Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

The effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro.Cortisol treatment in vivo reduced spleen cell numbers by 88% after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells.In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10⁶ cells occurred 24 hr later than in controls and averaged, respectively, 27% and 141% of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen.The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture.     

Long-term effects of splenectomy on immunocompetent cells of adult mice

The long-term effects of adult splenectomy on immunocompetent cells was studied in BALB/c mice during an observation period of 600 days following splenectomy in comparison to age-matched controls. The percentage and absolute number of circulating Thy 1.2-positive (T) cells diminished gradually, reaching about 50% of the level of T cells in age-matched controls, with a concomittant increase in the proportion of Ig-positive (B) cells. The in vitro response of peripheral blood lymphocytes (PBL) to T-cell mitogens showed a 60% decrease of [³H]TdR uptake following phytohemagglutinin stimulation without a significant impairment of the responsiveness to concanavalin A. Alloreactivity of PBL was unaltered by splenectomy since both the in vitro reactivity of PBL to allogeneic cells using mixed leukocyte reaction, and the in vivo assay, measuring a first set skin allograft survival, were similar to those observed in age-matched controls. Studies on the humoral antibody responsiveness to sheep red blood cells (SRBC) indicated a diminished primary, and normal secondary anti-SRBC responses in splenectomized mice. No IgG response was detectable in mice primed with SRBC starting at 160 days following splenectomy, whereas normal IgG titers were observed following SRBC boost in splenectomized mice. We conclude that the spleen seems to play a regulatory role on some immune functions during adult life in mice.     

Suppression of in vitro primary immune response by L1210 cells and their culture supernatant: Evidence for cytotoxic effects

L1210 cells and their culture supernatants were found to inhibit the generation of PFC in the in vitro primary immune response of spleen cells to SRBC. As few as 1% of L1210 cells and 1% of culture fluid were inhibitory. Inhibition of DNA or protein synthesis of L1210 cells did not abolish their immunosuppressive activity, excluding exhaustion of culture medium as a possible mechanism of inhibition of PFC. Heating of the supernatant completely abrogated the suppressive effect and resulted in a marked increase of PFC. Daily evaluation of cell viability in the cultures revealed that, in the presence of L1210 and supernatants, the fraction of surviving cells is markedly reduced. We conclude that a direct cytotoxic effect on splenic lymphocytes and macrophages is the predominant immunosuppressive mechanism of L1210 cells and their culture supernatants.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Spontaneous plaque-forming cells against autologous erythrocytes develop in cultures of normal rabbit appendix cells.

Cultured appendix and, to a lesser extent, mesenteric lymph node cells from normal, unimmunized rabbits spontaneously develop PFC against several erythrocyte species, including sheep erythrocytes (SRBC), trypsin-treated, autologous erythrocytes (TRRBC), and, most importantly, untreated, autologous erythocytes (RRBC). Cells from most other lymphoid tissues of the rabbit, including the spleen, fail to develop spontaneous, anti-autologous PFC in culture. This failure seems to be due to a lack of appropriate precursors among the cells comprising their populations, rather than to an inhibition by some suppressor cell population. The development of spontaneous PFC in vitro, and their virtual absence among appendix cells freshly removed from the rabbit implies an effective regulation on their expression in situ. This regulation may involve, in part, antigen itself. The development of the anti-autologous RRBC specificities may be a consequence of the intimate association of the gut-associated lymphoid tissue with the rich antigenic milieu in the intestinal lumen, part of which may present antigens cross-reactive with self antigens.     

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.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

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.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

Cellular immunity in the mouse. IV. Altered thymic-dependent lymphocyte reactivity in the chronic graft vs host reaction and leukemia virus activation.

The lymphocytes obtained from several F₁ strains undergoing chronic GVH reactions were studied for in vitro alterations of thymic-dependent lymphoid function. Spontaneous blastogenesis was increased. The in vitro response to nonspecific mitogenic stimuli (PHA and CON-A) and specific antigenic challenge (SRBC and allogeneic cells) was initially increased and subsequently impaired. The degree of alteration was related to the severity of the observed disease and dependent upon the F₁-parental combination employed. Thymic-dependent lymphocytes obtained from animals with GVH disease possessed the ability to suppress actively the response of normal mouse cells in vitro to various T-cell mitogenic stimuli and this suppressive activity was present in the supernatant culture fluid from such cells. The mechanism of this altered in vitro T-cell reactivity is not yet completely understood, but may in part be related to the immunologic activation of murine leukemia virus from mouse cells undergoing allogeneic stimulation.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

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.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

