Modulation of antibody synthesis by cholera exotoxin: Influence on helper thymocytes

This report demonstrates that a major biological influence of cholera exotoxin (CT) on antibody formation to sheep erythrocytes is mediated by the toxin's influence on the helper function of thymic lymphocytes. Hemolytic plaque-forming cell (PFC) responses for lethally X-irradiated mice repopulated with isologous thymus-marrow cell suspensions were stimulated three- to fivefold when 1.0 μg CT was administered at cell transfer. For mice given marrow cells only, CT did not elevate the PFC response; however, CT stimulated as many PFC in spleens of mice given marrow and 1 × 10⁷ thymus cells as mice given marrow and 4 × 10⁷ thymus cells. In other transfer experiments, depressed PFC responses were observed when irradiated mice were given marrow and thymus cells from donor mice inoculated with CT 24 hr prior to cell preparation and infusion, or given marrow cells from normal mice and thymus cells from CT-treated mice. In contrast, mice given marrow from CT-treated mice and thymus cells from normal mice responded as well as mice repopulated with normal thymus-marrow cell suspensions.     

In vitro anamnestic antibody response: differential cellular and calcium requirements for induction by antigen and regulation by cyclic AMP.

Lymph node cells from rabbits, immunized 6 clays previously with keyhole limpet hemocyanin (KLH) were fractionated on columns containing nylon fibers. The non-retained population (effluent cells) and the retained population (adherent cells) were subsequently characterized by various criteria. The addition of dibutyryl cAMP (DbcAMP) or cholera enterotoxin (CT) during induction by 1 and 100 μg KLH resulted in a >100% increase in antibody synthesis over the control (KLH only) responses in the unfractionated and adherent cell populations. In the effluent population CT and DbcAMP failed to enhance the 1 μg response, but did increase the 100 μg response. Antibody forming cells, as judged by ongoing antibody synthesis during the first 24 hr of culture, were deficient in the effluent population. Both the effluent and adherent cells responded to the mitogens concanavalin A, phytohemagglutinin, and goat anti-rabbit Fab'. The control, effluent, and adherent populations each contained approximately 45% surface Ig positive cells as judged by direct immunofluorescence. The removal of calcium from the medium during induction (0–24 hr) also demonstrated that induction of the antibody response by KLH was separable from the cAMP mediated enhancement of antibody synthesis.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

Cyclophosphamide inhibits antibody-dependent cellular cytotoxicity (ADCC) suppression exerted by lymph node cells

The effect of cyclophosphamide (Cy) on suppression of antibody-dependent cellular cytotoxicity (ADCC) by lymph node cells (LNC) was evaluated. The results show that the suppression of ADCC exerted by LNC was abrogated when mice had been treated with Cy. Moreover, it was shown that ADCC inhibition induced by LNC was mediated by soluble factor(s) and that treatment with a single dose of 200 mg/kg ip of Cy, significantly decreased its release. In addition, suppressor activity of normal LNC was enriched by depletion of adherent cells and was not affected by treatment with monoclonal anti-Thy 1.2 plus complement. These observations indicate that modulatory cells are nonadherent and lack characteristic T-cell markers. Thus, we conclude that this suppressor system, which normally controls ADCC activity, can be inhibited by treatment of mice with Cy and that this effect may explain the enhancement of ADCC observed in splenocytes of Cy-treated animals.     

Mechanisms of lymphocyte "deletion" by high concentrations of ligand. I. Cyclic AMP levels and cell death in T-lymphocytes exposed to high concentrations of concanavalin A

