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.     

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.     

Effect of rabbit interferon on immune responses

Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes in vivo nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon.It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

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.     

Primary and secondary antibody responses of rabbits to bacteriophage T2: kinetic and quantitative analysis and suppression by antimacrophage globulin.

Antibody synthetic capacity of popliteal lymph node cells removed from rabbits at various times after immunization with bacteriophage T2 was assayed by radioimmunoassay of tissue culture fluid after incubation with ¹⁴C-leucine. Antibody synthesis began on day 2; IgM synthesis peaked on day 3; IgG synthesis peaked on day 5 and again on day 14. Reinjection of T2 one month later elicited an enhanced response which peaked sharply on day 2. The primary and secondary responses, but not priming for the secondary response, were suppressed by injection of goat antimacrophage globulin (AMG), but only when AMG was injected 1 to 3 days before T2. AMG reacted strongly with rabbit peritoneal macrophages and only slightly with rabbit lymphocytes or erythrocytes. Thus, macrophages appear to participate in the induction of antibody responses of rabbit lymph nodes to T2 and their function inhibited by AMG apparently operates only during the early phase of induction.     

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.     

Lactoferrin-mediated modulation of mononuclear cell activities: I. Suppression of the murine in vitro primary antibody response

The effect of the iron-binding glycoprotein lactoferrin (LF) on the in vitro primary antibody response of mouse splenic cells to a T-lymphocyte-dependent antigen, sheep erythrocytes (SE), and a T-lymphocyte-independent antigen, trinitrophenolated Brucella abortus, was examined. Both iron-saturated and native LF (8% saturated) at 10^?10 to 10^?6M concentrations but not transferrin (an iron-binding glycoprotein similar to LF) significantly (P < 0.05) decreased the number of direct (IgM) plaque-forming cells (PFC) to SE and trinitrophenol (TNP) as determined by the hemolytic plaque assay. LF was equally effective in decreasing the PFC response to TNP in T-lymphocyte-depleted splenic cell cultures. Concentrations of LF which decreased the PFC response were noncytotoxic. A 1-hr exposure of splenic cells to LF at the beginning of the 5-day culture period but not 1 hr prior to assaying for PFC, or exposure of isolated macrophage-rich but not lymphocyte-rich populations to LF prior to reconstitution resulted in a significant decrease in the anti-SE response. These data suggest that LF which is synthesized and released by polymorphonuclear leukocytes and present in inflammatory lesions may play a role in modulating the antibody response through an effect on the macrophage.     

Regulatory function of Thy 1 negative cells. II. Spontaneous release of enhancing product by B cells cultured in vitro

The supernatant from non-antigen- and non-mitogen-stimulated spleen cells, cultured for 2 days, is effective in augmenting the IgM primary antibody response to TD antigens. The IgM and IgG secondary response to TD antigens, as well as the IgM response to TI antigens is not affected. Neither T cells nor accessory cells seem to be responsible for the release of the enhancing product. The soluble factor described in this paper (i) is not a polyclonal activator of B cells, (ii) does not substitute for thymus-derived cell functions in the in vitro response to heterologous erythrocytes, (iii) does not promote the response to optimal and suboptimal doses of Con A. It modulates the antibody production only in conjunction with T cells and in the presence of macrophages. Studies carried out with mouse myeloma cell lines confirmed present results obtained with normal spleen cells. A soluble product which exhibits the biological activities ascribed to the BEF and which prevents the activation of T suppressor cells was produced by a B-cell clone that does not express immunoglobulin heavy or light chains (P. del Guercio, S. Brugère, and M. F. Poirier, Cell. Immunol., in press).     

