Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.     

Suppression of IgM responses to type III pneumococcal polysaccharide by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) suppressed the primary IgM response of mice to Type III pneumococcal polysaccharide (S3). LPS and S3 had to be given together in order for maximum suppression to occur and suppression was not due to a change in the time of the peak response in mice which received LPS. Suppression was not due to an effect of LPS on T cells since S3 responses of nude mice were suppressed by LPS. LPS did not suppress S3 responses of C₃H/HeJ mice and base hyrolysis of LPS destroyed the suppressive effect, i.e., suppression was dependent of B cell mitogenic activity of the LPS. The suppressive effect of LPS was presumably directed against virgin B cells since LPS did not suppress the S3 response of primed mice and did not suppress the development of IgG memory responses to S3.     

Suppression of IgG memory responses by T cells activated with the type 2 antigen polyvinylpyrrolidone (PVP)

Although type 2 antigens, such as polyvinylpyrrolidone (PVP), generally do not prime for IgG memory responses or activate specific helper T cells (TH), previous studies have established that low doses of PVP (0.0025 microgram) can prime for IgG memory and induce TH in vivo. Doses of PVP that are optimally immunogenic for IgM antibody production (0.25-25 micrograms) do not prime for IgG memory responses and preferentially activate PVP-specific suppressor T cells (TS) which suppress IgG antibody production. The studies reported here further characterize PVP-specific TS and begin to investigate the mode of action of these TS. TS induced with high doses of PVP have a typical suppressor cell surface phenotype in that they are Lyt 2+, I-J+, L3T4-, I-A- T cells. PVP-specific TS are inducible in mice expressing the X-linked immune defect and are Igh restricted in their actions. These TS suppress PVP-specific IgG responses of PVP-HRBC (horse red blood cells)-primed B cells when the TH population is from low-dose PVP-primed mice but not when the TH population is from PVP-HRBC-primed mice. Thus the TS do not apparently directly suppress the B-cell responses but act indirectly to suppress IgG responses by preventing the expression of PVP-specific TH function. The TS induced by 0.25 microgram PVP also prevent the generation of PVP-specific memory B cells apparently by preventing the expression of functional TH which are required for induction of memory B cells. Elimination of TS activation by pretreatment of mice with cyclophosphamide at the time of priming with 0.25 microgram PVP results in the expression of TH function and priming of memory B cells.     

Amplified B lymphocyte CD40 signaling drives regulatory B10 cell expansion in mice

Background

Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154^TG) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22^−/−) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154^TGCD22^−/− mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation.

Methodology/Principal Findings

CD154^TGCD22^−/− mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154^TGCD22^−/− mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154^TGCD22^−/− mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10⁶±6 in CD154^TGCD22^−/− mice; 1.7×10⁶±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10⁶±3 in CD154^TGCD22^−/− mice; 6.1×10⁶±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells.

Conclusions/Significance

These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.     

Secondary IgG responses to type III pneumococcal polysaccharide. II. Different cellular requirements for induction and elicitation.

Mice primed with a thymus- (T) dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to erythrocytes (S3-RBC) produce S3-specific IgG antibody after secondary challenge with either S3 or S3-RBC. The production of IgG antibody by mice challenged with S3 was shown to be T independent since secondary responses were enhanced when mice were treated with anti-lymphocyte serum (ALS) at the time of secondary challenge with S3 and T-depleted spleen cells responded as well as unfractionated spleen cells to S3 in an adoptive transfer system. Secondary S3-specific IgG responses in mice challenged with S3-RBC were shown to be T dependent by the same criteria. The results obtained by using S3 as the antigen indicate that IgG-producing B cells (B lambda cells) can recognize and respond to antigen in the absence of helper T cells. On the other hand, T cells were required for the induction of S3-specific memory B lambda cells since mice depleted of T cells by treatment with ALS at the time of priming with S3-RBC failed to produce S3-specific IgG antibody after secondary challenge with either S3-specific IgG antibody after secondary chall-nge with either S3 or S3rbc. Since RBC-specific memory cells were induced in T-deprived mice the results suggest that T cell regulation of IgG antibody production may vary for different antigens.     

