The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

A study of the functional capabilities of human neonatal lymphocytes for in vitro specific antibody production

The ability of neonatal B and T cells to participate in the in vitro production of anti-influenza virus antibody was studied. Peripheral blood mononuclear cells from nearly all normal adults produce anti-influenza virus antibody when stimulated in vitro with type A influenza virus. Cord blood mononuclear cells, however, consistently failed to do so. Using Epstein-Barr virus activation or coculture with irradiated adult T cells in the presence of influenza virus to identify precursor B cells for anti-viral antibody production, newborns were found to have a decreased number of influenza-specific B cells as compared with adults. Thus, a paucity of precursor B cells for anti-influenza virus antibody was one factor contributing to the absent in vitro antibody response. Additional studies were undertaken to investigate the capabilities of newborn T cells in the in vitro response to influenza virus. Newborn T cells failed to proliferate when cultured with influenza virus. Irradiated newborn T cells were, however, able to provide help for specific antibody production in influenza virus-stimulated cocultures with allogeneic adult B cells, and newborn T cells proliferated when stimulated with alloantigens; their helper function in allogeneic coculture was, thus, likely mediated by T cells stimulated by alloantigens rather than by influenza virus. In the absence of T cell irradiation, no antibody was produced in cocultures of adult B cells and neonatal T cells, at least in part as the result of a radiosensitive suppressor T cell. Suppression in influenza virus-stimulated allogeneic cocultures was also observed with normal adult T cells and is, therefore, a property of both newborn and adult T cells. Thus, allogeneic help and suppression can both be manifested in exogenous antigen-stimulated allogeneic cocultures. In addition, both of these allogeneic effects can be mediated by neonatal T cells, indicating that these functions are present at the time of birth and do not require previous exogenous antigenic exposure for expression.     

The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens

Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

In vitro influenza virus-specific antibody production in man: antigen-specific and HLA-restricted induction of helper activity mediated by cloned human T lymphocytes

Cloned human T lymphocytes induced with influenza A virus (A/Texas/1/77) and maintained in continuous culture with T cell growth factor were assayed for helper function in the in vitro production of anti-influenza antibody. Helper function mediated by both cloned helper T cells and normal peripheral blood lymphocytes was highly antigen dose-dependent, requiring lower concentrations than that necessary to induce blastogenesis. Optimal help was observed with 1 X 10(2) cloned T cells per culture, whereas excess helper cells inhibited the response. After culture with influenza A virus-induced cloned helper T cells, the antibodies formed were directed against influenza A and not B virus. Furthermore, the cloned helper T cells despite being specific for matrix protein collaborated in the production of predominantly anti-hemagglutinin antibody, suggesting associative recognition of the two discrete antigens. Cellular interactions between cloned helper cells from an HLA-Dw1,3 DR1,3 individual and erythrocyte rosette-negative cells required HLA-Dw1; DR1 compatibility for the production of specific antibody. This was confirmed by using subclones. Finally, it was observed that supernatants of the cloned helper T cells contained functional activity capable of replacing the parent cells in the production of anti-influenza A virus antibody.     

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

Transforming growth factor-beta inhibits the production of IgG, IgM, and IgA in human lymphocyte cultures.

Transforming growth factor beta (TGF beta) has potent immunoregulatory effects acting on both T and B cells. It strongly inhibits secretion of IgG and IgM in human and murine B cell cultures, but has been shown to have an enhancing effect on IgA production in the mouse. We have studied the effect of TGF beta on the production of IgA in human lymphocyte cultures. The addition of TGF beta to pokeweed-stimulated peripheral blood lymphocytes resulted in a suppression of IgA production of both subclasses, similar in magnitude to the suppression of IgG and IgM production. Membrane IgA expression was not increased by culturing tonsillar lymphocytes with TGF beta. In conclusion, we find no evidence for a selective enhancing effect of TGF beta on IgA synthesis in humans, in contrast to the findings reported in mice.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

An analysis of in vitro B cell immune responsiveness in human lymphatic filariasis

