Relation of the formation of producers of antigen-dependent nonspecific immunoglobulins to the dose of T-dependent and T-independent antigens

The dependence of the production of antibody-forming cells (AFC) and non-specific immunoglobulin-forming cells (nIFC) on the doses of T-dependent (sheep red blood cells, SRBC) and T-independent (polyvinylpyrrolidone, PVP and pneumococcal polysaccharide SSS III) antigens was investigated. The immunization of BALB/c mice with immunogenic or subimmunogenic doses of SRBC and PVP induced a marked increase in the number of antigen-dependent nIFC. In contrast, the injection of any SSS III doses did not influence the amount of nIFC, although a specific immune response to SSS III was quite obvious. Thus, two T-independent antigens, type II, differ in their ability to induce non-specific immune reactions. The experiments on simultaneous administration of monoclonal anti-Thy-1.2 antibodies and PVP or SSS III to mice have demonstrated that these differences were not related to T-suppressor activity. The possible role of T helpers in the immune response to T-independent antigens is discussed.     

Effect of unilateral nephrectomy in mice on the level of the humoral immune response to T-independent antigen

The influence of unilateral nephrectomy on the degree of humoral immune response to T-independent (polyvinylpyrrolidone, PVP) and T-dependent (sheep red blood cells, SRBC) antigens was studied. The increase in the number in antibody-forming cells (AFC) and nonspecific immunoglobulin-forming cells (nIFC) was investigated by means of the adaptive transfer model. The lethally irradiated recipients were injected with the antigen and also the spleen cells of operated and intact donors. PVP did not induce significant alterations of antibody genesis in mice receiving spleen cells of unilaterally nephrectomized animals comparing with recipients of intact spleen cells. At the same time, the kidney operation induced the increase in the number of AFC and nIFC when the SRBC were used. Hence the activation of humoral immune response induced by kidney operation was related not to the direct activation of B-lymphocytes but to T-cells. The possible causes of this activation were analyzed. Spleen cells of operated animals enhance both specific and antigen-dependent nonspecific immune response.     

Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny of B-lymphocyte function: XIII. Kinetics of maturation of neonatal B lymphocytes to produce a heterogeneous antibody response to a T-independent antigen

The ontogeny of the capacity of a B-cell population to produce a heterogeneous, relatively high-affinity plaque-forming cell (PFC) response to the T-independent antigen trinitrophenylated-Ficoll (TNP-F) was studied in a cell transfer system. Lethally irradiated mice were reconstituted with liver cells from neonatal donors and were immunized with TNP-F at various times thereafter. In contrast to the results of our previous studies on the ontogeny of the response to T-dependent antigens, it was found that, in the cell transfer recipient, the response of an immature B-cell population to a T-independent antigen matures slowly (21–28 days). Furthermore, this maturation does not appear to require the presence of adult thymus cells as does the maturation of the response of a B-cell population to T-dependent antigens. Thus, it appears that the acquisition of the capacity of a B-cell population to produce a high-affinity, heterogeneous, PFC response to T-dependent and T-independent antigens occurs under different regulatory influences.     

Evolution of the lymphoid system. IV. Murine T-independent but not T-dependent antigens are very immunogenic in rainbow trout (Salmo gairdneri).

Adult rainbow trout (RBT) were found to respond well to murine T-independent antigens but not to T-dependent antigens. In addition, the ability of RBT to respond with adult intensity to at least one murine T-independent antigen occurred very early in their ontogenetic development. Finally, subsequent to antigenic challenge, it was observed that while large numbers of plaque-forming cells appeared in the spleen and anterior kidney, none were found in the thymus.     

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.     

Immunosuppression induced by respiratory viruses (influenza virus, adenovirus) in mice

The influence of respiratory viruses (adenovirus, influenza virus) on humoral immune response to heterologous T-dependent and T-independent antigens was studied. It was shown that inoculation of mice by the influenza virus (A/PR8/34-A/PR/8) 3 days before sheep red blood cells administration led to the inhibition of antibody forming cell (AFC) and immunoglobulin, forming cell (IFC) increase on 69% and 59% respectively. Adenovirus type 6 induced the similar suppression of AFC and IFC formation. Thus, viruses induced immuno-suppression, which was polyclonal. It was also shown that virus of one strain (type) could inhibit immune response to another strain (type) of virus. The immune response to T-independent antigen was not suppressed. The virus-induced immunosuppression was dependent on: 1) the infectivity of respiratory viruses, 2) the route of virus and heterologous antigen injection, and 3) the interval between the viruses and antigen inoculation.     

