Regulation of leukocyte cytokine production by inhibitors of intracellular signalling pathways

The activation of leukocytes during inflammation results in the engagement of many related intracellular signalling pathways. The role of two key proteins families, phosphodiesterase (PDE) and Src kinase, has been assessed in these signalling pathways using specific inhibitors. Inhibition of phosphodiesterase type 4 (PDE4) in lipopolysaccharide (EPS) stimulated human peripheral blood mononuclear cells (PBMC) resulted in the inhibition of TNF-a by an IL-10-independent mechanism. This is in contrast to other agents that elevate cAMP which in turn inhibit TNF-a synthesis by an IL-10-dependent mechanism. When PBMC were stimulated with EPS plus IFN-y, inhibition of PDE4 resulted in an altered effect on cytokine production, elevating IE-10 while still inhibiting TNF-a. Inhibition of PDE4 in activated T cells only weakly inhibited cellular proliferation. In contrast, PDE4 inhibitors were potent but non-selective inhibitors of T-cell cytokine production. In contrast to studies in murine cells, inhibition of T-cell activation correlates with inhibition of binding to a high-affinity Rolipram binding site on the PDE4 protein. Kinases from the Src family (Eck, Fyn and Eyn) have been implicated in signalling via the T-cell receptor (TCR) on lymphocytes and the high affinity IgE receptor (FceRI) on mast cells. Potent inhibitors of Src kinases blocked T-cell proliferation and cytokine generation in response to specific ligation of the TCR. However, activation of T-cells by multiple costimulatory pathways was much more resistant to Src kinase inhibition. Inhibition of Src kinase in human cord blood-derived mast cells inhibited IgE-dependent degranulation of these cells. This indicates that Src kinase inhibitors may be useful in down-regulating allergic responses. Conversely, inhibitors of PDE4 and PDE type 7 (PDE7) did not prevent mast cell degranulation. The observation that PDE4 inhibitors can regulate monocyte cytokine generation further supports the view that these agents could have therapeutic benefit in a number of inflammatory diseases such as asthma, arthritis and inflammatory bowel disease. The failure of Src kinase inhibitors to modify T cell signalling activated by multiple pathways indicates redundancy in these signalling pathways and therefore Src kinases may not represent good targets for immunomodulation. However, a selective Src kinase inhibitor could represent a good target for an anti-allergic drug

Small Molecules That Inhibit Tnf Signalling by Stabilising an Asymmetric Form of the Trimer

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn\u27s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions

Cytokines and Inflammatory Mediators [30-39]: 30. The LPS Stimulated Production of Interleukin-10 is not Associated with -819C/T and -592C/A Promoter Polymorphisms in Healthy Indian Subjects

Background: Interleukin-10 is a pivotal immunoregulatory cytokine with pleiotropic effects on the immune system. IL-10 promoter polymorphisms have been associated with disease susceptibility and the ability to secrete IL-10 in vitro. We suspected that the association of the widely studied -819C/T and -592C/A polymorphisms with the IL-10 production might vary between ethnic groups. Therefore, we examined the association of -819 C/T and -592 C/A promoter polymorphisms with in vitro LPS stimulated secretion of IL-10 in normal healthy Indian volunteers. Methods: Peripheral blood was collected from 103 healthy volunteers and diluted whole blood cultures were set up with 100 ng/ml of LPS as stimulant: supernatant was collected at 24 h and IL-10 levels were assayed by ELISA. Genotyping was done for -819C/T polymorphism in 101 individuals and -592C/A polymorphism in 68 individuals by polymerase chain reaction followed by RFLP. The differences in IL-10 production between the genotypes were analysed by ANOVA. Results: There were 30, 47 and 24 individuals with the CC, CT and TT genotypes with a minor allele (T) frequency of 47% for the -819C/T polymorphism. The CC and TT genotypes at position -819 were strongly associated with CC and AA genotypes at -592 position suggestive of strong linkage disequilibrium. There was no association between the -819 genotype and the in vitro LPS stimulated IL-10 levels. Conclusions: The -819C/T and the -592 C/A polymorphisms of the IL-10 promoter region are not significantly associated with LPS stimulated IL-10 production healthy Indian subjects. Disclosure statement: All authors have declared no conflicts of interes

