The cellular basis for the Ia restriction in murine experimental autoimmune thyroiditis

Susceptibility to experimental autoimmune thyroiditis (EAT) in the mouse is linked to the I-A subregion of the major histocompatibility complex. EAT can be induced in susceptible strains of mice by immunization with mouse thyroglobulin (MTg) and adjuvant. We have described a cell transfer system wherein spleen cells from EAT-susceptible CBA/J mice primed in vivo with MTg and lipopolysaccharide (LPS) can be activated in vitro with MTg to transfer EAT to naive syngeneic recipients. This cell transfer system was used to elucidate the cellular basis for the I-A restriction in EAT. While the cell active in transferring EAT was Thy 1+ I-A-, depletion of I-A+ cells from the in vitro culture prevented the activation of EAT effector T cells. MTg-pulsed mitomycin C-treated naive syngeneic spleen cells as antigen-presenting cells (APCs) could replace the I-A+ cells in vitro. Allogeneic (Balb/c) APCs were ineffective. Using APCs from several recombinant inbred strains of mice, it was shown that C3H/HEN and B10.A(4R) APCs were effective in activating MTg/LPS-primed CBA/J spleen cells to transfer EAT while B10.A(5R) APCs were ineffective. This maps the H-2 restriction to the K or I-A subregions. Addition of polyclonal anti-Iak or monoclonal anti-I-Ak or anti-L3T4 during in vitro activation inhibited both the generation of EAT effector cells and the proliferative response to MTg. Irrelevant anti-Ia reagents, monoclonal anti-I-Ek, and monoclonal anti-I-Jk were ineffective. Thus the I-A restriction in murine EAT appears to result from an I-A restricted interaction between Ia+ APCs and Ia- EAT effector T cells.     

The role of cellular proliferation in the induction of experimental autoimmune thyroiditis (EAT) in mice

Experimental autoimmune thyroiditis (EAT) can be induced in susceptible strains of mice by injection of mouse thyroglobulin (MTg) and adjuvant. Lymphocytes from immunized mice develop a proliferative response to MTg which generally correlates with the development of EAT. We utilize a cell transfer system wherein spleen cells from CBA/J mice primed with MTg and lipopolysaccharide (LPS) in vivo are activated by culture with MTg in vitro to transfer EAT to naive recipients. In vivo priming of CBA/J mice is required to develop an antigen specific proliferative response to MTg. This response is optimal between 48 and 90 hr of culture at an MTg concentration of 125-250 micrograms/ml. The correlation between proliferation and transfer of EAT is not absolute as primed Balb/c X CBA/J F1 and AKR lymphocytes do not proliferate detectably in response to MTg but can be activated to transfer EAT; primed Balb/c lymphocytes neither proliferate nor transfer EAT. Proliferation per se is not sufficient to activate cells to transfer EAT as culture with nonspecific mitogens is not effective in activating primed CBA/J spleen cells to transfer EAT. However, lymphoblasts generated during in vitro culture of primed CBA/J spleen cells with MTg are responsible for transfer of EAT; small lymphocytes are ineffective. We conclude that antigen specific proliferation in response to MTg is essential in activating lymphocytes in vitro to transfer EAT.     

Augmentation of transfer of experimental autoimmune thyroiditis (EAT) in mice by irradiation of recipients

Experimental autoimmune thyroiditis (EAT) can be adoptively transferred to normal syngeneic recipients using spleen cells from susceptible strains of mice primed in vivo with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) following in vitro activation of spleen cells by culture with MTg. Irradiation of recipient animals markedly augments the severity of thyroiditis induced in this system. Irradiation of recipients does not alter the time course of the development of thyroiditis, nor does it alter the requirement for both in vivo priming and in vitro activation of spleen cells for the development of EAT. Spleen cells from EAT-resistant strains of mice (e.g., Balb/c) do not induce EAT in irradiated recipients. Irradiated recipients develop significant levels of anti-MTg antibodies while unirradiated recipients have little detectable antibody response. The augmenting effect of irradiation can be substantially reversed by transferring naive spleen cells to recipients prior to the transfer of MTg/LPS-primed in vitro-activated spleen cells. In addition athymic CBA/Tufts nude mice develop more severe EAT than CBA/Tufts nude/+ littermates following transfer of activated CBA/J spleen cells. These data suggest that natural suppressor cells may regulate the development of EAT at the effector cell level.     

