Antibody formation by bone marrow cells in irradiated mice: I. Thymus-dependent and thymus-independent responses to sheep erythrocytes

Bone marrow-thymus cooperation experiments were carried out in lethally irradiated mice with sheep red blood cells (SRBC) as the antigen and direct plaque-forming cells (PFC) as the end point. Various parameters were altered, with the following results:

(1) Above 800 rad, the response by marrow cells alone, as well as the increase due to added thymus cells, was independent of irradiation dose.

(2) The response of marrow cells was greatest at high SRBC concentrations, but the co-operative effect of thymus cells was most evident at lower SRBC levels, and completely absent at high levels.

(3) Increasing the number of marrow cells, without thymus, gave increasing numbers of PFC, but the dose-response curve did not suggest cell synergism.

(4) Thymectomy and antithymocyte serum treatment of host or donor did not prevent the response by marrow cells alone. It was concluded that this was a true IgM response by antibody-forming precursors from the marrow, unaided by thymus-derived cells.

     

Modulation of primary antibody responses to sheep erythrocytes in Plasmodium chabaudi-infected resistant and susceptible mouse strains.

The in vivo primary antibody response to sheep erythrocytes (SRBC) was determined in genetically resistant C57BL/6 and susceptible A/J mice during the course of infection with Plasmodium chabaudi. Spleen cells from both strains of mice, immunized with SRBC and infected on the same day, showed significant increases in the number of direct plaque-forming cells. The response of malaria-infected C57BL/6 mice was significantly enhanced in comparison with the responses of both normal C57BL/6 and malaria-infected A/J mice. When mice were immunized at later times in the infection, the level of the response declined in both strains until it was less than 50% of the response of normal mice. Thus, suppression of the primary antibody response to SRBC does not correlate with the outcome of P. chaubaudi infection in genetically resistant and susceptible hosts.     

The in vitro immune response to sheep erythrocytes by fractionated spleen cells: biological and immunological differences

Normal spleen cells were fractionated using equilibrium density gradient centrifugation. Three fractions were taken and the ability of those fractions to produce antibody-forming cells (PFC) in vitro was examined. The three fractions were distinct from each other in susceptibility to anti-θ serum, nutritional requirements, requirements for radiation-resistant cells, antigen dose optima, kinetics and participation in cell cycle. The results are interpreted as showing a differentiation series with the immunocompetent cells moving from the dense to the less dense region of the gradient following antigen stimulation. The correlation between steps in the rate of production of PFC's by unfractionated spleen cells and the contribution of each fraction to the total response are discussed.     

Cellular and genetic control of antibody responses in vitro. I. Cellular requirements for the generation of genetically controlled primary IgM responses to soluble antigens.

Thrinitropheny1 (TNP)-specific primary IgM plaque-forming cell responses were generated in vitro by unprimed spleen cells to the soluble antigens TNP-keyhole limpet hemocyanin, trinitrophenylated poly-L(Tyr-Glu)-poly-D,L-Ala–poly-L-Lys[T,G)-A–L] and poly-L(His,Glu)-poly-D,L-Ala–poly-L-Lys[(H,G)-A–L].The T cell dependence of these primary lgm responses was esablished by (a) abrogation of responses after pretreatment of cells with a T cell-specific rabbit anti-mouse brain serum (RaMB)and C-treated spleen cells (B cells); and (c) absence of significant responses by spleen cells from congenic nude mice. A requirement for macrophages in these same antibody responses was also demonstrated:(a) responses were abrogated or strongly diminished in spleen cell populations depleted of macrophages by passage over Sephadex G-10 columns; and (b) these responses were fully reconstituted by the addition of small numbers of adherent, radioresistant, anti-Thy-1.2-treated spleen cells (macrophages) to the Sephadex G-10-depleted populations. In addition, H-2-linked Ir gene control has been demonstrated for the primary IgM responses to TNP conjugates of (T,G)-A–L and (H,G)-A–L. A system has thus been characterized which permits in vitro analysis of the cellular and genetic requirements involved in primary IgM responses to soluble antigens, including those under Ir gene control.     

