Despite the role of pigs as a source of new Influenza A Virus viruses (IAV) potentially capable of initiating human pandemics, immune responses to swine influenza virus (SwIV) in pigs are not fully understood. Several SwIV epitopes presented by swine MHC (SLA) class I have been identified using different approaches either in outbred pigs or in Babraham large white inbred pigs, which are 85% identical by genome wide SNP analysis. On the other hand, some class II SLA epitopes were recently described in outbred pigs. In this work, Babraham large white inbred pigs were selected to identify SLA II epitopes from SwIV H1N1. PBMCs were screened for recognition of overlapping peptides covering the NP and M1 proteins from heterologous IAV H1N1 in IFNγ ELISPOT. A novel SLA class II restricted epitope was identified in NP from swine H1N1. This conserved novel epitope could be the base for further vaccine approaches against H1N1 in pigs.

Licensed seasonal influenza vaccines induce antibody (Ab) responses against influenza hemagglutinin ( HA) that are limited in their ability to protect against different strains of influenza. Cytotoxic T lymphocytes recognizing the conserved internal nucleoprotein ( NP ) and matrix protein ( M 1) are capable of mediating a cross‐subtype immune response against influenza. Modified vaccinia A nkara ( MVA ) virus encoding NP and M 1 ( MVA‐NP+M 1) is designed to boost preexisting T ‐cell responses in adults in order to elicit a cross‐protective immune response. We examined the coadministration of HA protein formulations and candidate MVA‐NP+M 1 influenza vaccines in murine, avian, and swine models. A b responses postimmunization were measured by ELISA and pseudotype neutralization assays. Here, we demonstrate that MVA‐NP+M 1 can act as an adjuvant enhancing A b responses to HA while simultaneously inducing potent T ‐cell responses to conserved internal A gs. We show that this regimen leads to the induction of cytophilic A b isotypes that are capable of inhibiting hemagglutination and in the context of H 5 exhibit cross‐clade neutralization. The simultaneous induction of T cells and A b responses has the potential to improve seasonal vaccine performance and could be employed in pandemic situations.

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1) pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.

We have performed a series of studies to investigate the role of CD4+ T-cells in the immune response to foot-and-mouth disease virus (FMDV) post-vaccination. Virus neutralizing antibody titres (VNT) in cattle vaccinated with killed FMD commercial vaccine were significantly reduced and class switching delayed as a consequence of rigorous in vivo CD4+ T-cell depletion. Further studies were performed to examine whether the magnitude of T-cell proliferative responses correlated with the antibody responses. FMD vaccination was found to induce T-cell proliferative responses, with CD4+ T-cells responding specifically to the FMDV antigen. In addition, gamma interferon (IFN-γ) was detected in the supernatant of FMDV antigen-stimulated PBMC and purified CD4+ T-cells from vaccinated cattle. Similarly, intracellular IFN-γ could be detected specifically in purified CD4+ T-cells after restimulation. It was not possible to correlate in vitro proliferative responses or IFN-γ production of PBMC with VNT, probably as a consequence of the induction of T-independent and T-dependent antibody responses and antigen non-specific T-cell responses. However, our studies demonstrate the importance of stimulating CD4+ T-cell responses for the induction of optimum antibody responses to FMD-killed vaccines.

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.

In multiple myeloma (MM), malignant plasma cells reside in the bone marrow, where they accumulate in close contact with stromal cells. The mechanisms responsible for the chemotaxis of malignant plasma cells are still poorly understood. Thus, we investigated the mechanisms involved in the chemotaxis of MDN and XG2 MM cell lines. Both cell lines strongly expressed CCR9, CXCR3 and CXCR4 chemokine receptors but only migrated toward CXCL12. Activation of CXCR4 by CXCL12 resulted in the association of CXCR4 with CD45 and activation of PLCβ3, AKT, RhoA, IκBα and ERK1/2. Using siRNA-silencing techniques, we showed CD45/CXCR4 association is essential for CXCL12-induced migration of MM cells. Thymoquinone (TQ), the major active component of the medicinal herb Nigella sativa Linn, has been described as a chemopreventive and chemotherapeutic compound. TQ treatment strongly inhibited CXCL12-mediated chemotaxis in MM cell lines as well as primary cells isolated from MM patients, but not normal PBMCs. Moreover, TQ significantly down-regulated CXCR4 expression and CXCL12-mediated CXCR4/CD45 association in MM cells. Finally, TQ also induced the relocalization of cytoplasmic Fas/CD95 to the membrane of MM cells and increased CD95-mediated apoptosis by 80%. In conclusion, we demonstrate the potent anti-myeloma activity of TQ, providing a rationale for further clinical evaluation.