In vitro generation of K562 killers in human T-lymphocyte subsets

Group A streptococcal pyrogenic exotoxin (SPE) is a potent modulator of the immune system when used experimentally in mice. Typically, a late burst of plaque-forming cells (PFC) follows an early suppression of the antibody response in appropriately immunized and SPE-treated mice or their spleen cells in vitro. This altered response to antigen caused by SPE is termed a deregulated antibody response. The site of action of SPE was studied by use of cellular reconstruction and complementation experiments using the separated subpopulations of immunocytes which are required for full expression of mouse spleen PFC responses to sheep erythrocytes or to trinitrophenylated (TNP) rabbit erythrocytes in vitro. The SPE site was thus localized to the T-cell subpopulation. Recently SPE has been purified to a very high degree, making it possible to ascertain that SPE alone generates the deregulation of the immune system as described before and to limit the role of nondefined components of cruder preparations of SPE. A purified horse anti-scarlet fever antitoxin which recognizes highly purified SPE as being homogeneous also recognized a single component of crude SPE by agar-gel analysis. A rabbit anti-SPE immunoglobulin raised against crude SPE and absorbed with killed, strain NY5, Group A streptococci recognized the pure SPE and a major component of the homologous crude SPE similarly. Both of these antisera neutralized the capacity of SPE to deregulate the in vitro PFC response to TNP almost completely. A third antiserum raised in rabbits against a NY5 Group A streptococcal whole cell vaccine recognized a different component of crude SPE and totally failed to recognize pure SPE. This antiserum also recognized a purified Group A streptococcal peptidoglycan as being related to components contained in the crude SPE preparation. This antiserum, however, totally failed to neutralize the capacity of SPE to deregulate the PFC response to TNP. These results show that SPE-A is the active component of cruder preparations of SPE which deregulates PFC responses.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.     

Leishmania tropica: Association of a B-cell mitogen with hypergammaglobulinemia in mice

Infection of BALB/c mice with Leishmania tropica NIH S strain resulted in splenic enlargement, hypergammaglobulinemia, and polyclonal activation of B lymphocytes as measured by the splenic plaque-forming cell response (PFC) to trinitrophenyl (TNP) and sheep erythrocytes (SRBC). The peak anti-SRBC PFC response occurred 5 weeks after infection; both direct and indirect (facilitated) plaques were significantly increased. The in vitro primary immune response to trinitrophenyl haptenated lipopolysaccharide (TNP-LPS), as enumerated by the anti-TNP PFC response, was also increased on a per-spleen basis beginning 3 weeks after infection. The properties of a lysate of L. tropica promastigotes (LTL) was studied to determine whether polyclonal B-cell activation was related to a parasite-derived mitogen. A B-cell mitogen was identified in LTL which stimulated the proliferation of spleen cells in vitro from uninfected control and congenitally athymic (T-cell-deficient) but not from μ-suppressed (B-cell-deficient) animals. Preliminary characterization of the mitogen material indicated that it was a nonpyrogenic, heat-labile peptide or protein and was probably not bacterial lipopolysaccharide (LPS).     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Cellular immune response to viral infection: in vitro studies of lymphocytes from mice infected with Sindbis virus

The development and evanescence of cell-mediated immunity to Sindbis virus infection in the mouse was studied using in vitro lymphocyte transformation. Adult mice were inoculated subcutaneously with Sindbis virus, a group A arbovirus, and cells from the draining lymph nodes and spleen were examined temporally for their ability to incorporate ³H-Tdr in the presence of Sindbis virus antigen in vitro. Lymphocyte transformation was shown to be specific and dose-related. Better stimulation was obtained with live virus antigen than with inactivated virus antigen. Specific ³H-Tdr incorporation was markedly reduced when lymph node cells were pretreated with anti-θ and complement, but anti-mouse immunoglobulin also reduced the response. Specifically sensitized cells were present in the draining lymph nodes 3–4 days after primary Sindbis virus infection, peaked at 6 days, and returned to control levels by 16 days. The response in the spleen appeared later and disappeared later. Neutralizing antibody appeared by Day 4, rose rapidly, and plateaued at a high level. The secondary cellular response differed from the primary response by being somewhat earlier and being elicitable with an amount of inactivated virus antigen which was insufficient to produce a primary response.     

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin

Rabbits were injected once or twice into the hind foot pads with alum-precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of ¹⁴C-leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane-bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco-sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate-limiting step to occur.     

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.     

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.     

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.