DNA synthesis, cell survival, and cyclic AMP (cAMP) levels were compared in whole and purified lymph node cells (LNC) cultured with optimal (5 μg/ml) and excess (200 μg/ml) concentrations of native (N) or succinyl (S) concanavalin A (Con A) as possible models for antigen-induced lymphocyte activation and “high-dose” tolerance. Whole LNC cultured with optimal N-Con A or S-Con A showed continuing DNA synthesis and cell viability between 30 and 50% at 48 hr. In contrast, with excess N-Con A, they showed virtually no [³H]TdR uptake at this time and there was progressive loss of cell viability beginning at 8 hr; by 48 hr almost no viable cells remained. Excess S-Con A induced little cell death up to 24 hr, but by 48 hr only 20% of the cells initially placed in culture remained alive and sythesized DNA. Intracellular cAMP showed a transient rise in cultures stimulated with optimal N-Con A, peaking at 15 min, then returning to normal levels, to rise again between 24 and 48 hr. With excess N-Con A, cAMP rose within 15 min and continued to increase to a peak at 24 hr. cAMP levels in the presence of excess S-Con A remained at control levels for the first 24 hr and increased between 24 and 48 hr. LNC depleted of macrophages and B cells, when cultured in excess N-Con A, had an inhibition of DNA synthesis, elevated cAMP levels, and cell death comparable to whole LNC. It seems unlikely, however, that the increase in cAMP mediates cell killing since cAMP was not elevated yet cell death occurred in nylon wool-purified T cells exposed to excess N-Con A. Dibutyryl cAMP, and prostaglandin E₁, which markedly increase cAMP levels, failed to kill LNC at doses which totally inhibited DNA synthesis, and cells of the mouse T-lymphoma S49 and its cAMP-dependent protein kinase-deficient variant were killed equally by excess N-Con A. It is suggested that a sustained elevation of either cAMP or Ca²⁺ after early commitment may provide a significant mechanism of tolerogenesis.     

Effect of guinea pig thyroglobulin in incomplete Freund's adjuvant on experimental autoimmune thyroiditis induced by in vitro-activated lymph node cells

Guinea pigs injected with guinea pig thyroglobulin (GPTG) in incomplete Freund's adjuvant (IFA) have been shown to be unresponsive to challenge with GPTG in complete Freund's adjuvant (CFA). However, effector cells which transfer experimental autoimmune thyroiditis (EAT) can be demonstrated in cultured lymph node cells (LNC) of unresponsive animals, indicating that GPTG in IFA does not suppress the initial sensitization of EAT effector cells. LNC from unresponsive animals were unable to suppress the in vitro activation of effector LNC or to suppress EAT when cotransferred with effector cells. When GPTG in IFA was given to animals which were used as recipients of effector cells, the production of EAT was markedly suppressed. These results suggest that GPTG in IFA can suppress EAT either by preventing effector cells from interacting with the thyroid or by interfering with the function of a cell in the normal recipient which may interact with effector cells to result in the lesions of EAT.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin.

The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund's adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.     

Mitogen responsiveness and inhibitory activity of mesenteric lymph node cells

Summary Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of ³H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced ³H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

In vitro anamnestic immune responses and modulating factors

Summary The term anamnestic refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr.The antibody response was regulated by antigen-concentration. One g KLH evoked maximal antibody synthesis, 10 and 100 g KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 g KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 g KLH; 100 g inhibited antibody formation.The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 g KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca²⁺ was not required for induction of this response by KLH, but only its regulation by cAMP.Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This spontaneous antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity.Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.Abbreviations AP alum-precipitated - AFab goat IgG antibody to rabbit Fab - ATG goat IgG antibody to rabbit thymocytes - BGG bovine gamma globulin - Bsa bovine serum albumin - BAC bromo acetyl cellulose - B bursalequivalent lymphocytes - CT cholera enterotoxin - CRL complement receptor lymphocytes - DFA complete Freund's adjuvant-, - cAMP adenosine 3:5-cyclic monophosphate - cGMP guanosine 3:5-cyclic monophosphate - DbcAMP N⁶,O²-dibutryl cyclic AMP - EAC sheep erythrocytes sensitized with antibody and complement - FITC fluorescein isothiocyanate - HSA human serum albumin - KLH keyhole limpet hemocyanin - LNC lymph node cells - MEM minimum essectial Eagle's medium - medium; MIF m crophage migration inhibitory factor - Mb sperm-whale myoglobin - PHA phytohemagglutinin - PGE prostaglandins of the E series - PGF prostaglandins of the F series - PGSI inhibitors of prostaglandin systhesis - Slg surface immunoglobulin - T thymus-derived lymphocytes     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Mechanisms in opposite modulation of spleen cell and lymph node cell responses to mitogens following muramyl dipeptide treatment in vivo