Differentiation in the murine B cell lymphoma I.29: individual mu + clones may be induced by lipopolysaccharide to both IgM secretion and isotype switching

Cells from the monoclonal B cell lymphoma I.29 expressing surface IgM (mu +) are capable of differentiating in vitro to IgM secretion and of switching to IgA or IgE production in response to lipopolysaccharide (LPS) stimulation. To determine whether a single mu + B cell is capable of undertaking both differentiative pathways (isotype switch and plasma cell differentiation) I.29 mu + cells were cloned by limiting dilution and a panel of clones were analyzed by immunofluorescence, endogenous labeling and Northern blotting. While 100% of the clones could differentiate toward IgM secretion, only a proportion of them (greater than 70%) also switched to IgA and/or IgE production. Certain clones switched preferentially to a specific isotype. Taken together with the observation that C gamma genes were never the target of switching in our experiments, these data suggest that individual mu + clones from the I.29 lymphoma are "precommitted" as for their switching potentials. The subclones that showed a high frequency of switching to IgA transcribed the germ line C alpha gene(s), suggesting a role for chromatin structure in determining the isotype switch specificity. Switch variant clones expressing either IgA or IgE on the cell surface were isolated and found capable of further differentiating toward Ig secretion in response to LPS. On the contrary, we could not induce switch to IgA in IgE-producing cells. Unlike mu + and alpha + cells, all the switch variant clones expressing IgE tested by endogenous labeling constitutively secreted large amounts of IgE in the supernatants even in the absence of LPS stimulation.     

Antibody production studied by means of the LHG assay: I. The splenic response of CBA mice to sheep erythrocytes

By means of a modified plaque assay the numbers of cells producing 19S antibody (direct PFC) and 7S antibody (developed PFC) have been studied separately. Significant and somewhat surprising differences in the responses of the two kinds of antibody-producing cell have been noted. It is clear that the route of injection of the antigen is of importance in determining the response in the spleen. The response of both types of PFC was found to be biphasic. The total increase in spleen cell number after immunization was not readily accounted for by the increase in specific PFC.     

Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors. I. Preferential reactivity of in vitro generated secondary CTL with syngeneic tumor cells.

Incubation of spleen cells from mice having rejected a Moloney sarcoma virus (MSV)-induced tumor with syngeneic irradiated lymphoma or sarcoma cells bearing MSV-associated antigens in secondary mixed leukocyte-tumor cell cultures (MLTC) resulted in the generation of highly active cytolytic T lymphocytes (CTL) specifically directed against syngeneic target cells bearing MSV-associated antigens. When MSV-immune spleen cells from C57BL/6 (H-2b) and BALB/c(H-2d) mice were compared with respect to their ability to generate CTL in syngeneic secondary MLTC, it was found that both lymphoid cell populations were equally able to mount an anamnestic CTL response to MSV-associated antigens as assessed by a short-term 21Cr release assay. However, quantitative analysis of the activity of both CTL populations on either H-2b or H-2d tumor cells indicated that target cells sharing the same major histocompatibility complex (MHC) as the effector cells were lysed 10- to 100-fold more efficiently than allogeneic target cells. As suggested by the results of inhibition experiments using mixtures of 51Cr-labeled and unlabeled target cells, preferential lysis of syngeneic versus allogeneic tumor cells might be related to the establishment of effective adhesions between the former and CTL. Direct evidence for the role of MHC in determining the antigenic specificity of CTL directed against MSV-associated antigens was provided by results obtained using MSV-immune spleen cells from congenic resistant mice. Furthermore, studies of the response of F1 (H-2b/d) hybrid mice showed that stimulation of immune spleen cells with tumor cells from one parental strain or the other in secondary MLTC resulted in the generation of CTL capable of lysing tumor target cells of the same perental strain as the stimulating cells, but not of the other. The results thus suggested the presence of two sets of CTL precursor cells in F1 MSV-immune spleens, each set responding exclusively to tumor antigens associated with only one of the two parental phenotypes.     

An immunosuppressive murine leukaemia virus induces a Th1-->Th2 switch and abrogates the IgM antibody response to sheep erythrocytes by suppressing the production of IL-2.

Many retroviruses have tropism for cells in the immune system and have a propensity to induce immunosuppression in the host. Some of the effects of retroviruses on immune cell function are thought to be mediated through cytokines. Friend ImmunoSuppressive virus-2 (FIS-2) is a low oncogenic murine leukaemia virus (MuLV) that induces lymphadenopathy and immunosuppression in NMRI mice. The role of T cell cytokines during the generation of a primary antibody response in healthy and FIS-2-infected mice was studied following the antibody response to sheep erythrocytes by an in vitro immunization (IVI) technique. In cultures from FIS-2-infected mice, the antibody response was reduced compared with cultures from uninfected mice and the production of the Th2 cytokines IL-4 and IL-6 was elevated, whereas the Th1 cytokines IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were reduced. The suppressed anti-sheep erythrocyte antibody response in cultures from mice infected with FIS-2 seemed to be caused by an insufficient production of IL-2, since addition of recombinant IL-2 stimulated the antibody response. This effect was also observed in cultures depleted of T cells, indicating a direct effect of IL-2 on B cells. A switch to a Th2 cell response and suppression of IL-2 production might play a central role in the immune cell dysfunction induced by FIS-2.     

