Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

The ability of carbonate apatite (CO₃Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO₃Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO₃Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO₃Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO₃Ap and OVA-containing alumina salt (Alum), suggesting that CO₃Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO₃Ap.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response.

Immunization with protein-containing antigens results in two types of antigen-specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re-exposure to antigen. The issue of the inter-relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen-specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub-population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity-enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B-cell population contains few, if any, cells from the early response and is less stringently selected.     

Experimental Sepsis Impairs Humoral Memory in Mice

Patients with sepsis are often immune suppressed, and experimental mouse models of sepsis also display this feature. However, acute sepsis in mice is also characterized by a generalized B cell activation and plasma cell differentiation, resulting in a marked increase in serum antibody concentration. Its effects on humoral memory are not clearly defined. We measured the effects of experimental sepsis on long-term immunological memory for a defined antigen: we induced colon ascendens stent peritonitis (CASP) 8 weeks after 2 rounds of immunization with ovalbumin. Four weeks later, the antigen-specific bone marrow plasma cell count had doubled in immunized non-septic animals, but remained unchanged in immunized septic animals. Sepsis also caused a decrease in antigen-specific serum antibody concentration. We conclude that sepsis weakens humoral memory by impeding the antigen-specific plasma cell pool’s development, which is not complete 8 weeks after secondary immunization.     

Depletion of B cells rejuvenates the peripheral B‐cell compartment but is insufficient to restore immune competence in aging

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B‐cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B‐cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B‐cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B‐cell compartments. B‐cell depletion in old mice resulted in rejuvenated B‐cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"‐like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B‐cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"‐like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B‐cell compartment in aging, through B‐cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.     

2B4 Is Dispensable for T-Dependent B Cell Immune Responses,but Its Deficiency Leads to Enhanced T-Independent Responses Due to an Increase in Peritoneal Cavity B1b Cells

The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244 ^-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG₁ antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244 ^-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG₃ were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell responses due to a reduction in peritoneal cavity B cells, but has minimal impact on T-dependent B cell responses.     

Silibinin attenuates antigen-specific IgE production through the modulation of Th1/Th2 balance in ovalbumin-sensitized BALB/c mice

The effect of silibinin on antigen-specific antibody production and T-cell cytokine expression was investigated. BALB/c mice were either left untreated or administered daily with vehicle (VH; saline) and/or silibinin (200 or 400 mg/kg) by gavage for 3 consecutive days prior to sensitization with ovalbumin (OVA). The antibody production in the serum and T-cell-derived cytokine expression by splenocytes were determined 7 days post OVA sensitization. Our results demonstrated that the production of OVA-specific serum IgE and total IgE was significantly attenuated by silibinin treatment, whereas OVA-specific IgG_2a was markedly enhanced. In parallel with the differential modulation of the production of IgG_2a and IgE, treatment of OVA-sensitized mice with silibinin markedly increased and decreased the production of IFN-γ and IL-4, respectively, by splenocytes cultured in the presence of OVA. Together, these results suggest that silibinin treatment polarizes the Th1/Th2 immune balance toward the Th1-dominant direction, which may be beneficial against IgE-mediated allergy.     

Regulation of antigen-specific versus by-stander IgE production after antigen sensitization

IgE is critical in the pathogenesis of allergic disorders. In this report, we investigated the differential regulation of antigen-specific and by-stander IgE. Ovalbumin (OVA) immunization did not increase IgE producing cells in the spleen, but significantly enhanced the intracellular IgE content of all IgE+ cells. In contrast, OVA induced a significant increase of IgE+ cells in the draining lymph nodes (LN). Furthermore, OVA-specific IgE was detected only in the ex vivo cultures of the draining LN but not the spleen cells, while total IgE was increased in both cultures. These results indicated that antigen-specific IgE was mainly produced in the draining LN, while the spleen was a major source for by-stander IgE. Anti-IL-4, but not anti-IL-13, antibody blocked the expansion of IgE producing cells in the draining LN as well as systemic OVA-specific and total IgE levels, indicating IL-4 was important in both antigen-specific IgE generation and total IgE upregulation.     

The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells,but Not in the Production of Antigen-Specific IgG and IgM,in Response to T-dependent Antigens

The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2^−/− mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2^−/− mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG₁ plasma cells. There was also no difference in NP-specific IgM and class switched IgG₁ in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2^−/− mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.     

Improvement of adaptive immunity by antigen-carrying biodegradable nanoparticles

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying γ-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8⁺ T cells. Unlike complete Freund’s adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, γ-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

The capacity and mechanism of bone marrow antibody formation by thymus-independent antigens

Primary immunization of mice with certain thymus-independent (TI) antigens (i.e., TNP-LPS and DNP-Ficoll) leads to antibody formation in the bone marrow (BM). TNP-Brucella abortus, Pneumococcus pneumoniae organisms, and alpha-(1,6) dextran, on the other hand, do not induce a BM antibody-producing plaque-forming cell (PFC) response. This paper deals with the mechanism underlying antibody formation in the BM to TNP-LPS and DNP-Ficoll. The majority of the BM-localizing PFC induced by TNP-LPS are formed within the BM from the proliferating lymphocyte pool, because this response was found to be resistant to splenectomy and sensitive to treatment with hydroxyurea (HU) before immunization. This local activation of newly formed B cells requires in addition to the antigenic signal of TNP-LPS the mitogenic signal from the lipid A component of LPS. In contrast, the BM PFC response to DNP-ficoll was reduced in splenectomized mice and resistant to HU treatment before the primary immunization. Thus, antibody formation in the BM to DNP-Ficoll is mainly dependent on long-lived B cells that migrate from the peripheral lymphoid organs into the BM.     

Isotype specificity of antigen-specific helper clone in vivo

Inoculation of an immortalized clone of radiation leukemia virus (RadLV)-transformed antigen (ovalbumin, OVA)-specific T cells together with the relevant carrier (OVA) into unprimed syngeneic mice results in a preferential increase in the expression of anti-OVA antibodies of the immunoglobulin (Ig)G2b and IgG2a isotypes. Identical boosting of the clone-primed mice further augments the preferential production of anti-OVA antibodies of these two isotypes. The class-related helper activity is not due to nonspecific shift of class expression produced by the injected tumor cells, as a non-helper clone of RadLV-transformed T cells does not change the isotypic pattern of anti-OVA antibodies in the inoculated mice. A carrier-specific activation of the B cells is responsible for the class-restricted function of the helper clone. The isotypic profile of anti-hapten antibodies in mice injected with 2,4-dinitrophenyl (DNP)-bovine serum albumin and OVA-specific helper clone is not altered. On the other hand, mice inoculated with the OVA-specific helper clone and DNP-OVA respond with a preferential elevation of anti-DNP antibodies of the IgG2a and IgG2b isotypes. The preferential class augmentation may result from carrier-specific signals delivered by the helper clone which activate B cells in vivo toward certain CH expression. Alternatively, the observed class pattern may be induced by an isotype noncommited helper clone which triggers selected population of B lymphocytes of defined differentiation status toward secretion of a restricted array of isotypes. Regardless of the mechanism of the clone-dependent class expression, the isotypic profile in most of the experiments clearly demonstrates that an antigen-specific helper clone may be one of the elements which regulates the class of antibodies to be produced in vivo under normal physiologic conditions.