Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.     

Frequency analysis of immunoglobulin V-gene expression and functional reactivities in bone marrow B cells

Frequency of immunocompetent B cells in bone marrow has been determined in vitro under culture conditions that allow the development in vitro under culture conditions that allow the development of every growth-inducible B cell into a clone of IgM-secreting PFC. Three limiting dilution culture systems were employed: a specific helper assay with SRBC as antigen and using activated T helper cells, a nonspecific helper assay using Con A-induced factors as a source of help, and polyclonal activation with LPS. From unseparated, normal C57BL/6J bone marrow 1 in 2200 to 1 in 2820 B cells were induced to form a clone of PFC to SRBC in each of the 3 systems. This corresponds to a frequency of 1 SRBC-specific clone in every 900 IgM-secreting LPS-reactive clones. The frequencies of specific plaque-forming B cell clones in terms of LPS-reactive B cells was 1 in 36 for NNP1-SRBC, 1 in 58 for TNP30-SRBC, 1 in 75 for NIP1-SRBC, and 1 in 230 for TNP3-SRBC. These frequencies of v-gene expression in bone marrow B cells are of the same magnitude as the corresponding frequencies for splenic B cells. Bone marrow B cells are also fully susceptible to stimulation by antigen in combination with either specific or nonspecific T cell help, as well as polyclonal activation by LPS, since every 3rd Ig-positive cells in marrow could be induced to form a clone of IgM-secreting cells. There is thus no difference in immunocompetence between surface Ig-bearing B cells from bone marrow and spleen.     

Conversion of type III pneumococcal polysaccharide low responders to high responders by immunization with a thymus-dependent form of antigen

C57BL/Ks mice immunized with 0.6 μg Type III pneumococcal polysaccharide (S3) or with 10⁹ S3 conjugated sheep erythrocytes (S3-SRBC) produced 5–7 times fewer S3-specific plaque-forming cells than similarly immunized BALB/c mice. However, when mice were primed with the SRBC carrier prior to challenge with S3-SRBC the low responder C57BL/Ks mice responded as well as the high-responder BALBc strain. The cell activated by the carrier priming was shown to be a thymus-derived (T) cell and the antibody produced by primed mice was mercaptoethanol sensitive (presumably IgM). Nonspecific T cell activation by unrelated antigens did not enhance C57BL/Ks responses to the same degree as specific carrier priming. These findings are discussed in relation to the possible cellular basis for genetic control of the S3 immune response.     

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.     

Deterioration of cellular immunity during aging. The relationship between age-dependent impairment of delayed-type hypersensitivity reactivity, interleukin-2 production capacity, and frequency of Thy-1+,Lyt-2- cells in C57BL/Ka and CBA/Rij mice

The effect of aging on the delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in vivo and the interleukin-2 (IL-2) production capacity in vitro by spleen cells from young (17 weeks) and old (125 weeks) CBA/Rij and C57BL/Ka mice were investigated. For both CBA/Rij and C57BL/Ka mice an age-related decline in the DTH response to SRBC and the IL-2 production capacity was observed. Both parameters are mediated by Thy-1+,Lyt-2- spleen cells. For both mouse strains the proportion of Thy-1+,Lyt-2- spleen cells declined less strongly with aging than the DTH reactivity and the IL-2 production capacity. From this it was concluded that not only a quantitative but also a qualitative decrease of T-cell function occurs during senescence. It was also investigated whether the proportion of Thy-1+,Lyt-2- peripheral blood lymphocytes can be used as a predictive value with regard to the decline of DTH with aging of the corresponding mouse. This was indeed found to be the case in CBA/Rij mice, but not in C57BL mice.     

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 CD4- CD8- alpha beta bone marrow T cells with suppressor activity.

The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.     

