Studies of the immunological capacity of germ-free mouse radiation chimeras: I. Chimerism and humoral immune response

The genetic origin of both the functional lymphoid cell and progenitor cell populations of germ-free mouse radiation chimeras was assessed by indirect immunofluorescence (IIF), anti-H-2 cytotoxicity, and survival of lethally x-irradiated secondary recipients of chimera cell populations. These studies demonstrated that germ free C3H/He mice given 1000 R and 10⁷ DBA/2 bone marrow cells express H-2 antigens on their lymphoid and bone marrow cell populations characteristic of the DBA/2 donor. These cells persist for at least 14 months postirradiation and bone marrow transplantation. However, these allogeneic mouse radiation chimeras have a reduced humoral immune response to sheep erythrocytes (SRBC). This decreased humoral immune capacity as assessed by kinetic studies of the spleen plaque-forming cell (PFC) response is present throughout the life span of the chimera. The γ₁ PFC response shows extreme depression. The reduced humoral immune responsiveness to the thymusdependent SRBC antigen is considered to be due to the absence or malfunctioning of a thymocyte population.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

Separation of cells by velocity sedimentation

A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 10⁷ cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r²/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.     

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.     

Effects of estrogen on the lymphoid regeneration and immune response in irradiated and marrow-reconstituted mice.

Various doses of estriol (E3) were given to mice intraperitoneally, immediately after lethal irradiation and marrow reconstitution. The assessment of the plaque-forming cell (PFC) response to sheep erythrocytes in the spleen and the histological assessment of lymphoid tissues were carried out 30 days later. The effects appeared to be dose-dependent and resulted in a marked suppression of the PFC response. The depletion of lymphocytes was dramatic and dose-dependent in the thymus, and in the thymus-dependent and in the thymus independent areas of the peripheral lymphoid tissues. These results suggest that E3 acts on the differentiation of stem or precursor cells toweard both the populations of T and B lymphocytes. Although E3, given on day 7 after irradiation and marrow reconstitution, suppressed the lymphoid regeneration and PFC response markedly, E3 given on day 14 had no effect. On day 7 the majority of regenerating lymphoid tissues were large pyroninophilic cells and on day 14, small lymphocytes. These results suggest that the precursor or immature lymphocytes are sensitive to E3, while mature lymphocytes are resistant. Lymphoid regeneration and PFC response were retarded in mice irradiated and reconstituted with bone marrow cells from donors pretreated with E3. These results suggest that E3 acts on the stem or precursor cells capable to differentiate in the direction of lymphoid populations and reduce their number in the bone marrow.     

FCS-activated blast T cells inhibit the in vitro response of memory CTL precursors to alloantigens by removal of factor(s) from MLC supernatant

The inhibitory effect of spleen cells, precultured in the presence of FCS, was assayed on the memory cytolytic T lymphocytes (CTL) response to alloantigens. For this, we have used in vitro conditions in which both particulate alloantigen and MLC SN are required to allow the generation of CTL. It was shown that the CTL response was totally inhibited in the presence of 5 to 7 days precultured spleen cells. This inhibitory effect was partly due to removal, by those precultured cells, of relevant factor(s) contained in the MLC SN. After velocity sedimentation at unit gravity, it was shown that the T cells able to inhibit the cytolytic response and to remove MLC SN factor(s) are found in the fractions containing the large proliferating cells. It was further demonstrated that in the presence of inhibiting cells, a significant CTL response may be obtained after addition of concentrated MLC SN. However, in this way, this inhibitory effect was not totally circumvented, which suggests that the memory CTL response is also impaired by other mechanisms.     

Appendix and γM-antibody formation: VII. Rosette-forming cells in rabbits immunized with sheep erythrocytes

After intravenous immunization with sheep red blood cells the rabbit spleen shows a sharp rise in the number of plaque-forming cells but there is no detectable rise in PFC in the appendix or mesenteric lymph node of the same animals. Repeated immunization via an appendicostomy blind loop results in virtually no local PFC and only a small rise in splenic PFC.In lethally irradiated animals neither thymocytes nor appendix cells alone restore the splenic PFC response. Simultaneous injection of the two cell types restores both direct and indirect plaque formation. The injected cells were labeled with tritiated adenosine and a standard rosette assay for specific antigen-binding cells applied to recipients' spleen cells following immunization. Rosettes appeared by 3 days after immunization whether thymocytes or appendix cells were labeled. Labeled rosettes were observed only in animals receiving labeled appendix cells.This result demonstrates the presence of rosette forming cell precursors in rabbit appendix cell populations and suggests that the cells of gut-associated lymphoid tissues include antibody-forming cell precursors which are normally seeded to the spleen and draining lymph nodes.     

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.     

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.     

