Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

Cell-Cooperation in Anti-Sheep Red Blood Cell Antibody Response in Mouse Spleen Cell Cultures

Restoration of impaired antibody response to sheep red blood cells (SRBC) in spleen cell cultures from mice treated with heterologous antilymphocyte globulin (ALG) was studied by adding normal cells from various sources, to explore the problems of cell-cooperation in anti-SRBC antibody response and the target of ALG. When spleen cells from ALG-treated mice were separated into macrophage-rich and lymphoid cell-rich subpopulations, only the latter was found to be impaired in the ability for anti-SRBC antibody response. Addition of even a small number of normal allogeneic spleen cells sufficiently restored the impaired anti-SRBC antibody response of the spleen cells from ALG-treated mice. By use of allo-antisera, most hemolysin plaque-forming cells (PFC) generated in such cultures were proved to be derived from the cells of ALG-treated mice. Restoration was also achieved by adding thymus-derived cells, which were obtained from spleens of mice heavily irradiated and repopulated with syngeneic thymus cells, or lymphoid cells directly collected from thymuses. All results indicate that ALG selectively depletes the thymus-derived antigen reactive cells (ARC) in the spleen cell population, and that ARC supplied from normal spleen or thymus can interact with plaque-forming cell precursors (PFCP) that remain intact in the spleen cell population of ALG-treated mice. The results also suggest that a single ARC interacts with more than one PFCP and makes them develop into PFC.     

A membrane antigen of rabbit thymus cells

Rabbit cells, bearing a thymus-specific antigen, which we call rabbit thymus lymphocyte antigen (RTLA), could be detected with a suitably absorbed heterologous antiserum (goat). In the presence of complement, the RTLA antiserum lysed more than 95% of thymus cells, 70 ± 6% of lymph node cells, 46 ± 10% of spleen cells and 12 ± 7% of bone marrow cells. The number of direct or indirect hemolytic spleen plaques was not reduced by treatment with RTLA antiserum and complement, but was greatly diminished by an unabsorbed thymus antiserum which killed more than 90% of bone marrow cells. RTLA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from Ig receptor bearing subpopulations. The antiserum concentration could be so adjusted that the cytotoxicity against bone marrow was not manifested, while the cytotoxicity against other cell populations remained unchanged. The latter were identified by thymidine incorporation induced by treatment with antibody directed against rabbit light chain allotype. A small subpopulation of thymus cells did not have RTLA antigen and sedimented with a velocity distinct from that of the peak of RTLA-bearing cells.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Studies of mouse immunoglobulin allotypes by reverse plaque assay: Detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/6J mice

A reverse hemolytic plaque assay for the detection and enumeration of mouse spleen cells secreting immunoglobulins bearing a particular allotypic specificity is described. Sheep red blood cells (SRBC) coated with protein A or anti-mouse gamma globulin antibody were employed as indicator cells and an anti-allotype antibody was used as developer. A comparison of the efficiency of protein A, goat anti-mouse or rabbit anti-mouse gamma globulin antibody-coated SRBC as indicator cells in the plaque assay indicated that the rabbit anti-mouse gamma globulin-coated SRBC gave the best results in terms of number and morphology of the plaques. The number of indicator cells in the assay mixture also significantly affected the quality of the plaques formed. When the mouse spleen cells were assayed with the indicator cells and an anti-allotypic antibody as developer in presence of complement in a liquid medium, only those cells secreting the immunoglobulin of the given allotypic specificity formed hemolytic plaques.     

Rat T lymphocyte-specific antigens and their cross-reactivity with mouse T cells.

Anti-rat T lymphocyte serum (ATLS)2 was prepared by immunizing rabbits with purified T cells from rat mesenteric nodes and absorbed with rat red cells and syngeneic sarcoma cells. The specificity of ATLS for rat T cells was confirmed by the following reasons: a) ATLS was not toxic for bone marrow cells but lysed most of the thymocytes and a number of spleen and lymph node cells, which were inversely correlated to the percentage of cells with B cell characteristics in respective organs; b) anatomical localization of ATLS-reactive cells in lymphoid organs coincided to the thymus-dependent areas, i.e. the paracortex of lymph node and the periarteriolar region of spleen; c) spleen cells treated with ATLS and complement failed to respond to phytohemagglutinin but normally responded to bacterial lipopolysaccharide; d) those cells treated with ATLS and complement could not induce a graft-vs-host reaction in F1 hosts, whereas the same treatment did not affect direct plaque-forming cells. All of these data confirm the specificity of ATLS and indicate that ATLS recognizes rat T lymphocyte-specific antigens (RTLA). Absorption studies showed that RTLA were present in higher concentration on medullary thymocytes and peripheral T cells than on cortical thymocytes, but absent from bone marrow, liver, and brain tissues. When the cross-reactivity of RTLA with mouse T cells was studied by C-dependent cytotoxicity and immunofluorescence, it was found that mouse T cells shared at least one determinant of RTLA with rat T cells, and that distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RTLA in rat lymphoid organs.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

Reciprocal changes in interferon production and immune responses of mouse spleen cells fractionated over columns of insolubilized conjugates of histamine.