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

Strain differences in the development of auto-anti-idiotypic antibody regulation with age: genetic linkage to the Igh-C locus

The effect of age on the appearance of anti-idiotype (Id)-blocked, hapten-augmentable plaque-forming cells (PFC) in various strains of mice was investigated. Strains of mice at 2 and 6-11 months of age were immunized with 500 micrograms trinitrophenylated bovine gamma-globulin (TNP-BGG) in complete Freund's adjuvant (CFA) intraperitoneally. Splenic IgM and IgG anti-TNP PFC responses were assayed for anti-Id-blocked, hapten-augmentable PFC 14 days after immunization. It was found that strains differ with regard to the age at which they produce anti-Id-blocked, hapten-augmentable PFC. C57BL/6J (B6), DBA/1J, and C3H/HeJ mice produced a significantly high percentage of hapten-augmentable IgG anti-TNP PFC at 8-9 months of age as compared with the 2-month-old group. In contrast, 129/J, AKR/J, and C57L/J mice produced a significantly low percentage of hapten-augmentable PFC at 6-7 months of age as compared with the 2-month-old group. The CBA/J mice were high-hapten-augmentable plaque producers at both 2 and 7 months of age. SJL/J mice were, on the other hand, low producers at 2 and 11 months of age. Immune sera from high hapten-augmentable plaque-producing strains caused a hapten-reversible block of plaque formation by spleen cells from TNP-BGG-immune C57BL/6J mice and also revealed anti-(anti-TNP F(ab')2-IgG) titer as assayed by passive hemagglutination. This PFC-inhibiting activity in the immune sera of old C57BL/6J mice was an antibody of the IgG2a and IgG3 classes, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of C57BL/6J origin. Genetic analysis between high hapten-augmentable plaque production and allotypes in the (129/J X B6) crosses of the same H-2b haplotypes revealed that all of the backcrosses and F2 with high hapten-augmentable plaque production had the Igh-1a allele of the high-producer, 129/J mouse. In contrast, the crosses with low hapten-augmentable plaque production were homozygous for the Igh-1b allele of the low-producer, B6 mouse. The data suggest strain differences in the development of auto-anti-idiotypic antibody regulation with age which may be controlled by a gene(s) linked to the Igh-C locus.     

Dissociation between lymphoproliferative responses and virus replication in mice with different sensitivities to retrovirus-induced immunodeficiency.

Murine AIDS (MAIDS) is induced by a replication-defective virus (BM5d). In susceptible mice (C57BL/6J), inoculation with LP-BM5 murine leukemia virus, which consists of the BM5d virus and replication-competent B-tropic ecotropic (BM5e) and milk cell focus-inducing (BM5-MCF) helper viruses results in the polyclonal proliferation of T and B cells, immunodeficiency, and the expansion of B cells containing the BM5d provirus followed by the development of B-cell lymphomas. Several strains of mice that are resistant to LP-BM5-induced murine AIDS have been identified, and major histocompatibility complex genes as well as non-major histocompatibility complex genes were shown to play a role in this resistance. In the present study, we have examined and compared the replication of the BM5d and BM5e viruses after inoculation of LP-BM5 into sensitive (C57BL/6J) and resistant (C57BL/KSJ) mice. Using a specific polymerase chain reaction, we could detect the BM5d and BM5e proviruses as early as 1 week postinfection in the sensitive mice, and the levels of both viruses increased significantly with the progression of the disease. In contrast, in the resistant C57BL/KSJ mice, replication of BM5d and BM5e was restricted and no BM5d and only very low levels of the BM5e provirus could be detected either at early or late times postinoculation with the LP-BM5 virus mixture. Inoculation with LP-BM5 did not lead to the production of antibodies that could recognize the BM5d-encoded Pr60gag in either the sensitive or resistant mice; however, production of antibodies recognizing the env-related proteins of the helper virus was detected in the resistant but not in the sensitive mice at late times postinfection. Interestingly, inoculation with LP-BM5 increased polyclonal stimulation of spleen cells and decreased mitogen stimulation in both strains of mice. This stimulation of splenocytes persisted in the sensitive mice but decreased after a few weeks in the resistant mice. These results show an early block in BM5d and BM5e replication in the resistant C57BL/KSJ mice and indicate that resistance is a consequence of the inhibition of an onset of the BM5d virus infection and its expansion. However, initial responses to virus infection such as proliferation of spleen cells and response to mitogen are similar in both strains of mice and are therefore not necessarily related to the development of the disease.     