Clonal analysis of B-lymphocyte subpopulations separated on the basis of Lyb surface antigens

The aim of these experiments was to see whether antisera of the Lyb series could be used to identify B cells capable of responding differentially in T-dependent and T-independent systems. The antisera tested were against the alloantigens Lyb 1.1,2.1,3,4.1,5.2, and LyM 1. A polyvalent sheep anti-mouse immunoglobulin (Ig) antibody acted as a positive control for the identification of B cells. As a first step, all spleen B cells were treated to remove this surface Ig by a capping procedure. They were then washed, reacted with a mouse alloantiserum, and allowed to form rosettes with sheep erythrocytes to which a sheep anti-mouse IgG had been coupled. Rosetted fractions were prepared on a Percoll density gradient. After removal of erythrocytes by osmotic shock, the cells were tested for their capacity to respond to antigenic stimulation. To allow accurate estimation of functional potential, two B-cell cloning assays were used. To enumerate T-dependent B cells, the Klinman splenic microfocus assay was employed using haptenated KLH⁴ as antigen. To study T-independent cells, a limiting-dilution liquid microculture method employing hapten-polymerized flagellin as antigen was used. The results showed that none of the Lyb antigens clearly demarcated T-dependent from T-independent B cells. Rosetted fractions consisting of Lyb 1.1-, 2.1-, 3-, or 4.1-positive cells responded well in both assays. Fractions enriched for LyM 1-positive cells behaved like unfractionated spleen cells. Only the Lyb 5.2-rosetted fraction showed any discordance between the two assays, the fraction being enriched for cells responding in the T-dependent system and slightly depleted of cells responding in the T-independent system. Taken as a whole, the results suggest that these alloantigens will not serve as useful markers to characterize T-dependent and T-independent B-cell subsets. In fact, the experiments cast further doubt on whether such a distinction is valid.     

C-Reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition.

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.     

Regulation of clones responding to dextran B1355S. II. Response of T-dependent and T-independent precursors

The existence of precursors for TI and TD alpha 1 leads to 3 dextran antigens in BALB/c mice was demonstrated. A T-dependent dextran antigen was prepared by coupling dextran B1355S to hemocyanin and subsequent digestion with dextranase. The PFC response of BALB/c mice primed with hemocyanin to dextran-hemocyanin was found to be 8 times higher than in unprimed animals. The splenic focus assay was adapted for the analysis of precursors responding to T-dependent and T-independent dextran antigens. Pretreatment of recipients with anti-thymocyte serum abolished the response in fragment cultures to dextran-hemocyanin but did not affect the response to dextran B1355S. The frequencies of precursors in the adult BALB/c mouse responding to dextran and dextran-hemocyanin were determined by limiting dilution analysis. The frequency of T-dependent precursors was found to be almost 3 times greater than the frequency of T-independent precursors.     

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).     

Effects of cyclosporin A on humoral immune response and resistance against vesicular stomatitis virus in mice.

The effect of cyclosporin A (CS-A) on the antiviral humoral response was studied by using vesicular stomatitis virus (VSV); VSV provided the opportunity to simultaneously assess both T-independent and T-dependent antibody responses. The T-independent anti-VSV immunoglobulin M (IgM) response was virtually unaffected, whereas the T-dependent primary anti-VSV IgG response was suppressed by CS-A; in contrast, the secondary IgG response was highly resistant to CS-A. Moreover, once the switch from IgM to IgG had occurred, the primary response also became refractory to suppression by CS-A. We concluded that the effect of CS-A on the primary anti-VSV antibody response was mediated via impairment of a T-dependent mechanism; in contrast, memory T cells or memory B cells or both were quite resistant to the suppressive effects of CS-A. CS-A treatment rendered mice highly susceptible to VSV infection; under CS-A treatment, mortality was 100% after infection via footpads, whereas immunocompetent mice survived. Since CS-A does not impair induction of early T-independent anti-VSV IgM neutralizing antibodies, this high mortality in CS-A treated mice illustrates the crucial role of CS-A-sensitive cells in resistance against VSV.     

Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.     

Effects of antigens and lymphokines on early activation of single hapten-specific B lymphocytes

An assay was developed to monitor early activation of single fluorescein-specific B cells obtained from the spleens of nonimmunized adult mice by prefractionation on hapten gelatin. Early activation was assessed as a significant increase in the diameter of individual B cells after 24 hr in vitro. Significant enlargement of the single B cells was induced within 24 hr by either T-independent antigens acting alone or a crude source of B cell growth and differentiation factors (EL-BGDF-pik) acting alone. In contrast, T-dependent antigens acting alone were ineffective. When selected T-independent antigens and EL-BGDF-pik acted together, a greater number of B cells were induced to enlarge. B cell stimulatory factor 1 (BSF 1) behaved in a similar manner as EL-BGDF-pik, inducing early B cell enlargement both in the absence and more so in the presence of antigen. Both EL-BGDF-pik and BSF 1 enhanced the survival of single hapten-specific B cells during the 24-hr period. Interleukin 1 was unable to cause B cell enlargement when acting alone, although it was able to augment B cell enlargement induced by antigen. Interleukin 2 did not induce cell enlargement in either the presence or absence of antigen. Activation was demonstrated among cells of all sizes, regardless of the stimulus, although a greater response was demonstrated amongst the larger cell population. The addition of 3T3 filler cells enhanced early B cell activation and cell survival during the 24-hr period. The 24-hr B cell enlargement assay as applied to isolated single cells provides an unequivocal approach to the analysis of early B cell activation, adding a further parameter for the dissection of the precise roles of antigen and the various factors in the B cell differentiation pathway.     