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.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model

By the 1g sedimentation method using discontinuous gradients of Ficoll solution (concentrations of 6 to 14%), keyhole limpet hemocyanin (KLH)-primed spleen cells of C3H/He or DBA/2 mice were fractionated into 4 to 10 populations after IgG antibody-coated erythrocytes (EA gamma) rosetting and then treatment with anti-Thy-1.2 + complement (C). No significant difference was observed in the distribution of isotype specificities of surface immunoglobulins on B cells in each population thus fractionated, when determined by indirect immunofluorescence staining. The mixture of the 12 and 14% Ficoll fractions contained 95% of B cells bearing Fc receptor for IgG (FcR+ gamma) and 3.58% of antigen-binding cells (ABC) for KLH, while the 8% Ficoll fraction included 15% of FcR+ gamma B cells and 1.53% of ABC. Nevertheless, the FcR- gamma B-cell-enriched populations caused intensive plaque-forming cell (PFC) responses to dinitrophenol (DNP), whereas FcR+ gamma B-cell-enriched populations generated weak responses. Noteworthy is that 4 days preculture of a population containing 95% FcR+ gamma B cells resulted in the appearance of precursor activity which was ascertained by a further 4 days culture of these cells with antigen, DNP-dextran. These findings suggest that FcR gamma bearing B cells intrinsically possess precursor activity for IgM/IgG antibody-forming cells, but lose it transiently by binding immune complexes (IC). Moreover, the titer of a factor suppressing anti-DNP PFC responses (suppressive B-cell factor, SBF) was higher in the 24-hr culture supernatants of the FcR+ gamma B-cell-enriched fraction than of the FcR- gamma B-cell-enriched fraction, suggesting that SBF is produced by FcR+ gamma B cells themselves. Thus, IC seems to play an important role for the negative feedback regulation of antibody production by stimulating FcR gamma bearing B cells.     

Demonstration of hapten augmentable plaques in the bromelain treated anti-mouse red blood cell system: putative evidence for anti-idiotypic regulation of autoantibody secretion

Spleen cells from normal, nonautoimmune mice (C3H/HeN) spontaneously produce hemolytic plaques against autologous bromelain-treated red blood cells (BrMRBC). The number of anti-BrMRBC plaques detectable can be increased by including either a 3 M KCl extracted antigen from BrMRBCs or the hapten phosphorylcholine chloride (PC) as an antigenic analog in the plaque assay. Optimal PC concentration for augmenting the number of plaque forming cells (PFCs) was between 10(-7) and 10(-8) M. Incubation of spleen cells with an equal volume of 10(-7) M PC one, two, or three times resulted in the preparation of populations of cells which yielded increased numbers of PFCs. In addition, the number of anti-BrMRBC plaques of cells incubated three times could not be further increased by adding PC to the plaquing mixture. The eluate produced by the initial incubation of spleen cells with 10(-7) M PC specifically suppressed the anti-BrMRBC PFC response of these nonhapten augmentable cell populations (3 X eluted). These studies indicate that a naturally occurring autoantibody response is normally regulated by the presence of a molecule bound to the cell surface of autoantibody forming cells.     

Ontogenical studies on kinetics of lipopolysaccharide-induced response to bromelain-treated mouse erythrocytes in mouse spleen cells. II. Response of spleen cells with or without Fc receptors, C3 receptors, or Ia antigens

Lipopolysaccharide (LPS)-induced plaque-forming cells secreting IgM (IgM-PFC) and antibodies against bromelain-treated mouse erythrocytes (anti-BrMRBC PFC) on Days 1 and 2 of cultures were quantitatively estimated in spleen cells from mice of various ages. The concentrations of the four groups of PFC changed independently with age. The LPS dose dependency of the PFC response was markedly different between PFC on Days 1 and 2, but not different between anti-BrMRBC PFC and IgM-PFC or between 2- and 10-week-old mice. In a second experiment, spleen cells from 2- and 10-week-old mice were separated into subpopulations with or without Fc receptors, C3 receptors, or Ia antigens, and the LPS-induced PFC responses were quantitatively assessed in each subpopulation. Both the receptor-bearing and -lacking populations included LPS-reactive B cells, and the percentages of the LPS-reactive B cells recovered in the receptor-bearing population increased with age. However, the percentages of anti-BrMRBC PFC recovered in each receptor-bearing or -lacking population were different from those of IgM-PFC. In Ia- populations, the percentages of IgM-PFC on Day 2 were obviously higher than those on Day 1, and both of the percentages increased with age. These results suggest that the four groups of LPS-reactive B cells can be discriminated from each other by their LPS dose dependency and their cell surface markers, and that they develop differently during ontogeny.     

Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of ³H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.     

