Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function

Novantrone mitoxantrone, an antineoplastic agent with antiproliferative properties, is under investigation as an immunomodulating agent. The impact of mitoxantrone treatment on B lymphocyte reactivity is presented here. Administered i.p. in H2O at a dose of 0.5 mg/kg, daily for 14 days, mitoxantrone abrogated both the in vivo antibody response (to ovalbumin) and the in vitro plaque-forming cell (PFC) response (to SRC). In addition to the effects on thymus-dependent reactivity, PFC responses to the thymus-independent antigens TNP-LPS and TNP-Ficoll were also inhibited when tested in vivo or in vitro. B cells were identified as a target for the suppressive activity of mitoxantrone by using T cell-replacing factor to reconstitute the in vitro anti-SRC PFC response of a T lymphocyte-depleted spleen cell preparation. LPS-induced B cell mitogenesis was largely inhibited by mitoxantrone treatment. However, depletion of Sephadex G-10-adherent cells significantly restored the proliferative response. Flow cytometric analysis revealed a dramatic decrease in splenic B lymphocyte content. Therefore, mitoxantrone exerted a potent suppressive influence on the humoral immune system through a direct reduction in B cell number augmented by macrophage-mediated inhibition of B cell proliferation.     

Induction of an antibody response in vitro against synthetic antigens in guinea pigs. I. Culture and assay conditions

Guinea pig spleen and lymph node cells were found to produce anti-2,4-dinitrophenyl (Dnp) oligolysine PFC in vivo against 2,4-dinitrophenyl-β-alanyl glycyl glycyl (Dagg-SRBC) but not against trinitrophenyl-SRBC target indicator cells. Furthermore, when sensitized spleen cells or their purified B-cell fractions were cocultured with primed peritoneal exudate lymphocytes (PEL) but not splenic T cells they were able to generate a secondary PFC response in vitro to the synthetic antigens, Dnp oligolysines. PFC were not induced in vitro if these same cultures were pulsed with short-chain peptides (five lysines) or the complex antigen, dinitrophenyl-bovine γ-globulin (DnpBGG). Con A was able to substitute for PEL in triggering spleen cells to mount a secondary in vitro PFC response to homologous Dnp oligolysines. More importantly, the Con A-aided spleen cell cultures were not induced above background values when challenged in vitro with heterologous Dnp oligolysines. This study suggests that spleen cells may lack a nonspecific signal for the development of a secondary in vitro PFC response.     

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. I. Preferential inhibition of the induction of delayed-type hypersensitivity

Culture supernatants of spleen cells from susceptible CBA mice chronically infected with Trypanosoma cruzi were able to inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens as measured by 24-hr footpad swelling, bone marrow homing, and radioactivity accumulation assays. The suppressive activity, which was also present in the serum of these chronically infected mice, appears to be specific for the induction of DTH and had no effect on the 3-hr immediate-type hypersensitivity. It also failed to modify the expression of DTH in presensitized mice. Furthermore, it did not affect the synthesis in normal recipients of specific antibody or the induction of helper T cells or cytotoxic T cells. It also failed to induce DTH tolerance as recipient mice with markedly reduced DTH were able to develop a normal DTH response after secondary immunization. The suppressive activity was produced by an Ig- macrophage-depleted splenic T cell population, whose capacity to secrete the suppressive substance was completely abrogated by treatment in vitro with anti-L3T4 antibody and complement, but not with anti-Lyt-2 antibody and complement. These results therefore demonstrate that L3T4+ T cells from mice chronically infected with T. cruzi can produce substances which interfere with the induction of DTH. This finding may help to identify the differential antigenic stimulatory requirement for the activation of the various subsets of T cells.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Regulation of the in vitro secondary antibody response: I. Suppression by relatively high,but physiological levels of calcium ion

Secondary antibody responses generated in vitro with spleen cells from mice primed and boosted with SRBC or TNP-KLH antigen were found to be influenced by the amount of Ca²⁺ in the culture medium. Relatively low levels of Ca²⁺ (0.1 mM) were optimally supportive for the generation of PFC in vitro, with higher, more physiological levels of Ca²⁺ (1.0–1.7 mM) suppressing the generation of PFC by as much as 100%. Suppression by high levels of Ca²⁺ was most pronounced when the amount of antigen used to elicit the in vitro antibody response was high, whereas responses generated by lesser amounts of antigen were minimally affected by Ca²⁺ level. Ca²⁺-mediated suppression was localized to an intermediate phase (24–48 hr) of the response. Mitogenic and polyclonal antibody responses were not affected by high levels of Ca²⁺. The effect of Ca²⁺ concentration on the secondary, IgG-producing antibody response may be significant in terms of understanding the various control mechanisms interacting in regulation of IgG synthesis.     

