Immunological dynamics in response to two anthrax vaccines in mice

In order to understand the variation of humoral and cellular immune responses to A16R live spore and AVA vaccine and to identify efficient immunological parameters for the early evaluation of post immu- nization in mice, we dynamically monitored the antibody production and cellular responses after the vaccination of Balb/C mice with the anthrax vaccines. The results show that both anti-AVA and anti-Spore antibodies were detectable in the A16R live spore vaccinated group while high titers of anti-AVA antibodies but not anti-Spore antibodies existed in the AVA-immunized group. IgG1 and IgG2 were the major subtypes of IgG in both of the two groups. However, the IgG2a level was significantly higher in the A16R group than in the AVA group. At the cellular level, responses of antigen-specific TH2, TH1 and plasma cells were detected. The peripheral TH2 responses could be seen on day 5 after vac- cination, and remained at a high level throughout the experiment (from day 5 post primary immuniza- tion to day 60 post the tertiary immunization); the TH1 responses to A16R vaccine appeared on day 5, while the responses to AVA could only be detected by day 7 after the secondary immunization; a low level of TH1 responses could be observed at the end of the experiment. Antigen-specific plasma cells could be found in the peripheral blood of both the immunized groups, however, the responses in the A16R group appeared earlier, lasted longer, and shown an ascending tendency until the end of the ex- periment when the plasma cell responses in the AVA group were reduced to a very low level. The re- sults suggest that the multiple antigen containing A16R live spore vaccine induces better immune re- sponses than AVA. Combined with serum antibody titers, TH2, TH1 and plasma cell responses could be used as immunological parameters for the evaluation of vaccine efficacy. These findings may afford new insight into the early evaluation of vaccination as well as being a powerful strategy for vaccine development.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant—free condition

Trichosanthin(TCS) is a potent allergen in mice.It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum.TCS can bring out the IgE responses to ovalbumin(OVA),while OVA itself could not induce IgE responses to it .However,TCS only works when OVA immunization is given one day after TCS immunization.Either time lag in OVA immunization,or immunization of both antigens at the same time,or OVA immunization given first,all has no effect on the induction of IgE responses to OVA.Through analysis of the antibody specificity of hybridoma clones,it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones.The IgE antibldies showed no polyreactivity to different antigens.     

Chimeric antigen receptor engineered innate immune cells in cancer immunotherapy

Introducing chimeric antigen receptor into immune cells against malignancies has contributed to a revolutionary innovation in cancer immunotherapy. As an important type of adaptive immune cells, T cells first caught researchers' attention and became great success in chimeric antigen receptor-based immunotherapy. However, engineered T cells seem to hit their bottleneck when resistance of cancerous cells, less encouraging responses in solid tumors and unwanted toxicities to the host remain to be solved.Meanwhile, innate immune cells get to join the race. Representatives such as natural killer cells, natural killer T cells, γδT cells and macrophages also prove to be well redirected with chimeric antigen receptors. Compared to chimeric antigen receptor engineered T cells, these engineered innate immune cells may possess multiple targeting and killing mechanisms, have the potential to crack the barrier of solid tumors and have less side effects in the host. Besides, possible universal access to cell resources and improvements in expansion and transduction techniques make these cells promising candidates with huge potential in translational medicine. Therefore, innate immune cells claim a brand-new dimension and are likely to supplement T cells greatly in the field of chimeric antigen receptor-based immunotherapy.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Isolation and characteristics of autoreactive T cells specific to aggrecan G1 domain from rheumatoid arthritis patients

NTRODUCTIONRheumatoid arthritis (RA) is a common disease characterized by the chroniclesion of polyarthritis. The etiology and pathogenesis of RA remain unknown. Autoimmunity to cartilage alltigens may play a significant role in the pathogenesis ofchronic inflammatory polyarthritis. It is commonly accepted that cell mediated immune responses are involved in chronic inflammation since T and B lymphocytes andatigen presenting cells were observed to be enriched in the synovium fluid of RA…     

Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

Systemic lupus erythematosus （SLE） is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells （APCs） and an autoreactive T cell （ATLI） clone obtained from lupus-prone BXSB mice. ATLI cells, either before or after 7-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL 1 cells at a responder/stimulator （R/S） ratio of 1/2.5. Dendritic cells （DCs） were much more powerful stimulators for ATLI cells on a per cell basis. The T cell stimulating ability ofmacrophages and B cells, but not DCs, was sensitive to T-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱ and CD4 were able to block DC-mediated stimulation of ATL 1 proliferation, indicating cognate recognition between ATL 1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

Murine bone marrow IgA responses to orally administered sheep erythrocytes

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.     

