T cell-dependent hapten-specific and polyclonal B cell responses require release of interleukin 5

CD4+ T cells in the mouse have recently been subdivided into two major subpopulations which differ in their functional activities and in the lymphokines they produce. Although cloned T cells lines representative of both sets will activate B cells in polyclonal responses, only the subset producing interleukin 4 (IL-4) will activate antigen-specific B cells in linked recognition assays. This suggested that IL-4 was essential for such responses. In the present experiments, the requirements were compared for B cell activation in specific as opposed to polyclonal antibody responses by T cell clones of the helper (IL-4 producing) subset. It was found that specific responses involve primarily small B cells, whereas polyclonal responses activate exclusively the large B cells. Second, polyclonal B cell responses can proceed in the absence of T:B contact, whereas specific responses require physical interaction of the two cells. Third, it was found that interleukin 5 (IL-5, formerly known as T cell replacing factor/B cell growth factor II) is essential for these polyclonal responses by inhibition of such responses with monoclonal anti-IL-5 antibody. Anti-IL-5 also inhibits specific antibody responses involving direct T:B interaction. Thus, IL-5 is clearly a critical mediator of differentiation to immunoglobulin secretion of activated B cells, whether such B cells are obtained as large B cells from freshly isolated spleen cells or are initially activated in an IL-4-dependent fashion by cognate interaction by a helper T cell clone.     

Antigen-specific and polyclonal immunoglobulin production induced by a cloned tetanus toxoid-specific T cell line

This report describes the isolation and the phenotypic and functional characterization of a cloned, IL 2 dependent, TT-specific human helper T cell line (TCL), designated 1A1. 1A1 was derived by limiting dilution culture of a bulk IL 2-dependent TCL that was found to contain both TT and trinitrophenyl (TNP) altered self-reactive T cells. Specifically, 1A1 represents the outgrowth of one well of a microwell cloning plate initially seeded at 1 TCL/well, from which less than 4% (3/96) wells grew. Phenotypic analysis, utilizing a battery of monoclonal antibodies, demonstrates that all 1A1 cells are T cells belonging to a stable and discrete T cell subset: T3+, T4+, T17+, T8-. In proliferative assay, 1A1 responds specifically to TT but not to other soluble antigens against which the donor is sensitized, a panel of allogeneic stimulators, nor to TNP-modified-self. Moreover, 1A1 is HLA-DRw-restricted, proliferating only to TT in association with DRw3+ antigen-presenting cells. Of greater interest is the observation that 1A1 is an antigen-specific helper T cell line. Thus, by utilizing ELISA systems to quantitate class-specific immunoglobulin and antigen-specific antibody, it was determined that co-culture of autologous B cells, 1A1 cells, and a low concentration (1 to 10 ng/ml) of TT results in an IgG response that is predominantly, if not exclusively, antigen-specific antibody. In contrast, the presence of high concentrations of TT (4 micrograms/ml) triggers a polyclonal immunoglobulin response comprised of IgM with a small IgG component that is essentially devoid of anti-TT antibody. These results demonstrate that depending on the mechanism of activation, a cloned antigen-specific helper T cell line can mediate antigen-specific or polyclonal help for autologous B cells.     

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.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli.

