Role of dendritic cells in the immune response to T-independent antigens of type 2

Dendritic cells (DC) belong to the most effective antigen-presenting cells. Their role in the presentation of thymus-dependent antigens and initiation of primary immune response is well known. At the same time, participation of DC in the immune response to T-independent antigens of type 2 (TI-2 antigens) is poorly explored. In this work, the ability of DC to initiate the immune response to a TI-2 antigen α(1→3) dextran (Dex) is investigated. Mouse bone-marrow-derived DC were generated by culturing the precursors with GM-CSF and then DC were pulsed by TI-2 antigens. The pulse induced DC activation, as was verified by an increase in the number of CD80 and CD86 positive cells. Uptake of FITC-labeled Dex was examined by flow cytometry. At a concentration of FITC-Dex of 100 μg/10⁶ cells, the number of DC binding the antigen (Ag) reached “plateau”. DC charged by TI-2 antigens were mixed with normal mouse splenocytes and cultivated in RPMI-1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells were determined by ELISPOT method. The mixtures of splenocytes and naïve DC not charged by the Ag were used as control. It was shown that the increase in the numbers of AFC and IFC under the influence of naïve DC did not exceed 20%. On the contrary, the addition of DC pulsed by the Ag increased specific immune response more than twofold. The data obtained point to the direct interactions of DC with TI-2 antigens. Pulsed DC present TI-2 antigens to mouse splenocytes and induce specific and polyclonal B-cell activation, i.e., possess immunostimulating activity.     

Model system for study of cell interactions and mechanisms of immune response to T-independent antigens of type 2 in vitro

Cellular mechanisms of immune response to type 2 T-independent antigens (TI-2 antigens) are not fully elucidated up till now. In vitro system is the most convenient model for such studies. However, in vitro model requires relatively high cell density in the cultures. It hampers the study of minor lymphocyte subsets like CD5+ B-1 splenocytes, which play the main role in the immune response to TI-2 antigens. The use of cell mixtures of normal and immunodeficient congenic animals may help to resolve this problem. In this work, immune responses to TI-antigens of type 1 (TI-1 antigens) and to TI-2 antigens in vitro were studied in the mixtures of cells of normal (CBA) and congenic xid-mice (CBA/N). CBA/N mice lack CD5+ B-1 cells and do not respond to TI-2 antigens. Therefore, their splenocytes can be used as “filler” cells to create the optimal cell density in the cell cultures. Spleen and peritoneal cells of CBA mice and B-1 and B-2 lymphocytes isolated from peritoneum and spleen, respectively, were cultured in different proportions with CBA/N splenocytes with or without antigens. LPS and polyvinylpyrrolidone (PVP) were used as TI-1 and TI-2-antigens, respectively. Antibody- and immunoglobulin-forming cells (AFC and IFC, respectively) were determined by the ELISPOT method on the 4th day of cultivation. It was shown that CBA and CBA/N cells in mixed cell cultures retained their functional activity. Splenocytes of CBA mice responded to both TI-antigens. Splenocytes of CBA/N mice responded to TI-1 antigen (LPS) only. It means that in vitro B-1 cells play the main role in the immune response to TI-2 antigens, as they do in vivo. Thus, the developed model system can be used to study cellular mechanisms of immune response to TI-1 and TI-2 antigens in vitro.     

TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens

The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.     

Production of monoclonal antibodies to T-independent type 2 antigens with OptiMem-Iscove medium

To obtain monoclonal antibodies to T-independent antigens of type 2, having low immunogenicity and incapable of inducing the appearance of memory cells, the use of the medium OptiMem-Iscove (1:1) with 10% of fetal serum, glutamine (50 mM) and antibiotics (100 units/ml) is proposed. The main advantage of this medium is the possibility of cloning cells without the use of feeders. The method has been approved in the process of obtaining monoclonal antibodies (McAb) to TI-2 antigens, both bacterial (S3) and synthetic (PVP), as well as to McAb to T-dependent antigen (alpha-chains of human IgA).     

Different effects of cortisone on the humoral immune response to T-dependent and T-independent antigens.