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)     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Cell interactions in alveolar macrophage-mediated suppression of the immune response: an unusual suppressor pathway involving a population of T-cells that express Lyt-1, L3T4, and I-J

Studies from this laboratory have demonstrated that incubation of murine alveolar macrophages (AM) with SRBC-primed spleen cells (SC) results in suppression of the in vitro plaque-forming cell (PFC) response and that suppression is mediated by a soluble factor contained in supernatants obtained from cultures of AM and SC. In the present study, immunological techniques employing monoclonal antibody (MoAb) were used to isolate various T-cell subsets in order to determine the phenotype of the cells which interact with AM to produce suppression. Spleen cell populations depleted of Thy-1+-, Lyt-1+-, L3T4+-, or I-J+-bearing cells failed to generate suppressive supernatants when cultured with AM. Depletion of Lyt-2+ T-cells (the classical suppressor/effector subset) did not alter the ability of the remaining cell population to cooperate with AM for generation of suppressive supernatants. Direct suppression of the PFC response in cultures containing AM was abrogated after treatment of the spleen cells with anti-I-J, but not anti-Lyt-2 MoAbs. Reconstitution of the AM-mediated suppressive response with enriched populations of SC required the presence of T-cells which expressed Lyt-1, L3T4, and I-J. These results suggest the existence of an unusual suppressor pathway involving I-J restriction but which appears to be mediated by the interaction of AM with a population of T-cells that expresses surface markers characteristic of T-helper cells.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

Studies of the immunological capacity of germfree mouse radiation chimeras. III. In vitro reconstitution of the T-helper cell deficiency

The plaque-forming cell (PFC) response of long-term radiation induced allogeneic bone marrow chimeric (ABMC) mice has been shown to be markedly deficient. The nature of the cellular deficiency of the primary PFC response was investigated using in vitro culture techniques. Adherent spleen cells from ABMC or DBA/2 mice support equally well the development of PFC from nonadherent DBA/2 spleen cells. Nonadherent cells prepared from ABMC mice when cocultivated with DBA/2 adherent cells showed a minimal response. However, the addition of activated DBA/2 T cells to cultures containing adherent cells from DBA/2 mice and nonadherent cells from ABMC mice completely reconstituted the in vitro response to sheep erythrocytes. Therefore a cellular deficiency of the humoral immune system of ABMC mice was shown to be associated with the thymus-derived lymphocyte pool.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

The in vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A.

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to Br-MRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.     

Regulation of human in vitro anti-hapten responses: demonstration of a carrier effect

Human peripheral blood mononuclear cells, tonsillar lymphocytes, or mixtures of allogeneic or autologous B and T cells from these tissues were stimulated in vitro with the soluble hapten:carrier complexes TNP-OA, TNP-KLH, TNP-Myo, or TNP-Lac. These complexes were able to induce TNP-specific, direct PFC during 5–6 days in culture. The response involved proliferating PFC precursor B cells, nonproliferating T helper cells, and radiosensitive T-suppressor cell precursors. Exposure to high concentrations of free or carrier-bound hapten resulted in the inactivation of PFC precursor B cells. Carrier specific suppressor T cells could be induced by priming with nonhaptenated carrier protein and were able to block the PFC response when added to fresh target cultures. The use of hapten:carrier complexes permits the characterization of distinct T-cell functions independently of the assay system for B-cell activation.     

Hapten-Specific T Cell Response to Azobenzenearsonate-N-Acetyl-L-Tyrosine (ABA-tyr) in Lewis Rats: II. Induction and Suppression of ABA-Specific Helper Cell Activity for Antibody Production

Immunization of Lewis rats with azobenzenearsonate-N-acetyl-l-tyrosine (ABA-tyr) in complete Freund's adjuvant (CFA), produces a hapten-specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) prior to immunization with ABA-tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly l-glutamine-lysine-tyrosine (l-GLT); however, a d-amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA-tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA-tyr in IFA followed by boosting with ABA-tyr in CFA induced unresponsiveness to subsequent induction of hapten-specific help.