Epitope-specific antibody feedback regulation of the humoral immune response against sheep erythrocytes in vitro: specific effects of anti-antigen antibody vs nonspecific T cell activities

Antibody formed during a 1st in vitro anti-SRBC PFC response had previously been shown to inhibit the formation of PFC when added to a 2nd, freshly established test culture. This effect was to a large extent restricted to test cultures containing B cells sharing VH genes with the B cells producing the initial antibody, and this suggested that anti-SRBC antibody acted via triggering of an anti-idiotypic antibody of TS response. In the present studies this system has been further characterized. First, such antibody feedback occurred in cultures of purified and anti-Lyt2 antiserum and complement-treated surface Ig-positive cells in which TH were substituted for by T-replacing factor. Thus, T cells were not required. Moreover, T cells were always nonspecifically activated in cultures containing FCS. Secondly, anti-idiotypic antibody-like activity was not detected in the sense that generation of inhibitory antibody was never found to be dissociated from generation of anti-SRBC antibody, and LPS-dependent anti-SRBC PFC responses were not inhibited. However, feedback inhibition of SRBC-dependent responses was reversed at increased SRBC concentrations. Thirdly, the feedback mechanism was highly epitope specific, whereas in vitro anti-SRBC PFC responses of different mouse strains (B6 vs BALB/c) were directed to a large extent against different epitopes. These data strongly suggest that VH-restricted inhibitory activity of antibody in this system is a manifestation of epitope specificity of the antibody feedback and not of idiotype specificity, i.e., that anti-SRBC antibody acts via masking of epitopes.     

Acute hepatotoxin exposure effects lymphoid and accessory cell types in inbred mice

The effect of acute hepatotoxin exposure on in vivo and in vitro immune responses were investigated in inbred mice. Splenic anti-SRBC PFC responses were slightly enhanced by carbon tetrachloride or galactosamine administration 5 hr prior to immunization. Whereas splenic anti-SRBC PFC responses were slightly enhanced in euthymic mice exposed to carbon tetrachloride 5 hr prior to immunization, immune responses to the TI antigens, Fl-LPS, Fl-Ficoll, and TNP-LPS, were significantly suppressed. Athymic mice receiving similar hepatotoxin exposure elicited enhanced immune responses to the TI immunogens, thereby suggesting that the activities of B cells and macrophages are enhanced in treated animals and in euthymic mice, T suppressor cells are also activated. By admixture of purified B- and T-cell and macrophage populations from either carbon tetrachloride-treated or control animals, it was demonstrated that hepatotoxin exposure also induces suppressor T cells regulating immune responses to the T-dependent antigen, SRBC, and that macrophages from treated animals are more functional. Further, B-cell responsiveness is enhanced. In addition to these observations, an active factor could be demonstrated in sera from hepatotoxin-treated animals which augments immune responses to SRBC in normal mice and promotes immune responses to this antigen in athymic mice. These findings indicate that the effects of acute hepatotoxin exposure are multifocal, influencing the activity of lymphoid and accessory cells.     

Immune responses to hepatitis B surface antigen following epidermal powder immunization

Langerhans cells in the epidermis of skin are potent antigen-presenting cells that trigger the immune system to respond to invading microorganisms. We have previously shown that epidermal powder immunization with a powdered inactivated influenza virus vaccine, by targeting the Langerhans cell-rich epidermis, was more efficacious than deeper tissue injection using a needle and syringe. We now report enhanced humoral and cellular immune responses to recombinant hepatitis B surface antigen following epidermal powder immunization. We observed that epidermal powder immunization with unadjuvanted hepatitis B surface antigen elicited an antibody titre equivalent to that induced by the alum-adjuvanted vaccine delivered by intramuscular injection, suggesting that epidermal powder immunization can overcome the need for adjuvantation. We demonstrated that synthetic CpG oligonucleotides (CpG DNA) could be coformulated with hepatitis B surface antigen and delivered by epidermal powder immunization to further augment the antibody response and modulate T helper cell activities. Epidermal powder immunization of hepatitis B surface antigen formulated with CpG DNA formulations resulted in 1.5-2.0 logs higher IgG antibody titres than alum-adjuvanted commercial vaccines administered by intramuscular injection. Formulation of hepatitis B surface antigen with CpG DNA elicited an augmented IgG2a antibody response and increased frequency of IFN-gamma secreting cells. In addition, CpG DNA was found to activate epidermal Langerhans cells and stimulate the production of TNF-alpha and IL-12 cytokines by epidermal cells, explaining its strong adjuvant activity following epidermal powder immunization. These results show that epidermal powder immunization is a safe and effective method to deliver hepatitis B surface antigen and the addition of new adjuvants, such as CpG DNA, may further enhance the efficacy of this vaccine.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

Divergent Primary Immune Responses Induced by Human Immunodeficiency Virus-1 gp120 and Hepatitis B Surface Antigen Determine Antibody Recall Responses

