Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

T cell regulation of immunoglobulin class expression in the B cell response to TNP-Ficoll: characterization of the T cell responsible for preferential enhancement of the IgG2a response

Syngeneic T cells injected into athymic nu/nu mice cause a preferential enhancement in the amount of IgG2a anti-TNP Ab produced by these mice to TNP-Ficoll. This enhancement appears to be caused by T cell effects on the IgG switching pathway. Through the use of F1----parent chimeras, the helper T cells were shown to affect TNP-Ficoll-responsive B cells in an H-2-unrestricted manner. The ability of T cells to mediate this IgG2a enhancement did not appear to be unique to any particular murine genetic background, because it was observed with T cells and nu/nu mice of C57BL/10, BALB/c, CBA/Ca, and B10.D2 strains. Priming of T cell donors with Ficoll or TNP-Ficoll did not increase the ability of splenic T cells, on a per cell basis, to enhance the IgG2a Ab response to TNP-Ficoll. The T cell population responsible for modulating the isotypic response was found to be sensitive to C-mediated cytotoxicity with both anti-Lyt-2 and anti-Lyt-1 hybridoma Ab. Although T cells from both the thymus and the spleen expressed enhancing activity, splenic T cells were more effective, on a per cell basis, than were thymocytes. The observations suggest that T cells that appear to enhance the switch to IgG2a in TNP-Ficoll-responsive B cells are not effectively primed by the antigen and interact with TNP-Ficoll-activated B cells through an H-2-unrestricted mechanism.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.     

Induction of macrophage Ia expression by lipopolysaccharide and Listeria monocytogenes in congenitally athymic nude mice

Experiments were performed to analyze the mechanism by which lipopolysaccharide (LPS) modulates the expression of Ia by murine peritoneal macrophages in vivo. We investigated the effect of LPS on Ia expression in T cell deficient mice by using the congenitally athymic nude mouse model. Injection (i.p) of LPS into athymic (nu/nu) mice resulted in a dramatic increase in the expression and biosynthesis of Ia by peritoneal macrophages 7 days after injection. The magnitude and kinetics of this induction were equivalent to increases observed after LPS injection of euthymic (nu/+) mice. Viable Listeria monocytogenes also increased Ia expression in athymic mice, but in contrast to the induction observed in euthymic mice at 3 and 7 days after injection, increased Ia expression was not seen until 7 days. Ia induction by either LPS or L. monocytogenes in athymic mice was not due to the presence or development of mature T cell function as defined by assays for T cell mitogenesis and interleukin 2 production. We conclude that increased macrophage Ia expression by LPS and L. monocytogenes in vivo can occur in the absence of mature functioning T cells.     

A novel tumour model system for the study of long-term protective immunity and immune T cell memory

We present a novel non-transgenic system to be used for studies on anti-tumour adoptive immunotherapy (ADI) and long-term T cell memory. Tumour-reactive donor immune cells against lacZ-transfected syngeneic tumour cells (ESbL-Gal) were generated from a naíve T cell repertoire in DBA/2 mice by a well-established priming/restimulation protocol, and transferred to tumour-inoculated athymic nu/nu mice. The donor immune cells efficiently mediated protective anti-tumour immunity involving both CD4(+) and CD8(+) T cells, and anti-metastatic effects were stronger in 4.5 Gy pre-irradiated than in non-irradiated tumour-inoculated hosts. Long-term persistence of beta-galactosidase (Gal)-specific T cells was shown ex vivo by tetramer staining of CD8(+) T cells specific for an immunodominant Gal epitope. Resistance of treated nu/nu mice against tumour rechallenge revealed the existence of long-term protective immune memory.     

The effects of sodium metaperiodate on T and B lymphocytes.

The differential mitogenic response of T and B lymphocytes to sodium metaperiodate has been investigated. It was found that periodate treatment leads to lymphocyte stimulation in spleen cells from Balb/c mice but not in spleen cells from the congenitally athymic nu/nu mice. In addition, treatment of Balb/c spleen cells with anti-θ serum plus complement lowers the mitogenic response to periodate and to concanavalin A without affecting the response to lipopolysaccharide. These results suggest a requirement for the presence of T lymphocytes in the initiation of a response to periodate. Spleen cells from nude mice also react with periodate, and their ability to respond to B cell mitogens is impaired after treatment with the chemical reagent.     

