASCs at the onset of CIA significantly reduced clinical symptoms and joint pathology. Interestingly, ASCs protected periarticular and systemic bone loss in CIA mice by maintaining trabecular bone structure. We further observed that treatment with ASCs reduced osteoclast precursors in bone marrow, resulting in decreased osteoclastogenesis. Moreover, ASCs suppressed autoimmune T cell responses and increased the percentages of peripheral regulatory T and B cells. Thus, we provide strong evidence that ASCs ameliorate inflammation-induced systemic bone loss in CIA mice by reducing osteoclast precursors and promoting immune tolerance.

Leishmania donovani Infection of a Susceptible Host Results in Apoptosis of Th1‐Like Cells: Rescue of Anti‐Leishmanial CMI by Providing Th1‐Specific Bystander Costimulation

A protective immune response against Leishmania donovani infection is mediated by T‐helper type 1 (Th1) cells. Th1 induced cell‐mediated immunity (CMI), as assessed by anti‐leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down‐regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite downregulates the expression of a Th1‐specific costimulatory molecule, M150, on the surface of infected BALB/c mice‐derived macrophages. Th cells are rendered unresponsive to anti‐CD3 Ab‐mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL‐2, IL‐4 and IFN‐γ compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti‐CD3 Ab induced unresponsiveness and IFN‐γ but not IL‐4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.

The Th1-Specific Costimulatory Molecule, M150, Is a Posttranslational Isoform of Lysosome-Associated Membrane Protein-1

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4+ T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4+ T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.