Numerous changes have been reported to occur in T cell responsiveness of mice with increasing age. However, most of these studies have examined polyclonal stimulation of spleen cells from a limited number of mouse strains. This study investigated the influence of genetic background, source of lymphocytes, and type of stimulus on age-associated changes in T cells response. Con A-induced proliferation and IL-2 and IFN-gamma production by splenic lymphocytes (SL) was significantly greater in CBA/Ca mice compared to C57BL/6 mice, regardless of age. SL of both strains exhibited the predicted age-dependent decline in proliferative response and an increase in IFN-gamma production in response to Con A. In contrast, however, only SL from C57BL/6 mice demonstrated the predicted age-dependent decline in Con A-induced IL-2 production; Con A-induced SL of young and aged CBA/Ca mice produced comparable amounts of IL-2. Differences in age-associated responses to Con A were also observed between SL and inguinal lymph node (ILN) cells of CBA/Ca mice. In contrast to SL, ILN cells demonstrated an increased proliferative response to Con A. However, lymphokine production by Con A-stimulated ILN cells from aged CBA/Ca mice was similar to that of Con A-stimulated SL from aged CBA/Ca mice. To determine if aged ILN T cells respond similarly to polyclonal and antigen-specific stimuli, keyhole limpet hemocyanin (KLH) responses of T cells isolated from ILN of aged and young CBA/Ca mice were examined. KLH-specific T cells from aged mice cultured with KLH-pulsed macrophages (M phi) from aged mice were significantly reduced in their ability to proliferate compared to KLH-specific T cells of young mice cultured with young KLH-pulsed M phi. In contrast to the expected results, the defect was not at the level of the T cells; proliferation of young T cells cultured with aged KLH-pulsed M phi was equivalent to the proliferation of aged T cells cultured with aged M phi. These results suggest that aging has differential effects on polyclonal and antigen-specific T cell proliferation and on polyclonal stimulation of T cells isolated from different lymphoid organs and from different strains of mice.     

Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells

Dendritic cells (DC) are professional antigen-presenting cells which stimulate strong proliferative and cytolytic T cell responses. Stimulation of CD40 on dendritic cells by its ligands and anti-CD40 antibodies induces maturation and enhances DC stimulatory ability. In order to understand the mechanism by which ligand:CD40 interactions augment DC function, we assessed the role of T cell stimulatory cytokines IL-12 and IL-15 in the function of DC stimulated with soluble trimeric CD40L, a recombinant fusion protein incorporating three covalently linked extracellular CD40L domains (huCD40LT). Peripheral blood derived DC treated with huCD40LT and/or IFN-gamma were used to stimulate T cell responses in vitro to specific antigens. DC treated with huCD40LT or IFN-gamma/huCD40LT stimulated enhanced T cell proliferation to CASTA, a soluble protein from C. albicans, induced T cells with augmented antigen-specific lysis, and increased the yield of antigen-specific IFN-gamma-producing T cells. IL-15 production by DC was enhanced in cultures treated with huCD40LT and correlated with expansion of antigen-specific cytolytic T cells. Addition of a neutralizing anti-IL-15 monoclonal antibody inhibited the expansion of viral and tumor antigen-specific T cells stimulated by IFN-gamma and huCD40LT-treated DC. In contrast, this enhanced stimulatory ability of DC did not appear to depend on synthesis of IL-12 since huCD40LT treatment stimulated the generation of antigen-specific cytokine producing and cytolytic T cells without increased IL-12 production. Addition of anti-IL-12 monoclonal antibody did not inhibit expansion of these cells. These data suggest that production of IL-15 but not IL-12 is an important factor in the enhanced immunostimulatory ability of huCD40LT-treated DC.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection.

The kinetics of various specific and nonspecific immunologic responses were examined in BALB/c mice infected with 17X nonlethal Plasmodium berghei yoelii (a self-limiting infection). The sequence of events after infection was characterized by rapid sensitization of splenic T cells to malaria antigen and polyclonal B cell activation, followed by a period of depressed splenic proliferative responses in vitro to mitogens (PHA and LPS) and malaria (specific) antigen. At the same time, suppressed primary in vitro splenic PFC responses to trinitrophenyl-aminoethylcarbamylmethyl-Ficoll (TNP-F) were seen. This suppression was an active process requiring adherent cells. During this period, levels of antimalarial antibody also increased exponentially. As the infection was cleared, splenic malaria antigen-specific proliferative responses were again observed and splenic PFC and in vitro mitogen responses returned to preinfection levels after variable periods of time. Both splenic proliferative responses to malaria antigen and antimalarial antibody responses remained persistently elevated. In addition, some responses were examined in mice infected with 17X lethal P.b. yoelii (a fatal infection); in comparison to the early responses of mice infected with the nonlethal substrain, there was a decrease and delay in the development of a splenic T cell response to malaria antigen and a blunted antimalarial antibody response.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Immunoregulation by T cells. I. Characterization of the IEk-specific Lbd self-reactive T cell clone that helps, suppresses and contrasupresses B cell responses