ABSTRACT The use of heat shock proteins (HSP) to enhance activation of the immune response to chaperoned antigen is being explored for immunotherapy. Hsp110 chaperones large protein substrates more effectively than Hsp70, offering the potential to use complex antigens containing multiple epitopes in HSP-based vaccines. In this study, we investigated the ability of recombinant bovine Hsp110 to chaperone E2 glycoprotein, the major envelope protein of bovine viral diarrhea virus (BVDV) and the dominant target of neutralizing antibodies. Hsp110 formed complexes with E2, as demonstrated by immunoprecipitation. When monocytes from BVDV-immunized cattle were stimulated with these complexes and incubated with autologous CD4 + T cells, enhanced levels of proliferation were observed. To determine the ability of these complexes to improve immunogenicity in vivo , cattle were vaccinated with either Hsp110-E2 complex or E2 only, combined with Quil-A adjuvant. In contrast to the in vitro data, cellular and humoral responses to E2 were greater in the E2-only vaccination group, indicating that complex formation had actually reduced the immunogenicity of E2. This study highlights the need for further understanding of the means by which HSP complexes are endocytosed and processed in vivo to enable the design of successful vaccine strategies.

ABSTRACT The role of T-lymphocyte subsets in recovery from foot-and-mouth disease virus (FMDV) infection in calves was investigated by administering subset-specific monoclonal antibodies. The depletion of circulating CD4+ or WC1+ γδ T cells was achieved for a period extending from before challenge to after resolution of viremia and peak clinical signs, whereas CD8+ cell depletion was only partial. The depletion of CD4+ cells was also confirmed by analysis of lymph node biopsy specimens 5 days postchallenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs, and immune responses following FMDV infection. Three of the four CD4+ T-cell-depleted calves failed to generate an antibody response to the nonstructural polyprotein 3ABC but generated a neutralizing antibody response similar to that in the controls, including rapid isotype switching to immunoglobulin G antibody. We conclude that antibody responses to sites on the surface of the virus capsid are T cell independent, whereas those directed against the nonstructural proteins are T cell dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1135-156 on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralizing sites on the virus capsid, are T cell dependent. The depletion of CD4+ T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. Overall, we conclude that CD4+ T-cell-independent antibody responses play a major role in the resolution of foot-and-mouth disease in cattle.

The aim of the study was to build a comprehensive picture of the appearance in the blood stream of Ag-specific plasma cells and memory B cells in the bovine model. For this purpose, we have developed a method allowing the detection and quantification of both cell types within individual calves immunised with ovalbumin. During the primary response, we detected a burst of ovalbumin-specific plasma cells at days 6 and 7 post-immunisation, which was followed by the production of specific Ab, whereas a gradual increase of memory B cells was only detected from day 15. As expected, a boost immunisation performed 7 weeks later induced a quicker and stronger secondary response. Indeed, a burst of plasma cells was detected in the blood at days 3 and 4, which was followed by a strong increase in Ab titres. Furthermore, a burst of memory B cells, and not a gradual increase, was detected at days 5 and 6 post-boost immunisation. Importantly, we showed a strong correlation between the anti-ovalbumin-specific IgG titres detected 5 months after secondary immunisation and the plasma cell numbers detected in the blood at the peak response after secondary immunisation. The detection and quantification of plasma cells following an immunisation/vaccination strategy could constitute a very effective means for predicting the magnitude and longevity of an Ab response.

B-cell-activating factor of the TNF family, (BAFF), and a proliferation-inducing ligand (APRIL) regulate B-lymphocyte survival and activation. We report that BAFF, but not APRIL, increased the chemotactic response of primary human B cells to CCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxis was totally abolished by blockade of BAFF-R and was strongly dependent on the activation of PI3K/AKT, NF-kappa B, and p38MAPK pathways. BAFF had similar effects on the chemotaxis of naive and memory B cells in response to CCL21 but increased more strongly that of memory B cells to CXCL13 than that of naive B cells. Our findings indicate a previously unreported role for the BAFF/BAFF-R pair in mature B-cell chemotaxis. The synergy between CXCL13 and BAFF produced by stromal cells and follicular dendritic cells may have important implications for B-cell homeostasis, the development of normal B-cell areas, and for the formation of germinal center-like follicles that may be observed in various autoimmune diseases.