Effects of the adjuvants SGP and Quil A on the induction of experimental autoimmune thyroiditis in mice

In the present study, two adjuvants, SGP and Quil A, were assessed for their ability to induce experimental autoimmune thyroiditis (EAT) in mice. SGP (a synthetic copolymer of starch, acrylamide, and sodium acrylate) and Quil A (a plant saponin) were compared with lipopolysaccharide (LPS) and complete Freund's adjuvant (CFA) given together with mouse thyroglobulin (MTg) for their ability to induce EAT in CBA/J mice. Immunization with MTg and LPS, MTg and CFA, or MTg with SGP was effective in inducing anti-MTg antibodies and histologic EAT, while MTg with Quil A was ineffective in inducing either anti-MTg antibodies or EAT. MTg with LPS was able to prime mice for the development of an in vitro spleen cell proliferative response to MTg while MTg with SGP or with Quil A was unable to prime spleen cells to proliferate detectably in response to MTg. MTg with LPS given in vivo primes CBA/J spleen cells for further activation by in vitro culture with MTg to transfer EAT to naive CBA/J recipients. MTg with SGP was also effective in priming CBA/J spleen cells for in vitro activation and transfer of EAT while MTg with Quil A was ineffective. The effective adjuvant activity of SGP and its lack of toxicity relative to LPS should make it a useful agent for further studies in murine models of EAT.     

Induction of experimental autoimmune thyroiditis in IL-12-/- mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg and anti-IL-2R or MTg and IL-12. Previous work suggested that IL-12 was required in vitro for development of G-EAT. To determine whether IL-12 was also required during the induction and/or effector phases, DBA/1 mice with a disrupted IL-12-P40 gene (IL-12(-/-)) were used for EAT induction. Cells from MTg-sensitized IL12(-/-) donors activated in vitro by MTg or MTg and anti-IL2R induced severe EAT in recipient mice. Compared with effector cells from IL-12(+/+) donors, effector cells from IL-12(-/-) donors induced thyroid lesions dominated by lymphocytes with minimal granulomatous changes. Thyroids of recipients of IL-12(-/-) cells expressed less IFN-gamma mRNA and more TGF-beta, IL-4, and IL-10 compared with recipients of IL-12(+/+) cells. When IL-12 was added during in vitro activation, cells from both IL-12(-/-) and IL-12(+/+) donors induced severe G-EAT, and expression of all cytokines except IL-12 was comparable in thyroids of both IL-12(+/+) and IL-12(-/-) recipients. Transfer of cells from IL-12(+/+) or IL-12(-/-) donors into IL-12(+/+) or IL-12(-/-) recipients indicated that IL-12 expressed in thyroids was derived from recipients. Thus, endogenous IL-12 is not absolutely essential for the sensitization and activation of EAT effector cells to induce severe EAT, although it is required in vitro to promote activation of cells to induce severe granulomatous histopathology.     

Induction of granulomatous experimental autoimmune thyroiditis in mice with in vitro activated effector T cells and anti-IFN-gamma antibody.

Experimental autoimmune thyroiditis (EAT) can be induced in mice after the transfer of mouse thyroglobulin (MTg)-sensitized donor spleen cells that have been activated in vitro with MTg. CD4+ T cells are required for the transfer of EAT in this model. Because CD4+ T cells produce various lymphokines, such as IFN-gamma, that may be involved in the activation or regulation of the immune response to MTg and the development of EAT, the present study was undertaken to determine whether a neutralizing mAb to IFN-gamma could modulate the induction or expression of EAT. The anti-IFN-gamma mAb XMG-1.2 had no effect on sensitization of donor cells. However, addition of XMG-1.2 mAb during in vitro activation of MTg-primed spleen cells resulted in more severe EAT in recipient mice. The thyroid lesions in recipients of cells cultured with MTg and XMG-1.2 mAb also exhibited granulomatous changes, which differed qualitatively from the predominantly lymphocytic cell infiltrates in recipients of cells cultured with MTg alone. Recipients of MTg-activated spleen cells also developed severe granulomatous EAT when they were given injections of XMG-1.2 mAb. The effects of XMG-1.2 could be neutralized by IFN-gamma. Recipients of cells cultured in the presence of XMG-1.2 mAb had augmented autoantibody responses, although there were no apparent differences in the IgG subclass distribution of the anti-MTg autoantibody responses. These studies suggest that neutralization of endogenous IFN-gamma results in increased activity of cells capable of inducing granulomatous EAT in mice.     