It has been reported that a muramyl dipeptide (MDP) treatment regimen (200 micrograms MDP per mouse, Days -4, -3, -2, and -1) that, given prophylactically, affords protection against several infectious agents also induces lymph node hyperplasia, lymph node cell (LNC) hyperresponsiveness to mitogens, and spleen cell hyporesponsiveness to mitogens. The purpose of the present work was to extend those studies and delineate cellular mechanisms involved in these phenomena. It has been found that hyperresponsiveness of LNC was prolonged (7 days) posttreatment; in contrast, hyporesponsiveness of spleen cells was transient and rebounded by Day 4 posttreatment. Hyperresponsiveness of LNC and hyporesponsiveness of spleen cells actively enhanced and depressed normal lymphoid cell responses, respectively, in cell mixing experiments. Hyporesponsiveness of spleen cells was associated with the plastic-nonadherent, non-B-cell fraction and nylon wool-nonadherent subpopulations. Indomethacin (10(-6) M) did not abrogate hyporesponsiveness of spleen cells. These data suggest that splenic suppressor T cells result from MDP treatment and were responsible for spleen cell hyporesponsiveness. On the other hand, hyperresponsiveness of LNC was associated with the nylon wool-adherent cell subpopulations and a higher percentage of nonspecific esterase-positive cells. Hyporesponsiveness of spleen cells was associated with deficient production of interleukin 2 (IL-2), but not of interleukin 1 (IL-1). In contrast, hyperresponsiveness of LNC was not explained by enhanced IL-1 or IL-2 production.     

In vivo cyclophosphamide-mediated augmentation and aqueous HGG-induced suppression of murine delayed hypersensitivity to HGG are reflected by in vitro HGG-stimulated proliferation of lymph node cells from equivalent mice

The question addressed in this report was whether immunological processes which culminate in in vivo expression and augmentation as well as suppression of delayed effector cell activity are mirrored by events that can be quantified in vitro. For this purpose the previously characterized murine model of delayed hypersensitivity (DHS), which employs SJL/J mice immunized with aggregated human γ-globulin (AHGG) in complete Freund's adjuvant (CFA) was employed. The results indicated that lymph node cells (LNC) from cyclophosphamide (CY)-pretreated, AHGG-CFA immunized mice expressed increased proliferation in the presence of HGG and concanavalin A (Con A) but decreased LPS responsiveness compared with LNC from equivalently immunized but non-CY-treated animals. It was also found that LNC from CY-treated, AHGG-CFA immunized mice that were pretreated with aqueous HGG (aqHGG), but not aqueous bovine serum albumin (aqBSA), evidenced a markedly decreased capacity to proliferate in the presence of HGG compared with LNC from equivalent animals that were not pretreated with aqHGG. This suppressive effect was not attributable to antibody production. These findings support the conclusion that in vitro quantitation of antigen-induced proliferation by LNC from HGG-DHS mice appears to correlate with modulatory effects which are observed in in vivo expression of DHS responses.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Role of accessory cells in the in vitro growth of aggregate-derived murine T-cell colonies

C3H/HeJ lymph node cells (LNC) were seeded in 35-mm petri dishes containing 0.8% methylcellulose, 10% fetal calf serum, 2-mercapthoethanol, and supernatant from PHA or Con A-stimulated spleen cells. After 3–4 days incubation at 37 °C, colonies containing >50 cells appeared. The cells from individual colonies stained with a fluorescent anti-Thy-1 antiserum, and colony formation was prevented by treating the LNC with radiation or anti-T-cell serum + complement before culturing. When fewer than 1?2 × 10⁶ LNC were seeded, the number of colonies formed decreased exponentially; this observation suggested colony formation might require cell-cell interaction. Formation of cellular aggregates could be seen as early as 4–20 hr after plating. Colony formation of 2?5 × 10⁵ LNC was promoted by adding irradiated or anti-T serum + complement-treated LNC, and colony formation was inhibited by carbonyl iron treatment to remove adherent cells. Cell separation by velocity sedimentation showed colony promoting activity was associated with cells sedimenting at 4 mm/hr and also >6 mm/hr. These are properties similar to those of accessory cells that are required for immune responses in vitro and in vivo. Colony formation was also increased in LNC from tumor allograft immune mice, and in the uterine lymph nodes from mice bearing an allogeneic fetus. T-Cell colonies produced by direct plating of LNC in this system arise from proliferation of cellular aggregates, and are primarily a measure of accessory cell activity.     