The morphology of immune reactions in normal, thymectomized and reconstituted mice: I. The response to sheep erythrocytes

The response to sheep red blood cells has been studied in the lymph nodes draining their site of injection in normal mice, and in thymectomized, irradiated, bone-marrow injected mice with and without a reconstituting thymus graft. By using a chromosome marker to differentiate between cells derived from the bone-marrow and thymus graft it has proved possible to show that the immune response should be thought of in terms of at least two cell populations. Cells of thymic origin are stimulated to mitotic activity in the interfollicular cortex, and their activity precedes both antibody production and morphological signs of activity in the follicular regions. Mitotic divisions of cells of bone-marrow origin reached a peak a day later than did the thymic cells and their activity was sustained. Follicular enlargement and germinal centre production were coincident in time both with antibody production and bone-marrow cell mitotic activity. Lymph nodes of animals lacking a thymic influence showed only minor changes after antigenic stimulation and these were restricted to the follicular regions. There appeared to be only a small quantitative difference between the responses of normal and of reconstituted animals.     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

The immunological properties of haptens coupled to thymus-independent carrier molecules. III. The role of the immunogenicity and mitogenicity of the carrier in the induction of primary IgM anti-hapten responses

Hapten (DNP-lys) conjugates of two putatively nonimmunogenic polymers, hyaluronic acid and poly-γ-D-glutamic acid, induce significant primary IgM anti-DNP responses in C3H mice. Preparations of various immunogenic (Type 3 pneumococcal polysaccharide (SIII), levan, E. coli lipopolysaccharide) and nonimmunogenic (hyaluronic acid and poly-glutamic acid) polymers were tested for their ability to act as polyclonal mitogens in vitro. In serum-containing spleen cell cultures, only lipopolysaccharide stimulated substantial cell proliferation. In serum-free medium, and using high specific activity [³H]thymidine, lipopolysaccharide, levan, SIII and to a lesser degree hyaluronic acid induced significant thymidine incorporation. However, under the latter conditions cell survival and proliferation were much less impressive. There was no apparent correlation between the capacity of various polymers to induce lymphocyte proliferation and their ?potency”? as carriers for the generation of a primary IgM anti-DNP response. Furthermore while low doses of lipopolysaccharide elicited ?polyclonal”? antibody formation in vivo, high doses of SIII, levan and hyaluronic acid did not. These results indicate that T cell-independent B cell triggering is dependent on the polymeric nature of the antigen, and that polymers need not be immunogenic or mitogenic to act as carriers for the induction of primary IgM anti-hapten antibody responses.     

Graft-versus-host disease in murine bone marrow transplantation. I. Modification of GVHD by preimmunization of recipients with spleen cells of primary recipients undergoing GVHD

Lethally irradiated (1000 rad) CBA/J mice were transplanted with anti-Thy 1 treated BALB/c bone marrow. Under these conditions, we uniformly observed the development of pathology suggestive of acute graft-vs.-host disease (GVHD), i.e. weight loss, diarrhoea, hypogammaglobulinemia and thymic hypoplasia. If, 2 wk before irradiation, the recipients were preimmunized with spleen cells taken from mice undergoing acute GVHD, these symptoms were avoided. Instead, such animals seemed to show long term survival either with or without signs of chronic GVHD (hypergammaglobulinemia, splenomegaly, lymphoid hyperplasia). The ability to show long term survival with bone marrow allografts was dependent upon successful immunization of the recipient mice. Long term survivors contain a splenic population not bearing detectable host MHC antigens, which can elicit a memory anti-host cytotoxic response from a population of quiescent donor lymphocytes previously immunized in vitro against host MHC antigens.     

Immunodepression in Taenia crassiceps infection: restoration of the in vitro response to sheep erythrocytes by activated peritoneal cells.

The in vitro response to sheep erythrocytes of mesenteric lymph node cells from mice infected with the larval cestode Taenia crassiceps is significantly depressed and can be restored to control levels by addition of activated peritoneal cells depleted of functional T or B lymphocytes. Adherent mesenteric lymph node cells from infected mice are unable to reconstitute the in vitro response to sheep erythrocytes of normal nonadherent cells. The responses of mesenteric lymph node cells from infected mice to the T-lymphocyte mitogens concanavalin A and phytohemagglutinin and the B-lymphocyte mitogen lipopolysaccharide are normal. Mesenteric lymph node cells from infected mice do not suppress the in vitro response to sheep erythrocytes of normal mesenteric lymph node cells. These results suggest that the immunodepression in T. crassiceps-infected mice is primarily the result of alterations in functional accessory cells.     

Attenuated Salmonella vaccine-induced suppression of murine spleen cell responses to mitogen is mediated by macrophage nitric oxide: quantitative aspects.