Cultures of spleen cells from unimmunized and immunized BALB/c mice can support both direct and indirect anti-sheep erythrocyte plaque-forming responses. The responsiveness of spleen cells to this thymus-dependent sheep cell antigen can be altered by fractionation of the cells over insolubilized conjugates of histamines (H-R-S). Cells that do not adhere to H-R-S (i.e., those that pass through the columns) produce a significantly greater plaque-forming cell response than do non-chromatographed cells or cells that have passed through a control column of rabbit serum albumin-Sepharose (R-S). In contrast, the direct plaque-forming cell response of the same culture to Salmonella typhimurium lipopolysaccharide (a T-lymphocyte-independent antigen) was not significantly different in any of the groups of cells tested. In addition, spleen cell filtration over H-R-S also resulted in a significant increase in the blastogenic response to phytohemagglutinin as well as a significantly lower production of interferon in response to phytohemagglutinin. The possibility that suppressor cells (which were shown to adhere to H-R-S) produce their inhibitory effect via interferon is discussed.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

Immunosuppression by spleen cells from Moloney leukemia. Comparison of the suppressive effect on antibody response and on mitogen-induced response.

The inhibitory effect of cells from leukemic spleens on the immune functions of normal lymphocytes was studied. Suppressor cells were obtained as the nonadherent fraction (NA) from splenic tumors of mice infected with MuLV-Moloney. This fraction (NA MuLV- M) contained less than 10% membrane Ig-positive (Ig+) cells, 45 to 60% theta-positive cells (theta+) and 40 to 50% naught cells (theta-, Ig-). Similarly prepared fractions from normal control spleens (NAc) containing 75 to 90% theta+cells and less than 10% Ig+ and naught cells were utilized in control cultures. Addition of the NA MuLV- M cells into cultures (Marbrook system) of normal spleen cells with sheep red blood cells suppressed the specific antibody response determined by the number of hemolytic plaque forming cells (PFC). The PFC response was significantly suppressed at a suppressor cell to responder cell ratio of 1:100, and was completely abolished at a ratio of 1:10 or higher. The control NAc fraction showed some inhibitory effect only at high suppressor to responder ratios (1:2 or 1:1). In contrast, the suppressive effect of NAMuLV-M on mitogen-induced 3H-thymidine incorporation in normal B and T cells was much weaker. Very little, if any, suppression occurred at the ratio of 1:100 or 1:10, however, about 50% decrease in DNA synthesis was observed at the ratio 1:2 or 1:1. On the basis of this differential suppressive effect, it is suggested that leukemic spleen cells can suppress the function of immunocompetent cells by more than one mechanism.     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

Biochemical relationship of thymocyte mitogenic factor and factors enhancing humoral and cell-mediated immune responses.

Supernatants from concanavalin A-induced mouse spleen cells contain soluble factors which: 1) augment the anti-sheep erythrocyte hemolytic plaque-forming cell response of nude mouse spleen cells, 2) facilitate the generation of antigen-specific cytotoxic cells by alloantigen-stimulated thymocytes, and 3) enhance the mitogenic response of thymocytes in the presence or absence of phytohemagglutinin. The uninduced control spleen cell supernatants contained variable but significantly less biologic activity within the difference assay systems. The principle enhancing activity (apparent m.w. 35,000 to 38,000) in each of the assay systems was found to be inseparable after a multiple step purification scheme involving ammonium sulfate precipitation, two-cycle gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on Phenyl Sepharose. These results indicate that either a single factor (designated thymocyte mitogenic factor) augments both humoral and cell-mediated immune responses or, alternatively, the different biologic activities are mediated by different factors that share a common core structure.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.     

Suppression of heterologous immunity by Nematospiroides dubius antigens in vitro

The direct effect of the soluble antigens in the homogenate of adult Nematospiroides dubius (AH) on spleen cells from uninfected NIH mice was investigated using a Mishell-Dutton culture system. Parasite antigens were shown to reduce the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in a dose-dependent manner in vitro. A population of suppressor cells was demonstrated in the spleens of infected mice. Furthermore naive spleen cells cultured in the presence of AH gave rise to cells which depressed the PFC response of naive cells when subsequently cultured together in vitro. Treatment of these cell populations with anti-thy 1.2 plus complement did not impair suppressor activity, and it was concluded that cells expressing the T-cell phenotype were not involved.     

T-cell help for the IgA response: the function of T cells from different lymphoid organs in regulating the proportions of plasma cells expressing various isotypes

Differential distribution of IgA-specific primed Lyt 2- T cells (TH) in favor of gut-associated lymphoid tissue (GALT) has been proposed to account for the high proportion of IgA-producing plasma cells at mucosal versus nonmucosal sites. We find, however, that GALT TH primed enterically with sheep red blood cells (SRBC) contain no more help for IgA responses than peripheral lymph node (PN) TH primed subcutaneously. Moreover, GALT TH are only poorly primed by enterically administered soluble protein antigen and therefore provide less help for all isotypes than PN TH primed subcutaneously with the same antigen. On the other hand, supernatants of GALT TH stimulated with concanavalin A (Con A) in vitro do help higher IgA:IgG plaque-forming cell (PFC) ratios in cultures with 2,4, 6-trinitrophenyl-SRBC (TNP-SRBC) than supernatants from PN and spleen, indicating that, when appropriately stimulated, GALT TH are capable of promoting relatively higher IgA responses than TH from other sources. Responses elicited by either SRBC-primed TH or splenic Con A supernatants in the presence of TNP-SRBC contained higher IgA:IgG PFC ratios than those elicited by linked recognition in the presence of haptenated soluble protein carrier.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

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.