This paper describes an L3T4+, Lyt-2- cloned T cell line of CBA/N origin that is specific for IEk-encoded products. Splenic dendritic cells are the predominant stimulatory populations for Lbd cells, being approximately 100 times more effective than large activated B cells. Small B cells and T cells are nonstimulatory. Lbd cells help large B cells to make polyclonal antibody responses, but are unable to activate small B cells and to replace antigen-specific helper T cells in T cell-dependent responses to phosphorylcholine or sheep red blood cells. In the presence of antigen-specific helper T cells, Lbd can amplify, suppress, or contrasuppress T cell function, depending on the type and relative ratios of antigen-reactive cells. We explain our findings as a consequence of the self reactivity among helper T cells.     

Different requirements for Ia-bearing accessory cells in mitogen versus antigen induction of human B-cell responses

The accessory cell requirements for the induction of proliferative and specific antibody responses of human lymphocytes stimulated with either antigen or mitogen were examined. An Ia-negative human myeloid tumor cell line, K562, could substitute for monocytes in the proliferation of monocyte-depleted lymphocytes in response to pokeweed mitogen (PWM) stimulation. K562 cells could also act as accessory cells in the PWM-induced anti-keyhole limpet hemocyanin (KLH) antibody synthesis of cells from a KLH-immunized donor. In contrast, only monocytes and not K562 cells could function as accessory cells in antigen-induced lymphocyte proliferation as well as in antigen-induced, antigen-specific antibody production. However, K562 cells, like monocytes, were able to positively and negatively regulate polyclonal immunoglobulin responses. Thus, Ia-bearing accessory cells can function in antigen-induced proliferation and antibody responses while non-Ia-bearing cells can function in mitogen-induced, but not anti-geninduced responses. These studies indicate a dichotomy in the nature of required accessory cells in antigen-induced versus mitogen-induced human lymphocyte responses and strongly suggest an obligatory role of Ia or an Ia-related molecule on accessory cells in antigen-induced responses of human lymphocytes.     

Serum-mediated suppression of nonspecific B-cell activation. III. Selective inhibition of the polyclonal B-cell response by normal mouse serum

Normal, nonimmune adult serum is known to inhibit in vitro immune responses when present in sufficient amounts. The significance of inhibition of the immune response by serum, however, is not known. Previous work suggested that normal mouse plasma or serum (NMS) was selectively more inhibitory to nonantigen-specific (e.g., polyclonal) as compared to antigen-specific responses. This led to the hypothesis that constituents of serum (or plasma) may serve naturally to minimize the polyclonal type of antibody response, preserving immune specificity. The present study further examined the effect of NMS on polyclonal versus antigen-specific antibody responses. Under the in vitro assay conditions used, 0.5% NMS supported bacterial endotoxin (ET)-induced mitogenic and polyclonal B lymphocyte responses, antigen (SRBC, TNP-KLH)-specific antibody (IgM, IgG) responses, and antigen-induced or -specific T-lymphocyte proliferative responses, while 5% NMS inhibited all of these responses. However, antigen-specific T-lymphocyte responses could be restored by a 10-fold increase in the antigen concentration and antigen-specific antibody responses could be restored by the addition of ET (10 micrograms/ml) as adjuvant. On the other hand, the mitogenic response to ET remained suppressed regardless of ET concentration. Thus, despite significant reduction of the mitogenic and polyclonal properties of ET in 5% NMS (greater than 70% suppression), sufficient antigenic stimuli permitted optimal specific T- and B-cell responses. Many naturally occurring antigens, e.g., bacterial, fungal, and viral, have inherent B-cell mitogenic and polyclonal activity in addition to adjuvanticity and the presence of the serum inhibitory factor may serve to minimize their indiscriminate polyclonal stimulation of antibody.     