Vesicular stomatitis virus Indiana glycoprotein as a T-cell-dependent and -independent antigen.

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen froms of VSV, VSV antigen doses could be graded in vivo (infectious > > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.     

IgM-mediated enhancement of in vivo anti-sheep erythrocyte antibody responses: isotype analysis of the enhanced responses

The direct splenic anti-sheep erythrocyte (anti-SRBC) responses as well as the serum IgG1, IgG2a, IgG2b, and IgG3 anti-SRBC responses of CBA/CaJ mice were monitored 4-35 days after immunization with: (1) a suboptimal dose of SRBC, (2) a suboptimal dose of SRBC plus monoclonal IgM anti-SRBC, or (3) a high dose of SRBC. The direct plaque-forming cell (PFC) responses of mice in treatment group 2 were significantly higher than those in group 1 but similar to the responses in group 3. The serum anti-SRBC antibody responses of all IgG subclasses were significantly enhanced by IgM anti-SRBC and were generally even higher than the responses obtained with high doses of SRBC. The relative proportions of each serum IgG subclass were similar in all three groups. These data suggest that the enhancement of suboptimal anti-SRBC antibody responses by IgM anti-SRBC extends through IgM and all of the IgG subclasses and, further, that the isotype profile in antibody-enhanced responses is similar to that obtained with high doses of SRBC.     

Antibody-mediated modulation of the immune response

We have studied the ability of monoclonal IgM and IgG antibodies to enhance or suppress immune responses and attempted to dissect the underlying mechanisms. Both IgM and IgG1 antibodies increased the rate of clearance of antigen from the circulation. Monoclonal IgM antibody to SRBC was found to specifically increase antibody responses, enhancement being insensitive to low doses of irradiation (150 R). IgM antibody specifically depressed the delayed hypersensitivity response to SRBC in vivo. Following administration of IgM in vivo, in vitro responses to SRBC were also enhanced. This in vitro enhancement appeared to depend on both T cells and B cells. In contrast, monoclonal IgG1 antibody to SRBC specifically depressed antibody responses in vivo. Such depressed antibody responses were also seen in vitro following IgG1 in vivo and did not appear to be due to the induction of suppressor T cells.     

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG⁺ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS⁺ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice

The cytotoxic antibody response to the H-2D^b alloantigen has been investigated in ten strains of the C57BL/10 background. Three types of responses could be distinguished: no detectable response, an IgM response, and an IgG response. The IgG response is influenced by the D and probably the I-A region of the H-2 complex, whereas the IgM response is dependent on the allele for the E chain. The hypothesis is proposed that regulatory T cells, which recognize the antigen in context of self MHC molecules, determine the outcome of an anti-H-2D^b immunization in which the I-E molecule restricts the IgM response and the I-A molecule restricts the IgG response; the D molecule is probably responsible for activation of suppressor T cells which suppress only the IgG response.     

Conversion of type III pneumococcal polysaccharide low responders to high responders by immunization with a thymus-dependent form of antigen

C57BL/Ks mice immunized with 0.6 μg Type III pneumococcal polysaccharide (S3) or with 10⁹ S3 conjugated sheep erythrocytes (S3-SRBC) produced 5–7 times fewer S3-specific plaque-forming cells than similarly immunized BALB/c mice. However, when mice were primed with the SRBC carrier prior to challenge with S3-SRBC the low responder C57BL/Ks mice responded as well as the high-responder BALBc strain. The cell activated by the carrier priming was shown to be a thymus-derived (T) cell and the antibody produced by primed mice was mercaptoethanol sensitive (presumably IgM). Nonspecific T cell activation by unrelated antigens did not enhance C57BL/Ks responses to the same degree as specific carrier priming. These findings are discussed in relation to the possible cellular basis for genetic control of the S3 immune response.     