Studies on accessory cells in the adoptive antibody response to sheep erythrocytes in mice

CBA mice irradiated 3 days prior to injection of syngeneic nonadherent spleen cells and high numbers of SRBC contained approximately ten times more splenic direct plaque forming cells than mice irradiated immediately prior to transfer. This was not true of C57B1 mice. Increased responses in the CBA mice were shown to be dependent upon accessory cells (A cells). The results suggest that A cells are affected differently by irradiation in different strains of mice.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Allogeneic marrow transplantation after total lymphoid irradiation (TLI): effect of dose/fraction, thymic irradiation, delayed marrow infusion, and presensitization.

BALB/c mice infused with 30 x 10(6) C57BL/Ka bone marrow (BM) cells 1 day after treatment with fractionated total lymphoid irradiation (TLI) (17 fractions of 200 rads each) became stable mixed chimeras without clinical graft-vs-host disease (GVHD). Mice given 18 fractions of 100, 50, or 25 rads each followed 1 day later by C57BL/Ka BM did not become chimeric, indicating that a critical cumulative radiation dose is required for this effect. Animals given TLI with lead shielding placed over the thymus also developed stable chimerism without GVHD. Thus susceptibility to tolerance induction and protection from GVHD after TLI and allogeneic BM transplantation is not due to alteration of the thymic microenvironment by fractionated irradiation. A delay of 7 or 21 days between completion of TLI and BM administration resulted in a high incidence of graft rejection. Sensitization to minor histocompatibility antigens of the BM donor strain by blood transfusion either before or during TLI resulted in marrow graft rejection in a high percentage of animals.     

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.     

Antigenicity of erythrocytes as analyzed in terms of their cross-reactivity.

The antigenicity of human erythrocytes of four different ABO blood groups and sheep erythrocytes of unknown blood type from different individual sheep were analyzed in terms of their cross-reactivity with antibody-producing cells (plaque-forming cells, PFC) and serum antibody in immunized C57BL/6 and C3H/He mice. The antigenicity of human erythrocytes of different ABO blood groups in the C57BL/6 mice, as determined by the number of specific PFC, was, in decreasing order, AB = A greater than B = O (p less than 0.005). The efficiency of immunogenicity of the human erythrocytes in terms of their cross-reactivity with PFC was, in order, AB = A = B greater than O, and the degree of reactinogenicity was, in order, AB greater than A greater than B greater than O. The order of antigenicity of sheep erythrocytes from different animals, SRBC No. 1 - No. 6, was No. 1 (= No. 2) greater than No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 = No. 2 = No. 3 = No. 4 = No. 6 greater than No. 5 in C3H/He mice, determined by the number of specific PFC (p less than 0.01). The cross-reactivity of SRBC No. 1 - No. 6 with PFC demonstrates that the order of immunogenicity of SRBC was No. 1 = No. 2 = No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 = No. 2 = No. 3 = No. 4 = No. 6 greater than No. 5 in C3H/He mice, and that of their reactinogenicity was No. 1 greater than No. 2 =No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 greater than No. 4 = No. 6 greater than No. 2 = No. 3 greater than No. 5 in C3H/He mice. The cross-reactivity at the antibody level was indicative of the immunologic characteristics of blood cells of low antigenicity (human group O erythrocytes and SRBC No. 5 and No. 6). SRBC No. 5 and No. 6 were somewhat opposed to each other regarding antigenicity in C57BL/6 and C3H/He mice. This signifies the presence of different immunogenic components on SRBC No. 5 and No. 6. The production of anti-SRBC No. 1 antibody reached its peak on the third day after secondary immunization. That of anti- SRBC No. 1, cross-reactive with SRBC No. 6, occurred after a longer latent period, reaching its peak on day 6. This indicates that SRBC No. 1 possesses more than one kind of immunogenic component or immunogenic determinant group on its surface.     

In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

Suppressors of the graft vs graft reaction in tolerance to alloantigens

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.     

Colony-forming activity of hematopoietic stem cells in tolerant mice as affected by antigens

The effect of sheep red blood cells (SRBC) and human red blood cells (HRBC) on the amount of CFUs in the bone marrow and spleen of (CBA X C57BL/6) FI SRBC-tolerant mice was studied. The increase in the number of bone marrow and spleen CFUs was demonstrated in SRBC-tolerant mice injected with HRBC. Using SRBC test injection the increase in CFUs amount was observed in the spleen, but not the bone marrow, where the amount of CFUs remained unchanged.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.