Newborn and Wiskott-Aldrich patient B cells can be activated by TNP-Brucella abortus: evidence that TNP-Brucella abortus behaves as a T-independent type 1 antigen in humans

TNP-Brucella abortus (TNP-Ba) has been classified as a T-independent type 1 (TI-1) antigen in the mouse on the basis that it activates neonatal and CBA/N (X-linked immunodeficient) murine B cells in contrast to T-independent type 2 (TI-2) antigens. Therefore, it was of interest to determine whether human newborn and X-linked Wiskott-Aldrich syndrome B cells could be triggered by TNP-Ba. Previous studies had shown that human B cells from both these latter sources were relatively insensitive to stimulation with T-dependent and polysaccharide antigens (TI-2 in mouse). In this study, we show that TNP-Ba can trigger human cord blood B cells to differentiate into anti-TNP plaque-forming cells (PFC) in a hapten-specific and T-independent manner. The dose response and kinetics were similar to those previously seen with adult cells. The newborn responses, however, were lower than adult PFC responses. Precursor frequency and clone size analyses revealed that this lower response was not due to newborn cells containing fewer precursors but was the result of a reduced ability of these anti-TNP clones to expand. The ability of TNP-Ba to activate immature newborn B cells implies that this antigen can be used to assess B cell function in very young children. It also implies that TNP-Ba behaves as a TI-1 antigen in humans as well as in mice. This was supported by the finding that B cells from Wiskott-Aldrich patients, which were unreactive to polysaccharide antigens, were generally responsive to TNP-Ba. Therefore, it would appear that human newborn and Wiskott-Aldrich patients do possess a functionally competent B cell subset possibly equivalent to Lyb-5- immature murine B cells.     

Two processes for B-cell triggering by T-independent antigens as evidenced by the effect of azathioprine.

The effect of azathioprine (Az) on the in vitro antibody response induced by two T-independent antigens has been evaluated. The response to trinitrophenyl (TNP)-polyacrylamide is highly sensitive to Az whereas that to TNP-lipopolysaccharide (LPS) is Az resistant. A similar Az resistance is found for both the polyclonal B-cell activation and the B-cell proliferation induced by LPS, suggesting that Az resistance is related to the presence of a mitogenic carrier. These data suggest that at least two different processes may operate in B-cell triggering by T-independent antigens. On the basis of Az sensitivity, the response to T-independent antigens without a mitogenic moiety such as TNP-polyacrylamide is comparable to the responses to T-dependent antigens which are Az sensitive.     

Studies on Fc receptor function: II. Depressive effect of aggregated IgG2b on B lymphocyte activation by T-dependent and T-independent antigens

Mouse aggregated IgG2b, continuously present in mouse spleen cell cultures, markedly depressed, in a dose-dependent fashion, the direct plaque-forming cell response to sheep red blood cells (SRBC, T-dependent) and to DNP-Ficoll (T-independent). Pretreatment of mouse spleen cells with IgG2b for 30 min at 4 °C prior to culturing also markedly depressed the response to both antigens. Delayed addition of IgG2b to SRBC-immunized cultures caused an early and a late depression separated by a period when no significant depression was seen. Using DNP-Ficoll as antigen the late-occurring depression was observed only in Mishell-Dutton cultures, while it was not seen in microcultures. These data support a regulatory role of Fc receptors on the activation of B lymphocytes by antigens and suggest that Fc receptors may be important in at least two events during the differentiation of B lymphocytes into plasma cells: an early, short lived one and a later, longer-lasting event.     

Ribonucleic acid in the immune response

Summary In the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.     

The extracorporeal immunostimulating effect of terrilytin in staphylococcal infection]

The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin-treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients.     

The functional relevance of NK-cell-mediated upregulation of antigen-specific IgG2a responses

We have previously shown that activation of NK cells by poly(I:C) or tumor treatment of mice increases the level of antigen-specific IgG2a (1, 2). We have now assessed the functional relevance of this effect of the innate immune system on the specific immune response. We found that the increased IgG2a significantly augments antibody-dependent cellular cytotoxicity mediated by NK cells both in vivo and in vitro. Furthermore, we show that both IgG3 producing plasma cells induced by T-independent antigens and IgG2a plasma cells induced in the presence of activated NK cells may be just as long-lived as plasma cells induced by T-dependent antigens. These results indicate that if NK cells are activated early in the immune response, before T cells are recruited, they could exert long-lasting effects.