Prevention and reversal of experimental autoimmune thyroiditis (EAT) in mice by administration of anti-L3T4 monoclonal antibody at different stages of disease development

Experimental autoimmune thyroiditis (EAT) can be induced in CBA/J mice following the transfer of spleen cells from mouse thyroglobulin (MTg)-sensitized donors that have been activated in vitro with MTg. Since L3T4+ T cells are required to transfer EAT in this model, the present study was undertaken to assess the effectiveness of the anti-L3T4 monoclonal antibody (mAb) GK1.5 in preventing or arresting the development of EAT. Spleen cells from mice given mAb GK1.5 prior to sensitization with MTg and adjuvant could not transfer EAT to normal recipients and cells from these mice did not proliferate in vitro to MTg. Donor mice given GK1.5 before immunization did not develop anti-MTg autoantibody and recipients of cells from such mice also produced little anti-MTg. GK1.5 could also prevent the proliferation and activation of sensitized effector cell precursors when added to in vitro cultures. When a single injection of mAb GK1.5 was given to recipients of in vitro-activated spleen cells, EAT was reduced whether the mAb was given prior to cell transfer or as late as 19 days after cell transfer. Whereas the incidence and severity of EAT was consistently reduced by injecting recipient mice with GK1.5, the same mice generally had no reduction in anti-MTg autoantibody. Since EAT is consistently induced in control recipients by 14-19 days after cell transfer, the ability of mAb GK1.5 to inhibit EAT when injected 14 or 19 days after cell transfer indicates that a single injection of the mAb GK1.5 can cause reversal of the histopathologic lesions of EAT in mice. These studies further establish the important role of L3T4+ T cells in the pathogenesis of EAT in mice and also suggest that therapy with an appropriate mAb may be an effective treatment for certain autoimmune diseases even when the therapy is initiated late in the course of the disease.     

Synergism between mouse thyroglobulin- and vaccination-induced suppressor mechanisms in murine experimental autoimmune thyroiditis.

Previous studies have shown that genetically susceptible mice can be rendered resistant to the induction of experimental autoimmune thyroiditis (EAT) by pretreatment with deaggregated mouse thyroglobulin (dMTg). This resistance is mediated by CD4+ suppressor T cells (Ts) which suppress the afferent/inductive phase of EAT. Recent work has also shown that resistance to EAT can be achieved by vaccination with irradiated spleen cells previously primed in vivo with MTg and cultured in vitro with MTg (gamma SC). The gamma SC-induced resistance also inhibits the afferent phase of EAT but is mediated by both CD4+ and CD8+ Ts. To determine if dMTg- and gamma SC-induced suppression can cooperate to prevent EAT, we pretreated mice with suboptimal doses of dMTg and gamma SC before challenge with MTg and adjuvant. Mice receiving dMTg or gamma SC only showed suppressed in vitro response to MTg, but the development of thyroid lesions was unaltered. However, mice given one or two subtolerogenic doses of dMTg followed by gamma SC not only showed suppressed in vitro response to MTg, but also little or no thyroiditis, indicating cooperation between these two mechanisms. The cooperation was not reciprocal since reversing the order, giving gamma SC first followed by dMTg, was not effective in suppressing EAT. Thus, suppressor mechanisms activated by pretreatment with dMTg and gamma SC can act synergistically to suppress EAT induction; the two mechanisms may cooperate in vivo to maintain self-tolerance provided that MTg-specific CD4+ Ts are initially activated.     