Induction of Antiviral Antibodies by DNA Immunization Requires neither Perforin-Mediated nor CD8+-T-Cell-Mediated Lysis of Antigen-Expressing Cells

DNA immunization induces antibodies to the encoded protein, which indicates that the protein must gain access to the extracellular milieu, allowing it to interact with naïve B lymphocytes. It has been suggested that antigen release may be effected by cytotoxic-T-lymphocyte-mediated lysis of transfected antigen-expressing cells; this might be particularly important for the induction of responses to a noncytopathic, cytosolic protein. Here we show that the induction of antibody responses to one such DNA-encoded protein required neither perforin nor CD8⁺ T cells. In addition, there was no skewing of the immunoglobulin G isotypes in the absence of perforin.DNA immunization is unique in that antigenic proteins are synthesized within the transfected cell in the absence of any associated infectious agent, and the vaccine itself contains no soluble protein that could initiate humoral immune responses. Thus, if the immunizing plasmid expresses a cytoplasmic protein which is relatively noncytopathic and is unable to be effectively processed endogenously by the major histocompatibility complex (MHC) class II antigen presentation pathway, then it might be ineffective in inducing humoral immune responses. The nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) meets these criteria, yet intramuscular injection with a plasmid expressing the NP (pCMV-NP) has been shown by us and others to induce antibody responses in mice (13, 23, 24). Antibodies are also induced following DNA immunization with plasmids expressing other cytoplasmic antigens, such as the measles virus and influenza virus NPs (4, 18, 21). How might such antigens be released from the transfected cell? We hypothesized that the development of CD8⁺ antigen-specific cytotoxic T lymphocytes (CTL) could lead to the recognition and lysis of cells expressing plasmid-derived NP, resulting in the liberation of protein into the extracellular milieu, where it can then interact with B cells and antigen-presenting cells (APCs). CTL-mediated release of LCMV NP occurs during virus infection (12), and the possibility that it occurs following DNA immunization was strengthened by our observations of a profound myositis following intramuscular inoculation of pCMV-NP into LCMV-infected or -immune mice; the peak of the inflammatory infiltrate in acutely infected mice coincided with the development of anti-LCMV CTL, and destruction of muscle cells occurred (8, 22). Professional APCs, which are known to be a source of plasmid-expressed antigens (5) and which appear to be the cell type responsible for initiating immune responses following DNA immunization (6, 7), can also be recognized and lysed by CTL. Dendritic cells infected with human immunodeficiency virus are susceptible to lysis in vitro by CTL (11), and there is evidence to suggest that CD8⁺ T cells can limit the immune response by lysing APCs in vivo (1–3). Therefore, the possibility of lytic release of protein is not limited to NP-expressing myocytes but extends to most transfected somatic cells, including APCs. To assess what effect such lysis may have on the generation or maintenance of B-cell responses following DNA immunization, we analyzed humoral immune responses in DNA-immunized mice that lacked the cytolytic protein perforin (10, 20). Although antigen-specific CD8⁺-T-cell responses were induced in these mice by vaccination with pCMV-NP, they were unable to lyse NP-expressing cells in a perforin-dependent manner (data not shown).

Strong antibody responses are induced by DNA immunization of PKO mice.

