CD1a promotes systemic manifestations of skin inflammation

Inflammatory skin conditions are increasingly recognised as being associated with systemic inflammation. The mechanisms connecting the cutaneous and systemic disease are not well understood. CD1a is a virtually monomorphic major histocompatibility complex (MHC) class I-like molecule, highly expressed by skin and mucosal Langerhans cells, and presents lipid antigens to T-cells. Here we show an important role for CD1a in linking cutaneous and systemic inflammation in two experimental disease models. In human CD1a transgenic mice, the toll-like receptor (TLR)7 agonist imiquimod induces more pronounced splenomegaly, expansion of the peripheral blood and spleen T cell compartments, and enhanced neutrophil and eosinophil responses compared to the wild-type, accompanied by elevated skin and plasma cytokine levels, including IL-23, IL-1α, IL-1β, MCP-1 and IL-17A. Similar systemic escalation is shown in MC903-induced skin inflammation. The exacerbated inflammation could be counter-acted by CD1a-blocking antibodies, developed and screened in our laboratories. The beneficial effect is epitope dependent, and we further characterise the five best-performing antibodies for their capacity to modulate CD1a-expressing cells and ameliorate CD1a-dependent systemic inflammatory responses. In summary, we show that a therapeutically targetable CD1a-dependent pathway may play a role in the systemic spread of cutaneous inflammation

Inclusive jet cross sections and dijet correlations in D∗±D^{*\pm} photoproduction at HERA

Inclusive jet cross sections in photoproduction for events containing a $D^*$ meson have been measured with the ZEUS detector at HERA using an integrated luminosity of $78.6 {\rm pb}^{-1}$. The events were required to have a virtuality of the incoming photon, $Q^2$, of less than 1 GeV$^2$, and a photon-proton centre-of-mass energy in the range $130<W_{\gamma p}<280 {\rm GeV}$. The measurements are compared with next-to-leading-order (NLO) QCD calculations. Good agreement is found with the NLO calculations over most of the measured kinematic region. Requiring a second jet in the event allowed a more detailed comparison with QCD calculations. The measured dijet cross sections are also compared to Monte Carlo (MC) models which incorporate leading-order matrix elements followed by parton showers and hadronisation. The NLO QCD predictions are in general agreement with the data although differences have been isolated to regions where contributions from higher orders are expected to be significant. The MC models give a better description than the NLO predictions of the shape of the measured cross sections.Comment: 43 pages, 12 figures, charm jets ZEU

Dissociation of virtual photons in events with a leading proton at HERA

The ZEUS detector has been used to study dissociation of virtual photons in events with a leading proton, gamma^* p -> X p, in e^+p collisions at HERA. The data cover photon virtualities in two ranges, 0.03<Q^2<0.60 GeV^2 and 2<Q^2<100 GeV^2, with M_X>1.5 GeV, where M_X is the mass of the hadronic final state, X. Events were required to have a leading proton, detected in the ZEUS leading proton spectrometer, carrying at least 90% of the incoming proton energy. The cross section is presented as a function of t, the squared four-momentum transfer at the proton vertex, Phi, the azimuthal angle between the positron scattering plane and the proton scattering plane, and Q^2. The data are presented in terms of the diffractive structure function, F_2^D(3). A next-to-leading-order QCD fit to the higher-Q^2 data set and to previously published diffractive charm production data is presented

Study of deep inelastic inclusive and diffractive scattering with the ZEUS forward plug calorimeter

Deep inelastic scattering and its diffractive component, ep -> e'gamma*p ->e'XN, have been studied at HERA with the ZEUS detector using an integrated luminosity of 4.2 pb-1. The measurement covers a wide range in the gamma*p c.m. energy W (37 - 245 GeV), photon virtuality Q2 (2.2 - 80 GeV2) and mass Mx. The diffractive cross section for Mx > 2 GeV rises strongly with W; the rise is steeper with increasing Q2. The latter observation excludes the description of diffractive deep inelastic scattering in terms of the exchange of a single Pomeron. The ratio of diffractive to total cross section is constant as a function of W, in contradiction to the expectation of Regge phenomenology combined with a naive extension of the optical theorem to gamma*p scattering. Above Mx of 8 GeV, the ratio is flat with Q2, indicating a leading-twist behaviour of the diffractive cross section. The data are also presented in terms of the diffractive structure function, F2D(3)(beta,xpom,Q2), of the proton. For fixed beta, the Q2 dependence of xpom F2D(3) changes with xpom in violation of Regge factorisation. For fixed xpom, xpom F2D(3) rises as beta -> 0, the rise accelerating with increasing Q2. These positive scaling violations suggest substantial contributions of perturbative effects in the diffractive DIS cross section.Comment: 87 pages, 25 figure

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

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 the identification of antigen-specific mouse memory B cells from IL-25 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 47.6% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 99% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.8% of events) (C). CD19⁺ B cells were identified (gated population represented 99% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 1.49% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells, (gated population represented 91% of events) (F). Finally, dual-colour antigen staining allowed a gate (P1) to be drawn around the double-positive population (gated population P1 represented 0.283% of events) (G). Single cells from gate P1 were sorted into a 96-well plate for single-cell RT-PCR.</p

Gating strategy for identification of antigen-specific mouse memory B cells from TNFR2 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 34.9% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.1% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.4% of events) (C). CD19⁺ B cells were identified (gated population represented 97.2% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 0.899% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells (gated population (gate P1) represented 96.3% of events) (F). In order to demonstrate the importance of the antigen staining step, we sorted single cells from the total IgG⁺/IgD^- cell population (gate P1). Finally, dual-colour antigen staining allowed a gate (P2) to be drawn around the double-positive antigen-specific population (gated population P2 represented 0.95%of events) (G). Single cells from gate P2 were sorted into a 96-well plate for single-cell RT-PCR.</p

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

Diversity assessment of mouse anti-human IL-25 antibodies.

<p>For each antibody, VH CDR3 sequences were aligned in a pairwise manner to generate a sequence distance value. We performed a Principal Components Analysis (PCA)³⁴ as a means to reduce down the dimensionality of this data and generate an easy to interpret 2-dimensional data plot that illustrated the extent of diversity in our recombinant antibody panel. Data for principle component (PC) 1 and 2 are shown on the X and Y axis respectively. Clustering analysis was performed and families of closely related sequences were assigned on the basis of sequence identity in the VH CDR3 regions of 80% or higher. Each separate antibody family has been represented by a unique colour.</p