The effect of the administration of cortisone on the murine humoral immune response to either thymus-dependent (TD) or -independent (TI) antigens was studied in vivo. Whereas the thymus-dependent immune response was markedly suppressed, the thymus-independent immune response was preserved. The opposing effect of steroids on these two types of immune responses appears to be due to the relative independence of thymus-independent antigens of a radioresistant cortisone-sensitive accessory cell.     

Secretion and role of interleukin-1-like factor in the antibody response to dinitrophenyl dextran and other type 2 T-independent antigens

Splenic adherent cells (SAC) were found to produce a humoral factor when they were cultured with dinitrophenyl-dextran or some other type 2 T-independent (TI-2) antigens. The factor substituted adherent cells in in vitro antibody responses to TI-2 antigens, and acted in an antigen-nonspecific and H-2-nonrestricted manner. T cells were indicated not to participate in the production of the factor. The factor was eluted from a Sephadex G-75 column with interleukin-1 (IL-1) activity to promote a thymocyte proliferation response to phytohemagglutinin. The molecular weight of the factor was estimated to be 16,000 Da. Both activities of the factor were absorbed by LBRM-33-1A5 cells. These results indicate that SAC secrete IL-1-like factor on direct stimulation by TI-2 antigens and that the secretion of the factor represents a major function of SAC in the antibody response to these antigens.     

Novel vaccine strategies to T-independent antigens

T cell independent antigens do not require T cell help to induce an immune response, and are characterized by a lack of immunologic memory. These antigens can be divided into two classes, TI-1 or TI-2. TI-1 antigens, such as bacterial lipopolysaccharide, are potent B-cell mitogens, capable of non-specific, polyclonal activation of B cells. In contrast, TI-2 antigens can only activate mature B cells and consist of highly repetitive structures, such as capsular polysaccharides (CPS) from bacteria. Many vaccines currently in use consist of purified capsular polysaccharides from pathogenic bacteria such as Streptococcus pneumoniae and Neisseria meningitidis. These vaccines are efficacious in immune-competent adults, however, due to their TI-2 nature, are not effective in children <2 years of age. Converting polysaccharides into T cell dependent (TD) antigens, allows children, <2, to produce an effective immune response. This review focuses on various strategies used to convert the immune response to polysaccharide antigens from TI-2 to a TD response. Conjugate vaccines, anti-idiotypic antibodies, phage display library technology and DNA vaccines are discussed.     

Hypophysectomy and neurointermediate pituitary lobectomy decrease humoral immune responses to T-independent and T-dependent antigens

In rats, hypophysectomy (HYPOX) or neurointermediate pituitary lobectomy (NIL) reduce humoral and cell-mediated immune responses. However, to our knowledge, the differences in the effects of anterior versus posterior pituitary hormones on the immune responses have not been studied to date. We compared in rats, the effects of sham surgery (SHAM), HYPOX, and NIL on humoral immune responses to T cell-independent (TI) type 1 antigen DNP-LPS and to TI type 2 antigen DNP-FICOLL, as well as to T cell-dependent (TD) antigens ovalbumin (OVA) and bovine serum albumin (BSA). The results showed that: (1) both HYPOX and NIL induced a similar and significant decrease in IgM responses towards TI-1 antigens, (2) NIL but not HYPOX induced a decreased IgM response to TI-2 antigens, and (3) both HYPOX and NIL induced similar and significant decrease in IgG responses to TI-2 antigens. Compared with the SHAM group, IgM responses to both TD antigens did not change in HYPOX and NIL animals, whereas the IgG responses to OVA and BSA significantly decreased in HYPOX and NIL animals. These results indicate that hormones of the anterior and posterior pituitary play their own role in the regulation of humoral immune responses.     

