Immune Responses of Systemic and Mucosal Lymphoid Organs to Pnu-Imune Vaccine as a Function of Age and the Efficacy of Monophosphoryl Lipid A as an Adjuvant

A murine model system was established to study immune responses to the Pnu-Imune vaccine, which is made up of 23 different pneumococcal capsular polysaccharides. In this animal model, antibody-forming cell responses to 21 of 23 individual polysaccharides in the vaccine were detected. The Pnu-Imune vaccine elicited good antibody responses from the spleens and mesenteric lymph nodes (MLN) of young mice, whereas a variety of other peripheral lymph nodes were unresponsive. The immunoglobulin M plaque-forming cell (PFC) response in the spleen to the Pnu-Imune vaccine (given intraperitoneally or subcutaneously) decreased dramatically with increasing age. However, the spleen and MLN differed in their susceptibility to an age-associated decline in immune function. While the PFC responses in the spleen declined with age, the PFC response in the mucosa-associated MLN did not decline with age but instead remained constant over the entire age span of 4 to 28 months studied. These studies showed that the spleen, peripheral lymph nodes, and MLN did not demonstrate parallel age-associated defects in antibody responses to pneumococcal polysaccharides when the antigen was administered systematically. Also, the deficient splenic antibody response to Pnu-Imune vaccine in aged mice could be enhanced by injecting a combination of Pnu-Imune vaccine and the nontoxic adjuvant monophosphoryl lipid A. Moreover, an immunoglobulin G response was induced when the immunogen was a mixture of vaccine and adjuvant

Multiple Regulatory Mechanisms Control B-1 B Cell Activation

B-1 cells constitute a unique subset of B cells identified in several species including mice and humans. B-1 cells are further subdivided into B-1a and B-1b subsets as the former but not the later express CD5.The B-1a subset contributes to innate type of immune responses while the B-1b B cell subset contributes to adaptive responses. B-1 cell responses to B cell receptor (BCR) as well as Toll-like receptor (TLR) ligation are tightly regulated due to the cross-reactivity of antigen specific receptors on B-1 cells to self-antigens. B-1 cells are elevated in several autoimmune diseases. CD5 plays a major role in down regulation of BCR responses in the B-1a cell subset. Reduced amplification of BCR induced signals via CD19 and autoregulation of BCR and TLR responses by B-1 cell produced IL-10 appear to have a role in regulation of both B-1a and B-1b B cell responses. Siglec G receptors and Lyn kinase also regulate B-1 cell responses but their differential role in the two B-1 cell subsets is unknown

Primary Antibody Responses to Thymus-Independent Antigens in the Lungs and Hilar Lymph Nodes of Mice

B lymphocytes from the pulmonary lymphoid tissues were stimulated with a variety of thymus-independent (TI) antigens by intratracheal (i.t.) immunization. Immune responses in the lungs and hilar lymph nodes (HLN), which are part of the localized lymphoid tissue, as well as in the spleen, the systemic lymphoid organ, were studied. Thus, primary i.t. immunization of mice with the TI-1 antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) elicited both antigen-specific and polyclonal plaque-forming cell responses from HLN, lung, and splenic B lymphocytes. These responses appeared as early as 3 days after immunization and declined by day 7. Similar immunization with another TI-1 antigen, TNP-Brucella abortus, resulted in anti-TNP responses in both pulmonary and systemic lymphoid tissues, although the kinetics of the antibody response were different than those to TNP-LPS. Interestingly an i.t. immunization with a TI-2 antigen, TNP-Ficoll, failed to induce an anti-TNP PFC response from HLN and lung B cells, although there was good antibody formation from splenic B cells. Antibody response to TNP-Ficoll was restored in pulmonary tissues when mice were immunized with TNP-Ficoll mixed with unconjugated B. abortus. In conclusion, our results indicate that TI-1 and TI-2 antigens differ in their ability to induce antibody responses in the pulmonary lymphoid tissues. The inability of TNP-Ficoll to elicit an antibody response in pulmonary lymphoid tissues has significance in the development of vaccines containing bacterial polysaccharides

Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in \u3cem\u3eBorrelia hermsii\u3c/em\u3e Infection

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-kappaB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10-/- mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity

A Shared Gene Expression Signature in Mouse Models of EBV-Associated and Non-EBV-Associated Burkitt Lymphoma

The link between EBV infection and Burkitt lymphoma (BL) is strong, but the mechanism underlying that link has been elusive. We have developed a mouse model for EBV-associated BL in which LMP2A, an EBV latency protein, and MYC are expressed in B cells. Our model has demonstrated the ability of LMP2A to accelerate tumor onset, increase spleen size, and bypass p53 inactivation. Here we describe the results of total gene expression analysis of tumor and pretumor B cells from our transgenic mouse model. Although we see many phenotypic differences and changes in gene expression in pretumor B cells, the transcriptional profiles of tumor cells from LMP2A/λ-MYC and λ-MYC mice are strikingly similar, with fewer than 20 genes differentially expressed. We evaluated the functional significance of one of the most interesting differentially expressed genes, Egr1, and found that it was not required for acceleration of tumor onset by LMP2A. Our studies demonstrate the remarkable ability of LMP2A to affect the pretumor B-cell phenotype and tumorigenesis without substantially altering gene expression in tumor cells