To determine if a lack of perforin-mediated lysis by antigen-specific CTL resulted in an alteration in the temporal appearance or maintenance of antiviral serum antibodies, antibody levels were measured in PKO and C57BL/6 mice at 0, 2, 4, and 6 weeks after they received a single 50-μg intramuscular injection of pCMV-NP DNA. Serum immunoglobulin G (IgG) levels in individual mice were measured by enzyme-linked immunosorbent assay (ELISA), and the average for each group was calculated based upon the optical density measurement at a dilution of 1:200; the results are shown in Fig. ​Fig.1A.1A. Within 2 weeks of vaccination, anti-LCMV antibodies were demonstrable in both PKO and C57BL/6 mice, and the average levels in both groups were similar. The slight drop in antibody levels in the C57BL/6 mice at 4 weeks after DNA immunization is not statistically significant, but at 6 weeks, the difference is highly significant. In mice, the average half-life of an IgG molecule is approximately 6 to 10 days (17, 19). Therefore, the high level of antibodies present at 6 weeks postimmunization implies ongoing synthesis of NP-specific IgG in PKO mice, whereas the drop in antibody levels indicates decreased IgG synthesis in C57BL/6 mice. A representative analysis of the antibody response at 6 weeks postimmunization is presented in Fig. ​Fig.1B.1B. Anti-LCMV serum antibody levels were measured by ELISA in individual perforin-positive (C57BL/6, n = 6) or perforin-negative (PKO, n = 8) animals 6 weeks postvaccination. LCMV DNA-vaccinated C57BL/6 and PKO mice both produced anti-LCMV IgG. Together, these data clearly show that perforin-mediated release of plasmid-expressed LCMV NP is not required for the induction of humoral responses following intramuscular DNA injection. Open in a separate windowFIG. 1PKO mice mount strong antibody responses. (A) PKO and C57BL/6 mice (n = 4/group) were immunized with pCMV-NP and bled biweekly. Anti-LCMV serum antibodies were measured by ELISA. The mean optical density readings at 492 nm (OD₄₉₂) are shown for serum dilutions of 1:200, with standard errors. (B) Representative analyses of antibody responses at 6 weeks postimmunization. Anti-LCMV antibody levels in groups of DNA-immunized PKO (n = 8) and C57BL/6 (n = 6) mice were measured by ELISA. For controls, sera were derived from LCMV-immune or nonimmune C57BL/6 mice.

Isotype responses are unaltered in DNA-immunized perforin-deficient mice.

IgG responses to the LCMV NP require CD4⁺-T-cell help, and unlike the LCMV glycoprotein, endogenously synthesized NP cannot gain access to the MHC class II antigen presentation pathway (15). Thus, the CD4⁺ T cells required for immunoglobulin isotype class switching must be primed by APCs which have acquired extracellular NP. To determine if the isotype pattern was skewed in the absence of perforin, titers of LCMV-specific IgG, IgG1, IgG2a, and IgG2b were determined by ELISA in individual PKO and C57BL/6 mice 6 weeks after DNA immunization (Fig. ​(Fig.2).2). At this time point, all of the pCMV-NP DNA-immunized mice contained virus-specific immunoglobulin in their serum. Anti-LCMV IgG endpoint titers among PKO mice ranged from 1:4,000 to 1:15,000, with a geometric mean titer (Fig. ​(Fig.2)2) of 1:6,825. In contrast, among the C57BL/6 vaccines, the total IgG titers ranged from 1:1,700 to 1:3,400, with a geometric mean of 1:2,455. Detectable IgG1 and IgG2a were present in the sera of all mice of both strains with the exception of a single PKO mouse which lacked demonstrable levels of anti-LCMV IgG1. Therefore, the vaccinated PKO mice showed no skewing of their isotype classes, suggesting that CD4⁺-T-cell responses are appropriately induced in these mice and providing further evidence that perforin is not required for the release of NP from transfected cells. Open in a separate windowFIG. 2No skewing of antibody isotypes in PKO mice. IgG isotype titers were measured in individual PKO and C57BL/6 mice at 6 weeks after immunization with pCMV-NP. Triangles, individual mice; circles, geometric mean titers for each group. The LCMV-immune sample represents pooled sera from virus-immune C57BL/6 mice.

CD8⁺ T cells play no part in controlling humoral responses to DNA-encoded antigens.

