William F. Wade

Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10–20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)γ production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNγ, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co‐adoptive transfer of antigen‐pulsed DC and TCR‐transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40‐CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen‐pulsed DC in the draining lymph nodes when CD154‐CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40‐CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen‐presenting cell capacity are both dependent on CD40 signaling. While TNF‐α can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF‐α to rescue the immune deficiency of CD40–/– DC. Hence, the profound impact of CD154 deficiency on cell‐mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen‐specific T cells.

Induction of Protective Immunity by Synthetic Vibrio cholerae Hexasaccharide Derived from V. cholerae O1 Ogawa Lipopolysaccharide Bound to a Protein Carrier

Synthetic antigens that mimic the terminal hexasaccharide epitope of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, were conjugated to bovine serum albumin (BSA). Conjugates with carbohydrate-to-carrier molar ratios of 15.5:1, 9.2:1, and 4.6:1 were tested for immunogenicity and efficacy in mice. The role of preimmunity to BSA and the use of adjuvant in the generation of the serologic response to the O-specific polysaccharide and protection against virulent V. cholerae was examined. Preimmunity to BSA did not affect the anti-Ogawa titers but seemed to enhance the protective capacity of antiserum. All 3 conjugates were immunogenic, but adjuvant was effective at inducing higher and earlier antibody responses. In tertiary serum samples, a correlation was found between vibriocidal activity and protection. The protective capacity of antiserum was evident in serum from mice immunized with all conjugates, but it was highest in the groups that received the conjugate with the lowest level of substitution. Further studies are required to increase understanding of the reason for differential protection.

IgA Fc receptor (FcαR) cross-linking recruits tyrosine kinases, phosphoinositide kinases and serine/threonine kinases to glycolipid rafts

The human IgA Fc receptor (FcalphaR, CD89) triggers several important physiological functions, including phagocytosis, NADPH oxidase activation and antigen presentation. Efforts are underway to delineate FcalphaR signal-transduction pathways that control these functions. In a previous study, we demonstrated that cross-linking of FcalphaR increased its partitioning into membrane glycolipid rafts and was accompanied by gamma-chain-dependent recruitment and phosphorylation of the tyrosine kinases Lck/Yes-related novel protein tyrosine kinase (Lyn) and Brutons tyrosine kinase (Btk). Here we have performed a more extensive characterization of signalling effectors recruited to rafts on FcalphaR cross-linking. We demonstrate that in addition to tyrosine kinases Lyn and Btk, FcalphaR cross-linking also recruits B-lymphocyte kinase (Blk) and spleen tyrosine kinase (Syk) to rafts. We show recruitment of phosphoinositide kinases, including 3-phosphoinositide 3-kinase and phospholipase Cgamma2, and serine/threonine kinases such as protein kinase C (PKC) alpha, PKCepsilon, and protein kinase B (PKB) alpha. This suggests that lipid rafts serve as sites for FcalphaR-triggered recruitment of multiple classes of signalling effectors. We further demonstrate that tyrosine kinases and PKCalpha have a sustained association with rafts, whereas phosphoinositide 3-kinase and its downstream effectors have a transient association with rafts. This is consistent with temporally regulated divergence of FcalphaR signalling pathways in rafts. Furthermore, we suggest the spatial separation of signalling effectors by transport of phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, PKBalpha and PKCepsilon to endocytic compartments containing internalized FcalphaR.

Evaluation of Cholera Vaccines Formulated with Toxin-Coregulated Pilin Peptide Plus Polymer Adjuvant in Mice

ABSTRACT Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD50) challenge and 50% protection against a 10-LD50 challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.

Synthetic Fragments of Vibrio cholerae O1 Inaba O-Specific Polysaccharide Bound to a Protein Carrier Are Immunogenic in Mice but Do Not Induce Protective Antibodies

ABSTRACT Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody following infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide component of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin (BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody. We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective. Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate (CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1) were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked immunosorbent assay but not enough to be protective in vivo.