CD19 Signaling Is Impaired in Murine Peritoneal and Splenic B-1 B Lymphocytes

B-1 cells reside predominantly within the coelomic cavities, tonsils, Peyer\u27s patches, spleen (a minor fraction – ∼5%) and are absent in the lymph nodes. They are the primary sources of natural IgM in the body. B-1 cells express polyreactive B cell receptors (BCRs) that cross react with self-antigens and are thus implicated in auto-immune disorders. Previously, we reported that peritoneal B-1 cells are deficient in CD19-mediated intracellular signals leading to Ca2+ mobilization. Here, we find that splenic B-1 cells, like peritoneal B-1 cells, are defective in Ca2+ release upon B cell activation by co-cross-linking BCR and CD19. In the absence of extracellular sources of Ca2+, intracellular Ca2+ flux is similar between B-1 and B-2 cells. Moreover, the intracellular component of Ca2+ release in both subsets of B cells is mostly PI3K dependent. BCR and CD19 co-cross-linking activates Akt, a key mediator of survival and proliferation signals downstream of PI3K in splenic B-2 cells. Splenic B-1 cells, on the other hand, do not phosphorylate Akt (S473) upon similar treatment. Furthermore, BCR + CD19 cross-linking induced phosphorylation of JNK is much reduced in splenic B-1 cells. In contrast, B-1 cells exhibited increased levels of constitutively active pLyn which appears to have an inhibitory role. The CD19 induced Ca2+ response and BCR induced proliferation response were restored by a partial inhibition of pLyn with Src kinase specific inhibitors. These findings suggest a defect in CD19-mediated signals in both peritoneal and splenic B-1 B lymphocytes, which is in part, due to higher levels of constitutively active Lyn

Inhibition of Bruton Tyrosine Kinase Reduces Neuroimmune Cascade and Promotes Recovery after Spinal Cord Injury

Microglia/astrocyte and B cell neuroimmune responses are major contributors to the neurological deficits after traumatic spinal cord injury (SCI). Bruton tyrosine kinase (BTK) activation mechanistically links these neuroimmune mechanisms. Our objective is to use Ibrutinib, an FDA-approved BTK inhibitor, to inhibit the neuroimmune cascade thereby improving locomotor recovery after SCI. Rat models of contusive SCI, Western blot, immunofluorescence staining imaging, flow cytometry analysis, histological staining, and behavioral assessment were used to evaluate BTK activity, neuroimmune cascades, and functional outcomes. Both BTK expression and phosphorylation were increased at the lesion site at 2, 7, 14, and 28 days after SCI. Ibrutinib treatment (6 mg/kg/day, IP, starting 3 h post-injury for 7 or 14 days) reduced BTK activation and total BTK levels, attenuated the injury-induced elevations in Iba1, GFAP, CD138, and IgG at 7 or 14 days post-injury without reduction in CD45RA B cells, improved locomotor function (BBB scores), and resulted in a significant reduction in lesion volume and significant improvement in tissue-sparing 11 weeks post-injury. These results indicate that Ibrutinib exhibits neuroprotective effects by blocking excessive neuroimmune responses through BTK-mediated microglia/astroglial activation and B cell/antibody response in rat models of SCI. These data identify BTK as a potential therapeutic target for SCI

Identification of a Gal/GalNAc Lectin in the Protozoan \u3cem\u3eHartmannella vermiformis\u3c/em\u3e as a Potential Receptor for Attachment and Invasion by the Legionnaires\u27 Disease Bacterium

The Legionnaire\u27s disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire\u27s disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine–inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc–inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa

Role of Phosphoinositide 3-Kinase – Akt Signaling Pathway in the Age-Related Cytokine Dysregulation in Splenic Macrophages Stimulated via TLR-2 or TLR-4 Receptors

Age-associated defects in both B-lymphocytes and macrophages in elderly result in a reduction in the efficacy of vaccines to many Gram positive bacteria like Streptococcus pneumoniae. Splenic macrophages from aged mice have been shown to have a defect in production of pro-inflammatory cytokines (IL-6, IL-12, IL-1β, TNF-α) but exhibit increased production of IL-10 upon TLR4 ligation. Here we showed that aged macrophages demonstrate similar cytokine dysregulation phenotype upon stimulation with TLR2 ligands, or killed S. pneumoniae. We hypothesized that an age-associated increase in activity of phosphatidyl inositol 3-kinase (PI3K)-Akt signaling pathway may be playing a causal role in the age-associated cytokine dysregulation. We found that gene expression of both the regulatory (p85β) and the catalytic (p110δ) subunits of Class IA PI3K is higher in aged than in young splenic macrophages. The age-associated increase in the activity of PI3K was also demonstrated by an upregulation of P-Akt and its downstream target, glycogen synthase kinase-3 (GSK-3). Inhibition of PI3K enhanced induction of pro-inflammatory cytokines, by TLR-2/TLR-1, TLR-2/TLR-6 and TLR-4 ligands as well as heat killed S. pneumoniae (HKSP). Therefore, targeting PI3-Kinase could rescue cytokine dysregulation in aged macrophages and enhance the relevant pro-inflammatory cytokines needed to support B-cell activation and differentiation