The immunoregulatory mechanisms involved in B cell function in patients with varying clinical manifestations of bancroftian filariasis were examined by studying the ability of peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from patients with elephantiasis, asymptomatic microfilaremia (MF), and acute tropical pulmonary eosinophilia (TPE) to produce polyclonal and parasite-specific antibody in vitro, both spontaneously and in response to a mitogen (PWM) and to parasite antigen. When the spontaneous or mitogen-driven polyclonal responses were examined, all groups produced significant amounts of IgM and IgG; those with TPE produced extremely high levels. However, when in vitro parasite antigen-specific responses were examined, those with MF were unable to produce filaria-specific antibody either spontaneously or in response to PWM or parasite antigen; in contrast, patients with chronic lymphatic obstruction or TPE produced large quantities. Removal of neither adherent cells nor T8+ T cells affected the parasite-specific B cell anergy seen in those with MF. This absent or severely diminished capacity to produce antibody on parasite antigenic stimulation in patients with MF is likely responsible for the low levels of parasite-specific antibody seen in this most common clinical manifestation of bancroftian filariasis. Its inability to be reversed by the removal of "suppressor elements" suggests a state of B cell unresponsiveness to the parasite.     

Reactivity of Leu-1+ tonsillar B cells to a high molecular weight B cell growth factor

This work was focused on the responsiveness of B cells from blood and tonsils to typical B cell growth factors (BCGF) in the absence of anti-mu antibody. This direct responsiveness was not observed with small dense B cells from either organ. Large tonsillar B cells but not large blood B cells did respond directly to a high m.w. BCGF (m.w. 50,000 BCGF), whereas large B cells from both organs responded directly to the low m.w. BCGF (m.w. 20,000 BCGF). The tonsillar B cells directly responsive to the m.w. 50,000 BCGF express the 4F2 marker and thus belong to a population of in vivo-preactivated B cells. Moreover, this direct responsiveness to the m.w. 50,000 BCGF is localized in Leu-1+ tonsillar B cells. The Leu-1+ and Leu-1- tonsillar B cell subsets do not differ in their responsiveness to the m.w. 50,000 BCGF upon costimulation with anti-mu antibody and to the m.w. 20,000 BCGF regardless of the presence of anti-mu antibody. Thus, the Leu-1+ subset present in tonsils shows a particular reactivity to a high m.w. BCGF.     

In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

Follicular B helper T cells in antibody responses and autoimmunity

T-cell help for B cells is essential for high-affinity antibody responses and B-cell memory. Recently, the identity of a discrete follicular population of T cells that has a crucial role in this process has become clearer. Similar to primed CD4(+) T cells in the tonsils and memory CD4(+) T cells in the peripheral blood, this follicular population of T cells expresses CXC-chemokine receptor 5 (CXCR5). Owing to their distinct homing preferences and helper function, these T cells differ from T helper 1 and T helper 2 cells and have been denoted follicular B helper T cells. Here, we outline the central role of this subset in normal and pathological immune responses.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

Distinct ex vivo susceptibility of B-cell subsets to epstein-barr virus infection according to differentiation status and tissue origin

Epstein-Barr virus (EBV) uses tonsils as the portal of entry to establish persistent infection. EBV is found in various B-cell subsets in tonsils but exclusively in memory B cells in peripheral blood. The in vitro susceptibilities of B-cell subsets to EBV infection have been studied solely qualitatively. In this work, we examined quantitatively the in vitro susceptibilities of various B-cell subsets from different tissue origins to EBV infection. First, we established a centrifugation-based inoculation protocol (spinoculation) that resulted in a significantly increased proportion of infected cells compared to that obtained by conventional inoculation, enabling a detailed susceptibility analysis. Importantly, B-cell infection occurred via the known EBV receptors and infected cells showed EBV mRNA expression patterns similar to those observed after conventional inoculation, validating our approach. Tonsillar na?ve and memory B cells were infected ex vivo at similar frequencies. In contrast, memory B cells from blood, which represent B cells from various lymphoid tissues, were infected at lower frequencies than their na?ve counterparts. Immunoglobulin A (IgA)-positive or IgG-positive tonsillar memory B cells were significantly more susceptible to EBV infection than IgM-positive counterparts. Memory B cells were transformed with lower efficiency than na?ve B cells. This result was paralleled by lower proliferation rates. In summary, these data suggest that EBV exploits the B-cell differentiation status and tissue origin to establish persistent infection.     