Cell surface antigens and other characteristics of T cells regulating the antibody response to type III pneumococcal polysaccharide

The administration of a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) produces an antigen-specific T cell-dependent phenomenon termed low-dose paralysis (immunologic unresponsiveness). This form of unresponsiveness can be transferred by spleen cells obtained 5 to 24 hr after priming, and the suppressive activity of the transferred cells is abolished by prior treatment with monoclonal anti-Lyt-2 and anti-I-J antibody in the presence of complement, indicating that suppression is mediated by a distinct subset of T cells (suppressor T cells). If primed spleen cells are transferred 24 to 72 hr after immunization with SSS-III, however, the resulting antibody response of immunized recipients is enhanced. Greater enhancement is noted when transferred cells, pretreated with monoclonal anti-Lyt-2 antibody plus complement to remove suppressor T cells, are used; such enhancement is attributed to amplifier T cells. These findings indicate suppressor T cells regulate the antibody response to SSS-III by influencing the expansion of SSS-III-specific clones of B cells as well as the expression of amplifier T cell activity; the latter causes B cells to proliferate further in response to SSS-III.     

Stimulation of the synthesis and release of lipids in tumor cells under attack by antibody and C

Antibody-sensitized line-1 or line-10 tumor cells treated with GPC (TAC) incorporated fatty acids into complex cellular lipids and released increased amounts of fatty acids within 5 to 10 min after the addition of GPC as compared to control cells. This effect was dependent on the concentration of GPC used; however, under conditions where the cells were not killed, the enhanced synthesis and release of lipids were not dependent on the antibody concentration used to sensitize the cells. Treatment of the cells with antibody alone, GPC alone, or antibody plus heat-inactivated GPC did not result in enhanced synthesis or release of lipids. No enhancement in DNA, RNA, or protein synthesis in TAC was noted. Line-1 cells, which can be killed by GPC when sensitized with excess anti-Forssman IgM antibody, demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to the antibody-sensitized cells, before measurable killing of the cells had occurred. This effect persisted in the surviving cells when tested 5 and 10 min after the formation of TAC. Addition of GPC deficient in C4 to antibody-sensitized cells did not result in enhanced lipid synthesis or release. These data suggest that the synthesis of macromolecules of which lipids are a major component is of central importance for the ability of the cells to resist antibody-GPC mediated attack.     

Molecular events in the processing of avidin by antigen-presenting cells (APC)

The immune response of T lymphocytes to avidin was measured by proliferative assays, antibody production and delayed-type hypersensitivity. Mice ofH-2 ^k haplotypes were found to be low responders, whereas mice of other haplotypes, and particularly ofH-2 ^s , were high responders.Ir genes controlling this response were mapped to theI subregion ofH-2. Helper T cells were found to be responsible for the Ir phenotype of antibody production. These results indicate the feasibility of using the avidin-biotin complex as a tool for studying molecular mechanisms by which antigens underIr gene control are processed and presented to T lymphocytes.Abbreviations used in this paper Ir genes, immune-response genes - H-2 murine major histocompatibility complex - APC antigen-presenting cell - OA ovalbumin - BSA bovine serum albumin - DNP dinitrophenyl - DNP-OA DNP-ovalbumin - DNP-Av DNP-avidin - DNP-BSA DNP-bovine serum albumin - CFA complete Freund's adjuvant - PPD purified protein derivative - PBS phosphatebuffered saline - IP intraperitoneal - LNC lymph-node cells - DTH delayed-type hypersensitivity