Workshop cluster 1(+) gamma delta (WC1(+)gammadelta) T cells have been shown to play important roles in the immune response to infections. WC1 is a transmembrane glycoprotein, uniquely expressed on the surface of gammadelta T cells of ruminants and pigs. A role for WC1 in inducing a reversible growth arrest of gammadelta T cells has been previously demonstrated. WC1-induced growth inhibition has been shown to be overcome following gammadelta T cell activation with Concanavalin A (Con A). However, molecular mechanism(s) by which WC1 signalling might be modulated following activation have not been elucidated. In this paper we show that Con A activation of bovine lymphocytes induces the tyrosine phosphorylation of WC1 in a Src-family kinase-dependent manner. Src family kinases also phosphorylated WC1 in a COS-7 co-transfection system. Furthermore, a glutathione-S-transferase (GST)-WC1 cytoplasmic domain fusion protein was directly phosphorylated by recombinant Lck (rLck) in vitro. The Y(1303) of WC1 was identified by mutational analysis as the only one of the five WC1 tyrosine residues to be critical for Src family phosphorylation. The importance of activation-induced Src family activity for WC1 function was investigated with the Src-family specific inhibitor PP2. These studies show that the surface levels of WC1 are down regulated in a Src-family-dependent manner following activation of bovine lymphocytes. Down regulation of surface WC1 was accompanied by a Src-family-dependent accumulation of intracellular WC1. These data show that WC1 is modulated by activation-induced tyrosine phosphorylation thus providing a new insight into the signalling mechanisms by which WC1 and gammadelta T cell activation are regulated in this important and unique cell population.

Follicular dendritic cells (FDC) in the germinal centers (GC) of secondary lymphoid organs increase the survival and proliferation of antigen-stimulated B cells and are pivotal for the affinity maturation of an antibody response and for maintenance of B cell immunological memory. The dark zone (DZ) and the light zone (LZ) constitute distinct areas of the GC containing different subtypes of FDC as identified by their morphology and phenotype. Until now, most available FDC-specific reagents identify LZ FDC, and there are no reagents recognizing DZ FDC specifically. Here, we report a new mAb, D46, which stains FDC specifically in the DZ of bovine and ovine GC within the secondary follicles. We identify its ligand as bovine fibrinogen, and using commercially available anti-human fibrinogen antibodies, show that this inflammatory protein is also present on DZ FDC of human GC within palatine tonsils. In vitro, the addition of exogenous fibrinogen stimulates the proliferation and survival of BCR-stimulated L3055 cells, which constitute a clonal population of centroblastic cells and retain important features of normal GC B cells. Together, our results suggest that fibrinogen localized on DZ FDC could support the extensive proliferation and survival of GC B cells within the DZ in vivo.

In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN-alpha/beta), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN-alpha/beta in vitro. Similarly, cpBVDV induces IFN-alpha/beta in the early bovine fetus, but ncpBVDV does not. However, acute infection of naive cattle with ncpBVDV results in IFN-alpha/beta production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN-alpha in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN-alpha in the absence of detectable productive infection.

Data show that HIV-1 induces strong B-cell activation and the disorganization of lymphoid tissue. Besides impairing cell-cell interactions and cytokine production, the authors believe that these functional and phenotypic changes result from direct interactions between virions and B-cells. They have therefore studied over the last few years the mechanisms by which HIV-1 alters the various steps of B-cell differentiation and may actively participate in lymphoid tissue disorganization and disease progression.

http://bloodjournal.hematologylibrary.org/content/96/7/2338.full.html Updated information and services can be found at: (4939 articles) Immunobiology • (564 articles) Chemokines, Cytokines, and Interleukins • Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at:

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1β (MIP-1β) and MIP-1α, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1α/β in response to BCR triggering. In contrast to MIP-1α/β, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1α/β production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1β and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1α Ab suggesting that MIP-1β plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1α/β, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.