Mouse immune response to meningococcal antigens

The mouse immune response against Neisseria meningitidis was studied by using an extract from group Y (Slaterus) known to contain protein antigens common to other meningococci. By using a solid-phase radioimmunoassay, high titers of specific IgM and IgG class antibodies were measured which lasted over 2 months after immunization. These antibodies cross-reacted with similar extracts from other meningococci groups. Bactericidal antibodies directed against protein antigens were also elicited after immunization and they belonged to IgM, IgG2a, and IgG2b isotypes. Cellular immunity, expressed as delayed type hypersensitivity under the conditions tested, could be detected neither in homologous nor heterologous reactions.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Humoral immune response to filarial antigens in chyluria

Humoral immune parameters like total immunoglobulins and specific antibody levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly reduced in this group compared to normal controls, while IgA and IgM levels remained comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked immunosorbent assay also was significantly reduced. However, the total and specific IgE antibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could be related to their microfilaraemic status.     

Directing the immune response to carbohydrate antigens

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.     

Role of H-2 antigens in the host response to methylcholanthrene-induced tumors

H-2 loss variant sublines of a sarcoma (M-AS), induced by methylcholanthrene in an (A × A.SW)F₁ mouse, were used to study the role of the MHC products in the recognition of MC-TSTA. The two reciprocal variant sublines (M-A and M-S) were found to express the TSTA of the original tumor as shown by cross-reactions in graft rejection experiments performed in (A × A.SW)F₁ mice. In the A/Sn and A.SW mice the presence of the reciprocal parental H-2 antigens on the immunizing cells decreased the response against the tumor antigens. An admixture of lymphocytes derived from hyperimmune mice inhibited the outgrowth of the tumor cells. The growth inhibition was mediated by T cells and was H-2 restricted. Cells derived from hyperimmune syngeneic mice inhibited the outgrowth of the variant subline used for immunization but had no effect on the reciprocal variant subline.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Mitogens and T-independent antigens stimulate T lymphocytes to secrete La antigens.

Previous reports from this laboratory suggest that certain I region-associated (Ia) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125-fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes.All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125-fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.     

Cell-mediated immune response of marmosets to herpesvirus-associated antigens in vitro

Summary Immune responses in vitro of some species of marmosets to herpesvirus-associated antigens expressed on virus-transformed lymphoblastoid cell lines (LCL) were studied by determining lymphocyte proliferation, interferon production, and the induction of cytotoxic effector cells in mixed lymphocyte-LCL cultures (MLLC). Autologous Epstein-Barr virus (EBV)-transformed B-cell lines induced neither lymphocyte proliferation nor interferon production in MLLC, while autologous Herpesvirus ateles (HVA)-transformed T-cell lines stimulated responder cell DNA synthesis and interferon production. Both EBV-LCL and HVA-LCL failed to induce cytotoxic effector cells in autologous MLLC responder cells. These findings differ markedly from the human immune response to autologous EBV-LCL in vitro and may have implications for the unusual susceptibility of marmosets to the induction of lymphoproliferative disease following inoculation with oncogenic herpesviruses.     

Computational modeling of the immune response to tumor antigens

Vaccination protocols designed to elicit anti-cancer immune responses have, many times, failed in producing tumor eradication and in prolonging patient survival. Usually in cancer vaccination, epitopes from one organism are included in the genome or linked with some protein of another in the hope that the immunogenic properties of the latter will boost an immune response to the former. However, recent results have demonstrated that injections of two different vectors encoding the same recombinant antigen generate high levels of specific immunity. Systematic comparison of the efficacy of different vaccination protocols has been hampered by technical limitations, and clear evidence that the use of multiple vectors has advantages over single carrier injections is lacking. We used a computational model to investigate the dynamics of the immune response to different anti-cancer vaccines based on randomly generated antigen/carrier compounds. The computer model was adapted for simulations to this new area in immunology research and carefully validated to the purpose. As a matter of fact, it reproduces a relevant number of experimental observations. The model shows that when priming and boosting with the same construct, competition rather than cooperation develops amongst T cell clones of different specificities. Moreover, from the simulations, it appears that the sequential use of multiple carriers may generate more robust anti-tumor immune responses and may lead to effective tumor eradication in a higher percentage of cases. Our results provide a rational background for the design of novel strategies for the achievement of immune control of cancer.