Our data show that perforin is not required to prime B-cell responses against a plasmid-expressed, cytoplasmic antigen. Perforin is expressed by CD8⁺ CTL, some CD4⁺ T cells, and natural killer (NK) cells; we can therefore conclude that perforin-mediated lysis by any of these cell populations is not required for DNA-mediated antibody induction. However, other CD8⁺-T-cell functions, such as the Fas pathway (16) or the secretion of cytotoxic cytokines, can mediate target cell death and thus could liberate intracellular antigens (9). Our observation that PKO mice had elevated antibody titers compared to those of C57BL/6 mice (Fig. ​(Fig.11 and ​and2)2) also raised the possibility that perforin-mediated lysis might actually depress humoral responses to DNA immunization by killing cells expressing foreign plasmid-encoded antigens. Consistent with this idea, CD8⁺-T-cell-mediated lysis of APCs has been shown to suppress immune responses (1–3), and perforin has been shown to play a role in regulating immunity (14). If this hypothesis is correct, the absence of perforin might result in elevated and/or prolonged responses to DNA-encoded immunogens. To determine whether CD8⁺ T cells might play a perforin-independent role in antigen release and antibody induction, CD8⁺ T cells were removed from C57BL/6 mice by immunodepletion prior to and throughout the course of a DNA immunization experiment. C57BL/6 mice were treated with either an anti-mouse CD8⁺-T-cell monoclonal antibody or saline daily for 3 days prior to immunization with pCMV-NP and weekly thereafter. Nondepleted PKO mice served as controls. Mice were bled biweekly, and the average IgG response for each group is shown in Fig. ​Fig.3A.3A. Antibody responses were easily detected in CD8-depleted mice, showing that CD8⁺-T-cell functions are not required to release soluble NP for the initiation of humoral immune responses. Furthermore, at all time points, the antibody responses in CD8-depleted and nondepleted C57BL/6 mice were indistinguishable; therefore, it appears to be unlikely that CD8⁺ T cells play a role in down-regulating DNA-induced antibody responses. To confirm that the CD8⁺ cells had been successfully ablated by the monoclonal antibody treatment, splenocytes were obtained from mice immediately before DNA immunization (data not shown) and from three mice (two depleted and one sham-depleted) at the conclusion of the experiment and were analyzed for CD8 expression by flow cytometry (Fig. ​(Fig.3B).3B). The administration of anti-CD8 monoclonal antibody maintained CD8⁺-T-cell populations at almost undetectable levels throughout the course of the experiment. Thus, we conclude that the development of fully functional CD8⁺ CTL following DNA immunization neither inhibits nor enhances humoral immune responses. Open in a separate windowFIG. 3Depletion of CD8 cells has no effect on antibody induction by plasmid DNA. (A) Anti-LCMV antibody titers were measured over time in C57BL/6 mice depleted of CD8⁺ T cells (C57+anti CD8), nondepleted C57BL/6 mice (C57+saline), and nondepleted PKO mice (PKO+saline). Each group consisted of three animals, and the average antibody endpoint titer for each group at each time point is shown. (B) To confirm successful depletion of CD8⁺ cells, splenocytes were harvested from two mice treated with anti-CD8 antibody and from one control mouse. These cells were evaluated for CD8 expression by flow cytometry.These data argue against the hypothesis that lytic-antigen-specific CD8⁺ CTL have a profound effect on the generation of humoral immunity to plasmid-encoded antigens. Perforin is normally expressed not only in CD8⁺ T cells but also in some CD4⁺ T cells and in NK cells. Our observations therefore are not restricted to perforin-mediated CD8⁺-T-cell lysis but also show that perforin-mediated lysis by CD4⁺ T cells or NK cells plays no essential role in antigen release. Furthermore, the ratios of IgG1 to IgG2a were similar regardless of the perforin status of the mouse (Fig. ​(Fig.2).2). Thus, not only is perforin expendable in IgG induction but also its absence has no effect on the antibody classes—and, presumably, the ratio of Th1 to Th2 cells—induced.In conclusion, it is clear that plasmids encoding proteins which are relatively noncytotoxic and incapable of endogenous MHC class II antigen presentation can elicit strong IgG responses in the absence of CD8⁺ T cells and perforin. Thus, the mechanism of antigen release and B-cell stimulation remains to be determined. It is important that we identify the underlying mechanisms to permit the rational optimization of DNA vaccines.     