Adjuvant-induced nonspecific supressor cells: in vitro and in vivo studies

The in vitro mitogen responses of spleen cells from mice injected ip with the nonantigenic adjuvant, Al(OH)₃, are markedly depressed. This depressed reactivity was found to be mediated by a population of nylon wool adherent, Fc-receptor-bearing suppressor cells. Suppressor cells were detected only in the spleens of the adjuvant-treated mice, as the response of lymph node cells to mitogenic stimulation in vitro was found comparable to that of normal controls. Moreover, elevated levels of suppressive activity could be detected in sera of Al(OH)₃-treated mice during the first week after adjuvant administration, which, however, did not correlate with either the long-lasting presence of suppressor cells or the in vivo normal immune response of the adjuvant-treated animals. Studies designed to test the effect of suppressor cells on the generation of splenic PFC in vivo revealed that both the direct and indirect PFC responses against SRBC inoculated iv were enhanced rather than suppressed, as compared to those of the normal controls. Furthermore, the level of cytotoxic lymphocytes generated in spleens of Al(OH)₃-treated mice immunized with allogeneic tumor cells was equal to or higher than that of the normal controls. In view of the present results, we feel that the concept that splenic, nonspecific suppressor cells (macrophages) are immunosuppressive in vivo as well as the in vivo relevance of in vitro findings should be carefully reevaluated.     

Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Immunosuppression induced by staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) is a potent mitogen for both human and murine T lymphocytes. We report here studies which demonstrate that a suppressor cell population, capable of suppressing the primary immune response of normal syngeneic mouse splenocytes to heterologous sheep erythrocytes (SRBC), is activated by SEB. Enterotoxin concentrations ranging from 0.05 to 5.0 Mg ml^?1 are capable of activating this suppressor cell population, and significant suppression can be detected with relatively small numbers of SEB-primed spleen cells (SEB-PSC) in culture. Elimination of macrophages before or after priming splenocytes with SEB does not reduce the suppression of plaque-forming cell (PFC) responses when SEB-PSC are added to normal cells in Mishell-Dutton cultures. Treatment of cells with anti-Thy-1 serum plus complement, after priming with SEB, effectively eliminates the activity of the suppressor cell population. Enrichment for T cells before priming with SEB results in greater suppression of PFC responses than do SEB-PSC generated in cultures of nonfractionated splenocytes. Activation of suppressor cells with SEB in vitro appears to occur through the induction of the T-cell subpopulation expressing the Lyt-1^?,2⁺,3⁺ cell surface phenotype, since selective depletion of this T-cell subpopulation with monoclonal rat anti-mouse Lyt-2 antisera after priming cells with SEB virtually eliminates the suppressor activity.     

Analysis of lymphocytes reactive to histocompatibility antigens. II. Exponential alloantigen-dependent lymphocyte growth in vitro

Rat thoracic duct lymphocytes were maintained in continual blast transformation and cell division by repeated in vitro stimulation with allogeneic cells. This resulted in increases in responder cell numbers of up to 10,000-fold in 10-day periods. Growth of responder lymphocyte populations was dependent upon cell density, culture medium nutrients, and the presence of antigen in the form of allogeneic cells. A titration assay for mixed lymphocyte interactions (MLI) was used to relate absolute growth of cells in preparative cultures to [³H]thymidine incorporation in analytical MLI. Growth of lymphocyte populations derived by repeated stimulation with cells bearing a single foreign MHC haplotype was supported to lesser, variable degrees by stimulation with unrelated “third party” stimulator cells. The extent of this operational cross-reactivity was assessed by parallel line analysis of MLI titrations of responder lymphocytes enriched for specific alloreactivity.     

Forssman antigen and phase specific fetal antigens: an evaluation of their role in SV40 tumor immunity.

Forssman heterophile antigen was detected on hamster fetal cells which had been previously shown to be capable of eliciting transplantation resistance to syngeneic hamster SV40 tumors. The expression of Forssman antigen continued throughout fetal development and could be detected postpartum in the tissues of neonate hamsters. In contrast, fetal antigen(s) evoking immunity to SV40 tumors was also present on early fetal cells but, unlike Forssman antigen, was not expressed after the tenth day of gestation. Immunization of hamsters with guinea pig kidney cells or sheep erythrocytes which carry Forssman antigen failed to demonstrate significant protection against SV40 tumor development. Again by contrast, immunization with fetal cells was effective in evoking tumor immunity. Evaluation of serological responses to the FORSSMAN A ANTIGEN IN HAMSTERS INDICATED THAT THE HEMOLYTIC REACTIVITY PRODUCED BY IMMUNIZATION WITH GUINEA PIG KIDNEY CELLS OR SHEEP ERYTHROCYTES WAS ELICITED AGAINST ISOANTIGENS AND NOT THE Forssman antigen. A response to the Forssman determinant could only be detected in the serum from animals receiving exhaustive hyperimmunization with fetal cells or SV40 tumor cells. These data would eliminate a possible role of the Forssman heterophile antigen in the tumor protection evoked by immunization with fetal cells bearing embryonic antigens.