Inhibition of Human T Cell Activation by Novel Src Kinase Inhibitors Is Dependent upon the Complexity of the Signal Delivered to the Cell

ABSTRACT The activity of a novel series of protein tyrosine kinase inhibitors that are selective for the Src family has been assessed. The activity of these compounds [named CT-SKI (Celltech Src kinase inhibitors)] was investigated by assessing their potential to modulate T cell receptor activation, an event thought to involve the Src kinases Lck and Fyn. This series of compounds contained low-nanomolar inhibitors of Src kinases with selectivity over Csk, epidermal growth factor receptor kinase, protein kinase C, and -associated 70-kDa protein. These compounds were shown to attenuate anti-CD3-induced T cell proliferation and block interleukin (IL)-2, IL-4, and interferon-␥ production, and CD25 expression in anti-CD3-activated T cells. In addition, inhibition of anti-CD3-induced, but not phorbol ester and calcium ionophore-induced IL-2 production, correlated with inhi

Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM

Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the IL-25-specific IgG⁺ B cell population.

<p>Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the IL-25-specific IgG⁺ B cell population.</p

Schematic representation of the single-B cell sorting protocol used for antibody discovery from immunised mice.

<p>The following steps were undertaken: 1. Mice were immunised and a splenocyte suspension prepared. 2. B cells from the mouse splenocyte preparation were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. 3. Following enrichment, cells were stained with the following antibodies: rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences)(dump channel). Human TNFR2 extracellular domain was labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) and added to the cell suspension. 4. FACS was performed on a BD FACS ARIA III with single human TNFR2-specific IgG⁺ B cells being deposited into the well of a 96-well PCR plate. 5. cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR followed by a third round to generate transcriptionally-active PCR (TAP) products in a manner similar to that described in Clargo <i>et al</i>.⁹ employing an Aviso Onyx liquid handling robot to facilitate set-up. 6. Heavy and light chain TAP fragments were transiently co-transfected into Expi293 cells using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested for further characterisation.</p

Gating strategy for identification of antigen-specific rabbit memory B cells.

<p>Cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 59.8% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.7% of events) (B). 7AAD⁺ dead cells and T cells were eliminated in the “dump channel” (gated population represented 97.1% of events) (C). IgG⁺/ IgM^- B cells were identified and gated on (gated population represented 2.67% of events) (D). Finally, a gate (P1) was drawn around the double-positive mWISP-1 antigen-specific population (gated population represented 0.262% of events) (E). Cells from gate P1 were sorted into a 96-well PCR plate at either one or three cells per well for subsequent RT-PCR.</p

Schematic representation of the single-B cell sorting protocol used for antibody discovery from immunised mice.

<p>The following steps were undertaken: 1. Mice were immunised and a splenocyte suspension prepared. 2. B cells from the mouse splenocyte preparation were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. 3. Following enrichment, cells were stained with the following antibodies: rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences)(dump channel). Human TNFR2 extracellular domain was labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) and added to the cell suspension. 4. FACS was performed on a BD FACS ARIA III with single human TNFR2-specific IgG⁺ B cells being deposited into the well of a 96-well PCR plate. 5. cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR followed by a third round to generate transcriptionally-active PCR (TAP) products in a manner similar to that described in Clargo <i>et al</i>.⁹ employing an Aviso Onyx liquid handling robot to facilitate set-up. 6. Heavy and light chain TAP fragments were transiently co-transfected into Expi293 cells using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested for further characterisation.</p

Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the total IgG⁺ population and the TNFR2-specific subset of cells.

<p>Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the total IgG⁺ population and the TNFR2-specific subset of cells.</p