Previous reports from our laboratory have shown that 7 days after infection of C3HeB/FeJ mice with an attenuated strain of Salmonella typhimurium, there is profound suppression of responses to B- and T-cell mitogens and suppression of the capacity of spleen cells to mount a primary, in vitro plaque-forming-cell (PFC) response to sheep erythrocytes. Inhibition of the PFC response was shown to be mediated by nitric oxide (NO), as NG-monomethyl-L-arginine (NMMA) gave complete reversal of suppression. The experiments reported here examined the role of NO in suppression of the response to the mitogen concanavalin A (ConA). In contrast to the PFC system, it was found that addition of NMMA to ConA-stimulated immune spleen cells resulted in less than 20% reversal of suppression. However, addition to NMMA resulted in a 50% reversal of suppression in cocultures of immune and normal spleen cells at a ratio of 1:4. A complete restoration of ConA-induced responses was achieved in cocultures incubated in medium containing a reduced concentration of L-arginine plus 1.25 mM NMMA. Investigation of why NMMA alone was not 100% effective in reversing suppression showed that addition of ConA significantly augmented production of nitrite and gamma interferon (IFN-gamma) in cocultures containing immune cells. Addition of anti-IFN-gamma reduced nitrite levels in the cultures, although results with the combination of anti-IFN-gamma and NMMA were not significantly better than results with NMMA alone. These findings suggest that suppression in cultures stimulated with ConA is difficult to reverse completely with NMMA alone because of an overproduction of NO, which can be offset by either reducing the L-arginine concentration or blocking IFN-gamma. The quantitative relationship between nitrite levels and suppression in cocultures was examined. It was found that suppression did not correlate directly with the nitrite concentration but rather with the log10 of the nitrite concentration. Nitrite levels above 15 microM gave almost complete suppression, and levels between 1 and 10 microM gave a wide range of suppression. These results strongly support NO as the suppressor factor in Salmonella-induced immunosuppression of responses to ConA and, by inference, suppression of responses to mitogens induced by other microbes. The results show that involvement of NO cannot always be demonstrated by simple addition of NMMA to suppressed mitogen-stimulated spleen cell cultures.     

Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immuno-trapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies.

It has recently been proposed that the islet 64,000 Mr protein autoantigen (64K) of insulin-dependent diabetes mellitus (IDDM) is glutamic acid decarboxylase (GAD). We evaluated, by means of a newly developed immunotrapping enzyme activity assay (ITEAA), the prevalence of circulating GAD-autoantibodies (Ab) in a large population of IDDM patients (n = 168), blood donors (n = 87) and non-diabetic autoimmune patients (n = 40). The latter two groups were used as controls. Overall, GAD-Ab were found in 22% of IDDM patients, but in none of the two control groups (P = 0.007). These specificities were invariably associated with islet cell antibodies (ICA) (31.6% in IDDM with ICA vs 0 in IDDM without ICA, P = 0.0001), and this prevalence was higher in sera with high titer ICA (54.5% in IDDM with ICA greater than 80 JDF-units vs 22.6% of IDDM with ICA 5-80 JDF units; P = 0.002). Moreover, GAD-Ab were associated with the female sex (P = 0.002) and the concomitant presence of thyroid and/or gastric antibodies (P = 0.002). No correlation was observed between GAD-Ab and age of the patients, duration of IDDM, or associated non-organ specific antibodies. Our study indicates that GAD-Ab measured by ITEAA are: (1) detected in a proportion of IDDM patients; (2) strongly associated with ICA; (3) preferentially found in IDDM female patients with autoimmune polyendocrine serology; and (4) detected with lower frequency than that reported for 64K-Ab in IDDM.     

Female popliteal lymph node responses to H-Y antigen on male thymocytes in mice. I. Regulation of primary and secondary responses by H-2-associated Ir genes

H-2-linked genes which control popliteal lymph node (PLN) immune responses to the H-Y antigen were analysed. It was found that at least two genes or two groups of genes are involved in the genetic control and are responsible for the four variants of relations observed between the primary and the secondary response: +/-, +/+, -/+ and -/- ('+' and '-' stand for a high and a low response, respectively). The results obtained with H-2 recombinant haplotypes indicated that the genes controlling the primary and secondary responses map to the left and to the right of the E alpha locus, respectively. A high primary response was observed in the presence of b alleles at K, A beta, A alpha, and E beta loci, whereas a high secondary response occurred only in the presence of d alleles in the chromosomal segment between E alpha and D loci. From the experiments with F1 hybrids it is clear that low secondary responses are, for the most part, dominant and that the two seemingly separate control mechanisms for the primary and secondary responses may interact.     

A comparison of primary and secondary haemolysin responses to sheep erythrocytes in neonatally thymectomized, sham-thymectomized and normal Swiss mice: A time-course study of total, 19S and 7S antibody

A time-course study of total, 19S and 7S haemolysins showed that Swiss albino mice were able to give a normal secondary response to sheep erythrocytes at an age when the primary response was much reduced and delayed.