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.     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

Different Dynamics for IgG and IgA Memory B Cells in Adolescents following a Meningococcal Serogroup C Tetanus Toxoid Conjugate Booster Vaccination Nine Years after Priming: A Role for Priming Age?

Background

Antibody levels wane rapidly after Meningococcal serogroup C conjugate (MenCC) vaccination in young children, rendering the need for an adolescent booster dose. It is not clear whether circulating memory B cells are associated with persistence of MenC-specific antibody levels.

Methods

Measurement of MenC-specific IgG and IgA memory B cells and levels of serum and salivary MenC-specific IgG and IgA in healthy 10-, 12- and 15-year-olds prior to and one month and one year after a MenCC booster vaccination. All participants had received a primary MenCC vaccination nine years earlier.

Results

The number of circulating MenC-specific IgG memory B cells prior to booster was low and not predictive for MenC-specific IgG responses in serum or saliva post-booster, whereas the number of MenC-specific IgA memory B cells pre-booster positively correlated with MenC-specific IgA levels in saliva post-booster (R = 0.5, P<0.05). The booster induced a clear increase in the number of MenC-specific IgG and IgA memory B cells. The number of MenC-PS-specific IgG memory B cells at 1 month post-booster was highest in the 12-year-olds. The number of MenC-specific memory B cells at one month post-booster showed no correlation with the rate of MenC-specific antibody decay throughout the first year post-booster.

Conclusions

Circulating MenC-specific IgA memory B cells correlate with IgA responses in saliva, whereas circulating MenC-specific IgG memory B cells are not predictive for MenC-specific IgG responses in serum or saliva. Our results are suggestive for age-dependent differences in pre-existing memory against MenC.     

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.     

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose,short-term experimental model

Background

The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.

Objectives

This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses.

Methods

Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 μg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 μg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE.

Results

Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 μg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses.

Conclusions

The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.     

Pretargeted radioimmunotherapy using 131I-labelled bivalent hapten-bearing peptides

The advantages of bivalent hapten-bearing peptides for the detection oftumours pretargeted with bispecific antibodies have been demonstrated. Thistechnology is now considered for radioimmunotherapy and bivalent haptensdesigned to target ¹³¹I are needed. We thus synthesised aseries of tyrosine-containing peptides bearing the histamine-hemisuccinatehapten. These molecules were tested for their ability to bind simultaneouslytwo anti-hapten antibody molecules. One of these bivalent haptens, AG3.0,with a lysyl-d-tyrosyl-lysine connecting chain, was found to have optimalbinding characteristics and was thus selected for further investigations.AG3.0 was shown to efficiently deliver radioactive iodine to humancolorectal tumours grafted in nude mice using an anti-carcinoembryonicantigen×anti-histamine-hemisuccinate bispecific antibody. AG3.0 wasalso targeted to human B lymphoma cells pretargeted with a bispecificantibody specific for membrane IgM. In this system, bivalent ligands such asF(ab)₂ or IgG are rapidly internalised and covalentlylinked radioactive iodine is released from target cells as a result ofintracellular catabolism. With the pretargeted iodine-labelled bivalenthapten, a fivefold increase in the intracellular activity retention time ascompared to ¹²⁵I-labelled F(ab)₂ and IgGwas observed. The radiolabelled hapten did not undergo any degradation afterinternalisation. These results have been confirmed in vivo with ananti-BCL₁ IgM idiotype bispecific antibody and¹³¹I-labelled AG3.0. These reagents injected as a single 300µCi dose, 7 days after inoculation of 10⁴BCL₁ lymphoma cells in BALB/c mice, cured 14/16 of the animalsand the treatment was well tolerated. Comparatively, the same dose oflabelled IgG cured 13/16 of the mice but three mice died of haematologictoxicity. The same dose of labelled F(ab)₂ orFab was completely inefficient. was completely inefficient. ¹³¹I-labelled bivalenthaptens are now used in phase I radioimmunotherapy clinical trials.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.