The T4 molecule differentially regulating the activation of subpopulations of T4+ cells

It has been demonstrated that the T4+2H4+ subset functioned as a suppressor inducer cell, whereas the reciprocal T4+2H4- subset provided help for B cell Ig production. In the present studies, a series of monoclonal antibodies to cell surface structures expressed on these subsets of cells were examined for their effects on the proliferative and immunoregulatory functions generated in AMLR. We demonstrated that anti-T4 antibody preferentially inhibited the proliferative response of the T4+2H4+ but not T4+2H4- cells against self-MHC antigens. In contrast, anti-T3 and anti-Ia antibodies inhibited the response of both subsets of cells. This subset preference of anti-T4 antibody was not attributable to either the isolation procedures used or a shift in the kinetics of proliferation to autologous self-MHC antigens. Moreover, both IL 2 production and the immunoregulatory function of the T4+2H4+ subset was profoundly inhibited by anti-T4 antibody, whereas the T4+2H4- subset was minimally influenced. In the absence of Ia molecules, T4+2H4+ but not T4+2H4- cell proliferation was inhibited with anti-T4 antibody. Together, these results suggest that the T4 molecule plays a distinct functional role in the differential triggering of subsets of T4+ cells.     

T cell regulation of polyclonal B cell responsiveness. I. Helper effects of T cells.

Polyclonal activation of murine splenic B lymphocytes to secrete immunoglobulin was shown to be subject to regulation by splenic T cells. By admixture of separated B and T cell populations it was demonstrated that normal fresh splenic T cells were able to augment polyclonal B cell responsiveness to LPS up to several-fold. Optimal collaboration between these two cell types ensued when they were co-cultured in equal numbers. T cell-mediated enhancement of polyclonal B cell responses was dependent upon the ability of T cells to divide and was manifested upon T cell interaction with B cells soon after culture initiation. Originally expounded as a one-signal phenomenon, polyclonal activation of lymphocytes by LPS is, under the circumstances described, attributable instead to two distinct, nonspecific signals acting in concert. The observation that T cells from LPS-nonresponder (C3H/HeJ) mice were deficient in the capacity to enhance polyclonal B cell responsiveness of B cells derived from responder (C3H/HeN) mice implied a direct action of LPS on the involved T cells as well as an active role for the T cell signal in this immunoregulatory event. The novel observation of a functional T cell defect in LPS responsiveness in the C3H/HeJ mouse is discussed in terms of its other cellular defects.     

Role of antigen-specific B cells in the induction of SRBC-specific T cell proliferation

In this study, we ask whether antigen presentation can be effected by antigen-activated B cells. Antigen-dependent in vitro proliferation of T cells from mice primed with SRBC or HoRBC occurs in the presence of B cells primed to the relevant antigen. B cells prepared from lymph nodes of mice primed with irrelevant antigens are not effective antigen-presenting cells for RBC-specific T cell proliferation over a wide range of SRBC doses. This is true even when both RBC and the antigen to which the B cells are primed are included in the culture. In contrast, B cells specific for a hapten determinant coupled to SRBC are able to support proliferation of T cells specific for SRBC determinants. We conclude from these data that antigen-specific B cells play a role in the induction of T cell proliferative responses to SRBC and HoRBC antigens. Two models are proposed: either B cells, upon antigen interaction with surface antibody, are able to act as accessory cells to induce Ia-dependent proliferation of immune T cells; or B cells augment the T cell proliferative response by secretion of antibody, leading to opsonization of the antigen for macrophage uptake and presentation.     

Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways

Although the existence of autoreactive T cells has been widely reported, the functional capacities of these populations have been less well defined. Studies were therefore carried out to characterize the relationship of autoreactive T cells to antigen-specific major histocompatibility complex (MHC)-restricted T cells in their ability to act as helper cells for the induction of immunoglobulin synthesis by B cells. A number of autoreactive T cell lines and clones were isolated from antigen-primed spleen and lymph node cell populations. Autoreactive T cells were found to proliferate in response to direct recognition of syngeneic I-A or I-E subregion-encoded antigens in the absence of any apparent foreign antigen. It was shown that cloned autoreactive T cells were capable of activating B cell responses through two distinct pathways. After appropriate stimulation by syngeneic cells, autoreactive T cells polyclonally activated primed or unprimed B cells to synthesize IgM antibodies. These activated T cells functioned in these responses through an MHC-unrestricted pathway in which polyclonal responses were induced in both syngeneic and allogeneic B cells. These cloned autoreactive T cells were also able to activate IgG responses by primed B cells through a different activation pathway. In contrast to the polyclonal activation of IgM responses, the induction of IgG antibodies by the same cloned T cells required primed B cells and stimulation with the priming antigen. The activation of B cells to produce IgG was strongly MHC restricted and required the direct recognition by the autoreactive T cells of self MHC determinants expressed on the B cell surface, with no bystander activation of allogeneic B cells. These results indicate that cloned autoreactive T cells resemble antigen-specific MHC-restricted T cells in their ability to function as T helper cells through distinct MHC-restricted and MHC-unrestricted pathways.     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Antigen-specific and -nonspecific mitogenic signals in the activation of human T cell clones

We have directly compared the signals required for: induction of the [Ca+2]i response, expression of Tac antigen, and proliferation in antigen-specific human T cell clones. We have previously shown that antigen-specific activation of cloned T cells under conditions leading to proliferation is accompanied by a rapid increase in [Ca+2]i. Cloned T cells showed increased [Ca+2]i, enhanced Tac expression, and proliferated in response to specific antigen in the presence of viable, genetically appropriate antigen-presenting cells. Paraformaldehyde fixation of antigen-presenting cells after "pulsing" with antigen prevented proliferation, but did not affect MHC-restricted [Ca+2]i or Tac responses. Treatment of cloned T cells with monoclonal anti-T3 antibody also increased [Ca+2]i and Tac expression but did not induce proliferation. Proliferation was restored by viable autologous or allogenic APC or exogenous IL 2, but not by IL 1. In contrast to resting T cells, T cell clones were insensitive to the mitogenic effects of lectins or of ionophores and phorbol esters. These results suggest that activation of antigen-specific T cells requires the sequential action of at least two signals. The first is MHC restricted and is mediated by interaction of antigen + MHC class II products with the T cell receptor (T3-Ti) complex. This leads to Tac expression and increased [Ca+2]i, but is not sufficient for proliferation. This signal can be bypassed by anti-T3 monoclonal antibodies. Proliferation requires a second, nonantigen-specific, non-MHC-restricted antigen-presenting cell signal, which cannot be replaced by IL 1 in our system. This signal can be bypassed, however, by the addition of exogenous IL 2 to cells that have received the first signal and express Tac, suggesting that it is required for IL 2 synthesis and secretion. T cell clones therefore provide a useful model for studying antigen-dependent and -independent events in cell activation.     

Selective effects of anti-Ia serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulfate and lipopolysaccharide.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody; cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.     

cAMP is an essential signal in the induction of antibody production by B cells but inhibits helper function of T cells

Dibutyryl cAMP and IL 1 were found to stimulate antigen-specific and polyclonal antibody production when added together to cultures of highly purified B cells. We propose that IL 1 and an elevation in cytoplasmic cAMP represent minimal signal requirements for B cell activation. In contrast to its effect on B cells, dibutyryl cAMP inhibited helper T cell activity. Cyclic AMP suppressed the production of IL 2 and T cell replacing factor (TRF) by T cells and thus abrogated the ability of helper T cells to enhance SRBC-specific antibody production by B cells. Cyclic AMP did not inhibit the generation by T cells of B cell growth factor (BCGF). BCGF, not normally detected in Con A supernatant, was found in the culture supernatant of spleen cells that were stimulated with Con A in the presence of cAMP. Our findings indicate that cAMP blocks the production of an inhibitor of BCGF activity. cAMP had no effect on the production by macrophages of IL 1.