The development of a vaccine based on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) that elicits potent protective antibodies against infection has been challenging. Recently, we compared the antibody production patterns of HIV-1 Env gp120 and hepatitis B virus surface antigen (HBsAg) to provide insights into how we may improve the protective efficacy of Env-based immunogens. Our previous study showed that HIV Env and HBsAg display different mechanisms of antibody elicitation and that T cells facilitate the responses to repeated immunizations. Here, to elucidate the detailed roles of primary immunization in immune memory response formation and antibody production, we immunized C57BL/6 mice with each antigen and evaluated the development of T follicular helper (Tfh) cells, germinal centers, and the memory responses involved in prime and boost immunizations. We found that after prime immunization, compared with HBsAg, gp120 induced higher frequencies of Tfh cells and programmed death (PD)-1⁺ T cells, greater major histocompatibility complex II expression on B cells, comparable activated B cells, but weaker germinal center (GC) reactions and memory B cell responses in the draining lymph nodes, accompanied by slower antibody recall responses and poor immune memory responses. The above results suggested that more PD-1⁺ T cells arising in primary immunization may serve as major contributors to the slow antibody recall response elicited by HIV-1 Env.     

Effect of passive administration of alloantiserum containing antibody against putative acceptor(s) for T cell-replacing factor (TRF) in the neonatal stage on development of B cell activity responsive to TRF

Previously, we showed that the antiserum raised in male (DBA/2Ha X BALB/c)F1(DCF1) mice (T cell-replacing factor [TRF]-low response animals) by immunizing them with activated B cells from BALB/c mice (TRF-high-responders) contained antibodies against putative TRF-acceptor site(s). We have now evaluated the hypothesis that neonatal treatment of mice with the above antiserum suppresses the development of B cells responsive to TRF. Male DCF1 mouse anti-BALB/c B-cell antiserum or normal DCF1 mouse serum as a control was injected into BALB/c mice within 24 hr after birth. In the antiserum-treated mice, no augmented primary immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) were observed under the conditions in which markedly augmented IgM anti-SRBC responses were induced in control BALB/c mice, suggesting that development of B cells reacting with male DCF1 mouse anti-BALB/c B-cell antiserum is suppressed by the neonatal treatment with the antiserum. Furthermore, the development of B cell activity responsible for helper factors derived from T cells, such as TRF, was markedly suppressed in the neonatally antiserum-treated mice, whereas activity of B cells capable of interacting directly with helper T cells through antigen-bridges was not significantly affected by the same treatment. Such suppression of the B cell activity could be induced only when the antiserum was administered within 48 hr after birth. Moreover, neonatal treatment of mice with the antiserum induced suppressed responsiveness of B cells to a T-independent type 2 antigen, TNP-Ficoll. Neither serum-borne suppressive serum components nor suppressor cells were detected by the system employed. These results support the hypothesis that TRF responsive B cells constitute a subpopulation distinct from the other B cells capable of cooperating with helper T cells via cognate interaction.     

Complement Receptors 1 and 2 in Murine Antibody Responses to IgM-Complexed and Uncomplexed Sheep Erythrocytes

Early complement components are important for normal antibody responses. In this process, complement receptors 1 and 2 (CR1/2), expressed on B cells and follicular dendritic cells (FDCs) in mice, play a central role. Complement-activating IgM administered with the antigen it is specific for, enhances the antibody response to this antigen. Here, bone marrow chimeras between Cr2(-/-) and wildtype mice were used to analyze whether FDCs or B cells must express CR1/2 for antibody responses to sheep erythrocytes (SRBC), either administered alone or together with specific IgM. For robust IgG anti-SRBC responses, CR1/2 must be expressed on FDCs. Occasionally, weak antibody responses were seen when only B cells expressed CR1/2, probably reflecting extrafollicular antibody production enabled by co-crosslinking of CR2/CD19/CD81 and the BCR. When SRBC alone was administered to mice with CR1/2(+) FDCs, B cells from wildtype and Cr2(-/-) mice produced equal amounts of antibodies. Most likely antigen is then deposited on FDCs in a way that optimizes engagement of the B cell receptor, making CR2-facilitated signaling to the B cell superfluous. SRBC bound to IgM will have more C3 fragments, the ligands for CR1/2, on their surface than SRBC administered alone. Specific IgM, forming a complex with SRBC, enhances antibody responses in two ways when FDCs express CR1/2. One is dependent on CR1/2(+) B cells and probably acts via increased transport of IgM-SRBC-complement complexes bound to CR1/2 on marginal zone B cells. The other is independent on CR1/2(+) B cells and the likely mechanism is that IgM-SRBC-complement complexes bind better to FDCs than SRBC administered alone. These observations suggest that the immune system uses three different CR1/2-mediated effector functions to generate optimal antibody responses: capture by FDCs (playing a dominant role), transport by marginal zone B cells and enhanced B cell signaling.