The ABCs (Antibody, B Cells, and Carbohydrate Epitopes) of Cholera Immunity: Considerations for an Improved Vaccine

Cholera, a diarrheal disease, is known for explosive epidemics that can quickly kill thousands. Endemic cholera is a seasonal torment that also has a significant mortality. Not all nations with extensive rural communities can achieve the required infrastructure or behavioral changes to prevent epidemic or endemic cholera. For some communities, a single‐dose cholera vaccine that protects those at risk is the most efficacious means to reduce morbidity and mortality. It is clear that our understanding of what a protective cholera immune response is has not progressed at the rate our understanding of the pathogenesis and molecular biology of cholera infection has. This review addresses V. cholerae lipopolysaccharide (LPS)‐based immunogens because LPS is the only immunogen proven to induce protective antibody in humans. We discuss the role of anti‐LPS antibodies in protection from cholera, the importance and the potential role of B cell subsets in protection that is based on their anatomical location and the intrinsic antigen‐receptor specificity of various subsets is introduced.

Fcα Receptor Cross-Linking Causes Translocation of Phosphatidylinositol-Dependent Protein Kinase 1 and Protein Kinase Bα to MHC Class II Peptide-Loading-Like Compartments

A20 IIA1.6 B cells cotransfected with FcαR and wild-type γ-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or FcαR and γ-chain, in which the wt-ITAM was substituted with the FcγRIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of FcαR-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented FcαR-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that FcαR may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B α (PKBα) activation. Cross-linking FcαR on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKBα. Furthermore, FcαR cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKBα to capped cell surface FcαR irrespective of the γ-chain ITAM. Although FcαR endocytosis was accompanied by translocation of PDK1 and phospho-PKBα to FcαR-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKBα remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKBα translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.

Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.

Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5 microm 1/e2 radius. The effective spot area represents only 1/500 of the total surface of an 8-microm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100 molecules. This limits the precision and reproducibility of FPR measurements. We describe a method for interferometric fringe pattern FPR that permits simultaneous interrogation of the entire cells surface. Fringe patterns are generated interferometrically within the optical path of an FPR system. Methods for interpreting fluorescence recovery kinetics on cells and for determining the protein mobile fraction are presented. With fringe FPR, the murine major histocompatibility complex class II antigen I-Ak expressed on M12.C3.F6 cells has 100-fold improved fluorescence signals relative to spot FPR, with corresponding improvements in signal-to-noise ratios of recovery traces. Diffusion coefficients (+/- standard deviation) of (2.1 +/- 0.4) x 10(-10) and (1.8 +/- 1.0) x 10(-10) cm2 s-1 with corresponding mobile fractions of I-Ak of 66.1 +/- 7.8% and 63.4 +/- 18.0% were obtained by fringe and spot methods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be substantial variation from cell to cell in protein dynamics, and this method may permit the assessment of such variation.

Protein Kinase C-α and -δ Are Required for FcαR (CD89) Trafficking to MHC Class II Compartments and FcαR-Mediated Antigen Presentation

Studies have demonstrated that receptor‐mediated signaling, receptor/antigen complex trafficking, and major histocompatibility complex class II compartments (MIIC) are critically related to antigen presentation to CD4+ T cells. In this study, we investigated the role of protein kinase C (PKC) in FcαR/γγ (CD89, human IgA receptor)‐mediated internalization of immune complexes and subsequent antigen presentation. The classical and novel PKC inhibitor, Calphostin C, inhibits FcαR‐mediated antigen presentation and interaction of MIIC and cargo vesicle (receptor and antigen). PKC‐α, PKC‐δ, and PKC‐ε were recruited to lipid rafts following FcαR crosslinking, the extent of which was determined by the phenotype of the γ chain. Mutant γ chain with an FcγRIIA ITAM (immunoreceptor tyrosine‐based activation motif) insert was less able to recruit PKC and trigger antigen presentation. Both PKC isoform‐specific peptide inhibitors and short interfering RNA (siRNA) showed that PKC‐α and PKC‐δ, but not PKC‐ε, were required for association of cargo vesicle and MIIC and for FcαR‐mediated and soluble antigen presentation. Inhibition of PKC (classical and novel) did not alter major histocompatibility class II biosynthesis, assembly, transport, or plasma membrane stability. PKCs role in facilitating interaction of cargo vesicle and MIIC is likely due to regulation of vesicle biology required for fusion of cargo vesicles to MIIC.