The role of membrane gangliosides in murine alveolar macrophage-mediated suppression of the immune response

Generation of aldehydes on cell membranes of viable alveolar macrophages (AM) by mild oxidation with sodium periodate was previously shown to result in total abrogation of AM-mediated suppression of the plaque-forming cell (PFC) response of spleen cells previously primed with sheep erythrocytes (SRBC). These results suggested a possible role for macrophage sialoglycoconjugates, such as gangliosides and sialoglycoproteins, in suppression. In the present report, it is shown that a purified mixture of gangliosides suppressed the PFC response of SRBC-primed spleen cells in a dose-dependent manner. Addition of rabbit anti-mouse brain antiserum (RAMB), which reacts with the gangliosides, reversed both ganglioside- and AM-mediated suppression of the PFC response. Pretreatment of AM but not spleen cells with RAMB also resulted in the reversal of AM-mediated suppression. The expression of gangliosides on the membrane of AM was detected with RAMB in an enzyme-linked immunosorbent assay (ELISA). The results suggest that membrane gangliosides may play an important role in the AM-mediated suppression of the PFC response. Since paraformaldehyde-fixed AM were not suppressive, it is speculated that AM release the suppressive gangliosides into the culture medium and rabbit anti-mouse brain antibody either prevents their release and/or neutralizes the suppressive function of released gangliosides.     

Specifically depleted spleen cell populations recover responsiveness by a concomitant unrelated delayed hypersensitivity reaction.

Spleen cells from nonimmunized CBA mice were specifically depleted of cells able to react spontaneously to pigeon erythrocytes (PRBC) by the formation of either rosettes (RFC) or hemolytic plaques (PFC). Spontaneous RFC were eliminated by centrifugation on a Ficoll-Isopaque gradient whereas spontaneous PFC were removed by filtration through a PRBC-coated column. RFC-depleted populations transferred into lethally irradiated syngeneic recipients and stimulated with PRBC failed to develop any significant response during the first 7 days after transfer but developed a definite anti-PRBC reaction on the eighth day. PFC- depleted populations remained unresponsive to PRBC throughout the 12-day observation period. When the spleen cells were taken from mice whose skin had been painted with picryl (trinitrophenyl, TNP) chloride 12–15 days before and the recipients of cell populations depleted of anti-PRBC were challenged with picryl chloride and stimulated with PRBC, they recovered the responsiveness to PRBC in an accelerated fashion. Under these conditions both anti-PRBC and anti-TNP RFC and PFC were found, and some cells simultaneously reacting to both PRBC and TNP were also detected.     

Reversal of murine alveolar macrophage-mediated suppression of plaque-forming cell response by sodium periodate

Murine alveolar macrophages (AM) have been shown to suppress the in vitro plaque-forming cell (PFC) response of spleen cells previously primed with sheep erythrocytes (SRBC) in a dose-dependent manner. Mild oxidation of cell membranes on viable AM with sodium periodate resulted in total abrogation of AM-mediated suppression of the PFC response, while periodate treatment of spleen cells resulted only in partial reduction of the suppression. Pretreatment of AM with sodium periodate followed by addition of the aldehyde blocking agent, hydroxylamine, resulted in restoration of the PFC-suppressing activity of AM. Periodate treatment of AM also resulted in significantly increased macrophage-T-cell binding and cluster formation. These observations suggest that the generation of aldehyde moieties on AM membrane sialoglycoconjugates promotes positive macrophage-lymphocyte interactions, resulting in abrogation of AM-mediated suppression of the PFC response.     

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocytes reaction in vitro.

Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described.Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.     

Immunological aspects of retinoids in humans. II. Retinoic acid enhances induction of hemolytic plaque-forming cells

The effects of retinoic acid (RA) on the induction of antibody-producing cells from human tonsillar lymphocytes sensitized to sheep erythrocytes (SRBC) have been evaluated. Our results indicated that 10(-5) to 10(-7) M RA caused up to a three-fold increase in the number of plaque-forming cells (PFC) and a qualitative increase in the size of the plaques during the induction of PFC in 5- to 7-day cultures. Enhancement also occurred when tonsil cells were preincubated with RA for 24 hr and then washed, or when RA was added any time in the first 4 days after initiation of the culture. When T- and B-cell fractions were pretreated with RA for 24 hr, washed, and recombined with SRBC, RA-induced augmentation of PFC occurred only in conjunction with RA treatment of the B-cell fraction. Pretreatment of the T-cell fraction had no effect on PFC induction or on the RA-enhanced response when the B-cell fraction was simultaneously treated with RA. Other experiments suggested that RA did not modulate PFC induction by influencing regulatory functions of adherent accessory cells. Our study demonstrates that RA can enhance human antibody responses and shows that this effect is not caused by increased activity of T cells or adherent accessory cells, but is instead the result of a direct effect of RA on B-cell populations.