Characterization of helper T cells induced by the type 2 antigen polyvinylpyrrolidone

Type 2 antigens are usually unable to prime for IgG memory responses or to activate helper T cells (TH) necessary for memory B cell generation. Previous studies from this laboratory have established that low doses (0.0025 microgram) of polyvinylpyrrolidone (PVP) or a T-dependent form of PVP, PVP-coupled horse red blood cells (PVP-HRBC) can activate PVP-specific TH. The present study was undertaken in order to determine some of the characteristics of the TH activated by PVP and to compare their properties with those of classical TH1 and of TH2 cells described in many T-dependent systems. TH activated with either 0.0025 microgram of PVP or PVP-HRBC were characterized with respect to cell surface antigens, Igh restriction and generation in mice expressing an X-linked immune defect (xid mice). PVP-specific TH are similar to TH1 cells in that they are required for the production of IgG subclasses absent in primary responses and have the Lyt-1+, L3T4+, I-J-surface phenotype. These TH may not be identical with TH1 cells, however, since they are I-A+ and Igh restricted. PVP-specific TH can be generated in xid mice which do not produce antibody in a primary anti-PVP response and do not develop a memory response to PVP, regardless of whether it is presented as a type 2 or T-dependent antigen.     

Frequency and specificity of precursors of interleukin 2-producing cells in nude mice

The frequency and specificity of precursors of interleukin 2-producing cells (IL 2-P) in congenitally athymic (nude) N:NIH(s)II mice was investigated. IL 2-P were detected and quantitated in a sensitive limiting dilution microassay in which Lyt-2-depleted lymphoid cell populations were first cultured for 12 days with irradiated allogeneic (DBA/2) stimulating cells and a source of IL 2 and then washed and restimulated with irradiated T cell-depleted stimulating cells for an additional 24 hr. Supernatants from restimulated cultures were assayed for IL 2 activity on CTLL indicator cells, and IL 2-P frequencies were calculated. The results indicated that IL 2-P were undetectable in young (6-wk-old) nude mice, but increased in frequency with age to eventually reach levels five to 10-fold lower than their euthymic (nu/+) littermates. In specificity studies, microcultures established originally with limiting numbers of nude or nu/+ responding cells and DBA/2 stimulating cells were split into three aliquots and restimulated with T cell-depleted stimulating cells of DBA/2, BALB/c, or C57BL/6 origin. Analysis of IL 2 production in these restimulated microcultures clearly demonstrated different patterns of cross-reactivity in individual nude mice that were not seen in nu/+ controls. These results are discussed in the context of a model proposing that the T cell repertoire in athymic mice is oligoclonal in nature.     

Carrier-specific immune memory to a thymus-independent antigen in congenitally athymic mice.

Immune memory to the DNP epitope coupled to a nonmitogenic thymus-independent carrier, Ficoll, was demonstrated in congenitally athymic outbred Swiss mice. Strong IgM and modest IgG components of this memory were detected. Moreover, this memory was carrier specific since it was elicitable only when DNP-Ficoll primed mice were challenged with DNP-Ficoll and not when similarly primed mice were challenged with DNP coupled to pneumococcal polysaccharide or to keyhole limpet hemocyanin. Ficoll primed mice also demonstrated a memory response when challenged with DNP-Ficoll. These findings indicate that a non-T cell, presumably a B cell, is capable of recognizing the carrier epitopes of this thymus-independent hapten-carrier complex. Unlike their athymic counterparts, euthymic mice of the same genetic background failed to demonstrate the IgM component of this memory, but they did demonstrate modest carrier-specific IgG memory. These results strongly suggest that suppressor T cells are either directly or indirectly important in regulating the IgM memory response of these mice to DNP-Ficoll. Indirect regulation could possibly occur via an antibody-mediated specific immune suppression mechanism.     