Non-specific polyclonal antibody response induced by Mycoplasma pneumoniae

The ability of heat-killed Mycoplasma pneumoniae (MP) organisms to induce polyclonal antibody production in cultures of blood lymphocytes of healthy subjects was studied. MP induced both IgM and IgG production, with a predominance of IgM. Supernatants of MP-stimulated lymphocyte cultures were tested by an enzyme-linked immunosorbent assay for antibodies to measles, rubella, and herpes simplex virus. MP as well as pokeweed mitogen induced production of viral antibodies of IgG class in lymphocytes of donors who had serum antibodies to the corresponding viral antigens. The MP-induced non-specific antibody response was T-cell-dependent. Lymphocytes from four patients with MP pneumonia, collected nine to 13 days after onset of illness, were tested for in vitro Ig production in the absence of MP. These lymphocytes spontaneously produced increased amounts of IgM and/or IgG. Lymphocytes from three of these four patients spontaneously produced viral IgG antibodies to measles and/or varicella antigens, indicating that MP had induced non-specific activation of memory B cells in vivo. Spontaneous viral antibody production was not found in lymphocyte cultures of healthy donors. The non-specific activation of blood B cells in vitro is probably induced by non-specific helper factors from MP-activated T cells. It is possible that in vivo MP also may have a direct activating effect on B cells.     

Damage to an ancient parchment document by Aspergillus

Paracoccidioidomycosis (PCM) is often associated with hypergammaglobulinemia and increased serum levels of circulating immune complexes (CIC). In order to investigate whether polyclonal B lymphocyte activation (PBA) is a current process in PCM, we measured the numbers of IgG secreting cells (IgG SC) in the peripheral blood of 16 patients and of 8 healthy controls. The numbers of IgG SC were found to be significantly elevated in PCM patients. We also observed increased serum levels of IgG, IgA and CIC. These data reflect an activation of B lymphocytes in PCM patients.Abbreviations CIC circulating immune complexes - E-PtnA protein A- coupled sheep red blood cells - IgG SC immunoglobulin G secreting cells - PBA Polyclonal B cell activation - PBMC peripheral blood mononuclear cells - PCM paracoccidioidomycosis - PFC plaque forming cells assay - PtnA-BA protein A- binding, polyethyleneglycol precipitation immunoradiometric assay     

Passive transfer of local immunity to influenza virus infection by IgA antibody

Secretory IgA is presumed to be the mediator of mucosal immunity based on many studies that show a correlation between protection and secretory IgA titers; however, a causal relationship has not yet been established. Classically, passive transfer of antibody has been used to demonstrate causality, but the passive transfer of local immunity with physiologically transported IgA has not been previously reported. In this study mice were injected intravenously with polymeric IgA (pIgA), monomeric IgA (mIgA), or IgG1 mAb specific for the H1 hemaglutinin of PR8 influenza virus. pIgA was shown to be specifically transported into nasal secretions relative to the mIg. The transported pIgA was functional, as evidenced by its ability to bind to virus in an ELISA assay and to protect nonimmune mice against intranasal infection with H1N1 but not H3N2 influenza virus. Intravenous injection of similar virus-neutralizing doses of anti-influenza IgG1 mAb did not protect against nasal viral challenge. IgA-mediated protection could be abrogated by the intranasal administration of antiserum against the alpha chain of IgA. These data demonstrate the passive transfer of local immunity by the i.v. administration of pIgA antibody and show that the IgA in secretions can protect against influenza virus infection. This general approach could provide a model for the evaluation of the role of local IgA in host defense against other pathogens.