Characteristics of amplifier T cells involved in the antibody response to the capsular polysaccharide of type III Streptococcus pneumoniae

Amplifier T cell activity can be transferred by spleen cells harvested 72 hr after priming with type III pneumococcal polysaccharide (SSS-III) and can be abolished by treating the transferred cells with monoclonal anti-Lyt-1, or anti-Thy-1 antibodies in the presence of complement; thus, amplifier cells represent a distinct subpopulation of T cells. Amplifier T cells were found to be sensitive to irradiation but not to treatment with cyclophosphamide. When amplifier cells were transferred to athymic nude (nu/nu) mice, the enhancement obtained was much greater than that produced in thymus-bearing (nu/+) mice; this is presumably due to the lack of suppressor T cell activity in nu/nu mice that enables amplifier T cell activity to be expressed more fully. Amplifier T cells also were found to be present in peripheral blood; these amplifier T cells were Lyt-2- in phenotype. Although the induction and activation of amplifier T cells appear to be antigen-specific, the product made by amplifier T cells may not be antigen specific in its mode of action. Because amplifier T cells can be induced and activated by exposure to immune B cells, specificity is presumably due in whole or in part to the ability of amplifier T cells to recognize the idiotypic determinants of B cell-associated antibody specific for SSS-III.     

Suppression of IgG memory responses by T cells activated with the type 2 antigen polyvinylpyrrolidone (PVP)

Although type 2 antigens, such as polyvinylpyrrolidone (PVP), generally do not prime for IgG memory responses or activate specific helper T cells (TH), previous studies have established that low doses of PVP (0.0025 microgram) can prime for IgG memory and induce TH in vivo. Doses of PVP that are optimally immunogenic for IgM antibody production (0.25-25 micrograms) do not prime for IgG memory responses and preferentially activate PVP-specific suppressor T cells (TS) which suppress IgG antibody production. The studies reported here further characterize PVP-specific TS and begin to investigate the mode of action of these TS. TS induced with high doses of PVP have a typical suppressor cell surface phenotype in that they are Lyt 2+, I-J+, L3T4-, I-A- T cells. PVP-specific TS are inducible in mice expressing the X-linked immune defect and are Igh restricted in their actions. These TS suppress PVP-specific IgG responses of PVP-HRBC (horse red blood cells)-primed B cells when the TH population is from low-dose PVP-primed mice but not when the TH population is from PVP-HRBC-primed mice. Thus the TS do not apparently directly suppress the B-cell responses but act indirectly to suppress IgG responses by preventing the expression of PVP-specific TH function. The TS induced by 0.25 microgram PVP also prevent the generation of PVP-specific memory B cells apparently by preventing the expression of functional TH which are required for induction of memory B cells. Elimination of TS activation by pretreatment of mice with cyclophosphamide at the time of priming with 0.25 microgram PVP results in the expression of TH function and priming of memory B cells.     

Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.     

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.     

Mice can recover from pulmonary influenza virus infection in the absence of class I-restricted cytotoxic T cells.

Intranasal exposure of athymic (nu/nu) BALB/c mice to influenza virus leads to a persistent infection of the respiratory tract from which the mice die, usually within 3 to 4 wk with symptoms of general cachexia. However, if these nude mice were injected 1 day after infection, with approximately 10(6) cells from individual virus-specific MHC class II-restricted Th cell clones, they showed greatly reduced mortality and the titers of infectious virus in their lungs were reduced, often to undetectable levels. By coinfecting mice with pairs of antigenically distinct viruses and subsequently determining the extent of clearance of each type of virus, it could be shown first that the clearance mechanism was immunologically specific but did not display the typical crossreaction of class I-restricted cytotoxic T (Tc) cells. In addition, neither primary nor memory Tc responses could be detected in these mice. Second, Th cell clones promoted clearance solely of those viruses that contained the specific Th cell determinant, i.e., Th cell-nonreactive bystander viruses were not cleared. These findings were compatible with virus clearance being effected either directly after recognition of infected class II-positive cells by the transferred Th cells or indirectly via promotion of a glycoprotein-specific antibody response. The latter seems to be the case because transfer of Th cells into infected T and B cell-deficient SCID mice did not result in virus clearance, although transfer of an anti-hemagglutinin antibody cocktail did. Thus, a virus-specific Tc cell response is not a requirement for recovery from a pulmonary influenza virus infection.     

Effects of tobacco glycoprotein (TGP) on the immune system. I. TGP is a T-independent B cell mitogen for murine lymphoid cells

It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.     

Essential role of extrathymic T cells in protection against malaria

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1(-)TCR(int) cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 x 10(3) per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1(-)TCR(int) cells (IL-2Rbeta(+)TCRalphabeta(+)). Unconventional CD8(+) NKT cells (V(alpha)14(-)) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.     

Extrathymic clonal deletion of self-reactive cells in athymic mice

The repertoire of T cells present in congenitally athymic mice was studied by flow cytometric analysis on populations of T cells expanded polyclonally in vitro. Athymic (BALB/c x C57BL/6)F1 mice have levels of potentially autoreactive V beta 3- and V beta 11-bearing T cells that are significantly higher than those of euthymic CB6F1 mice. Examination of potentially autoreactive cells in athymic AKR mice, however, yielded contrasting results. V beta 6+ cells, which are deleted intrathymically in normal AKR mice, are present in the repertoire of young (less than 6-wk-old) AKR nu/nu mice. Isolation of a cloned CD4+V beta 6+ cell line with Mls-1a reactivity from young AKR nu/nu mice indicates that the correlation between TCR usage and specificity is consistent with that described in euthymic mice and that this population contains autoreactive T cells that are not anergic. By 6 mo of age, however, cells expressing V beta 6 are no longer detectable. Inability to detect these cells is not simply caused by failure to expand these cells in culture, because freshly isolated populations from old nude mice exhibit the same selective absence of V beta 6-bearing cells. The data strongly suggest that extrathymic deletion, rather than clonal anergy, accounts for the apparent absence of autoreactive V beta 6-bearing cells in aged AKR nu/nu mice.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.

Spleens and lymph nodes of nu/nu, congenitally athymic, mice have 16–32% and 72–76%, respectively, of large, brain-associated-T (BAT) antigen positive cells. These BAT positive large cells were isolated from nu/nu spleens with a fluorescence-activated cell sorter (FACS-II) and then cultured on thymic reticuloepithelial cell (TRC) monolayers. Both concanavalin A and mixed lymphocyte reactivity was induced during this culture period. We concluded that these BAT positive cells are pre-T cells which can he induced to at least some T cell functions by an inductive factor produced by TRC.     

Mechanisms of the adjuvant effect of nystatin on in vitro antibody response of mouse spleen cells: indication of nystatin as a B-cell mitogen and as a stimulant for polyclonal antibody synthesis in B cells.

Adjuvanticity of nystatin, one of the polyenic antifungal antibiotics having as its primary target the membrane sterol of eukaryotic cells, was investigated by examining its effect on several functions of mouse spleen cells relevant to immunological phenomena in vitro. Nystatin was found to stimulate significantly DNA synthesis in thymus-independent (B) cells but not in thymus-dependent (T) cells. Like the other B-cell mitogens such as bacterial lipopolysaccharide (LPS), nystatin elicited nonspecifically polyclonal antibody synthesis in mouse spleen cell cultures, and also restored antibody response of T cell-deficient spleen cells of congenitally athymic nude mice to heterologous erythrocytes (RBC; thymus-dependent antigen). Thus, nystatin and LPS appeared to cause similar changes in the functions of spleen cells relevant to immunological events. However, antagonism but no additive effect in the adjuvanticity was revealed between the two adjuvants. As an interesting finding, the polyclonal generation of anti-RBC antibody-forming cells (AFC) in the spleen cell cultures by stimulation with B-cell mitogen, i.e., either nystatin or LPS, was not inhibited at all by inclusion of any anti-RBC antiserum, whereas, as is well known, the generation of AFC by stimulation with the antigen was specifically suppressed by the corresponding antiserum, indicating a difference in the genesis between the mitogen-induced AFC and the antigen-induced AFC.