Suppression in experimental autoimmune thyroiditis: the role of unique and shared determinants on mouse thyroglobulin in self-tolerance.

Previous studies have shown that T cells from mice genetically susceptible to experimental autoimmune thyroiditis (EAT) recognize determinants shared between mouse thyroglobulin (Tg) and heterologous Tgs. Some shared determinants are thyroiditogenic; lymphocytes from mice immunized with mouse Tg (MTg) or human Tg (HTg) and reciprocally restimulated in vitro with either Tg can transfer EAT. Studies on the mechanisms of self-tolerance have shown that pretreatment with soluble MTg suppresses in vitro proliferation to MTg and EAT induction with MTg. To determine the role of share epitopes in maintaining tolerance, mice were pretreated with soluble HTg and immunized with HTg or MTg and adjuvant. Cells from HTg-pretreated. HTg-immunized mice showed suppressed in vitro proliferative response to HTg. Following MTg immunization, the cells showed suppressed in vitro response to MTg. However, in contrast to MTg pretreatment, the subsequent development of EAT in vivo was unaltered in severity following HTg pretreatment. Thus, determinants shared between HTg and MTg can induce suppression of in vitro responses to HTg and MTg, but not inhibit the onset of thyroiditis, suggesting that T cells recognizing MTg-unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique and shared epitopes on MTg are essential for the overall maintenance of self-tolerance.     

In situ analysis of T cell subset composition in experimental autoimmune thyroiditis after adoptive transfer of activated spleen cells

T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro. The in vitro-activated cells adoptively transfer EAT as well as differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine the kinetics of T cell subset infiltration and distribution in situ after adoptive transfer, we applied the avidin-biotin-peroxidase labeling technique to thyroid sections, utilizing rat monoclonal antibodies followed by a biotinylated rabbit anti-rat antibody. Female CBA donor mice were immunized with MTg and lipopolysaccharide. Their spleen cells were obtained 7 days later, cultured with MTg, and transferred into recipient mice. The thyroids were removed on Days 7, 10, and 14 after transfer and serially sectioned. The early phase of transferred EAT showed a higher percentage of L3T4+ cells compared to Lyt-2+ cells, yielding a ratio of 2.3 and total T cells of about 35%. By Day 10, both T cell subsets had increased to a total of about 56%. However, the relative increase was greater in the Lyt-2+ subset; the nearly doubled percentage was statistically significant, resulting in a downward shift in the subset ratio to 1.7. Little change in the in situ distribution was seen on Day 14. The percentages of F4/80+ (macrophage) population in lesions examined on Days 10 and 14 were fairly constant and B cell involvement was minimal. These findings illustrate the pathogenic role of both T cell subsets in adoptively transferred EAT and the time-dependent changes in their relative proportions leading to thyroid gland destruction.     

Characterization of the in vitro murine T-cell proliferative responses to murine and human thyroglobulins in thyroiditis-susceptible and -resistant mice

The in vitro proliferative response to autoantigenic mouse thyroglobulin (MTg) of lymph node cells (LNC) from thyroiditis-susceptible (high-responder) CBA/J (H-2k) mice was further characterized. The relatively weak response was enhanced by adding irradiated spleen cells from normal syngeneic mice to cultures of responding LNC. Furthermore, the adjuvant used for immunization was found to influence the magnitude of the response. Results of experiments varying both the adjuvant and the route of immunization (footpad versus subcutaneous) demonstrated that marked proliferative response to MTg in vitro was not necessarily a predictor of the severity of disease. However, the capacity to proliferate in response to MTg correlated with disease susceptibility, as reported previously. The response to MTg was dependent on Thy-1+, Lyt-1+2- cells and was inhibited by monoclonal I-A antibodies. Thus, proliferation is mediated by T cells of the helper/amplifier phenotype recognizing the autoantigen in association with Ia molecules. The determinants on human thyroglobulin (HTg) and MTg stimulating the proliferative responses of LNC from thyroiditis-susceptible and thyroiditis-resistant (low-responder) BALB/c (H-2d) mice were found to differ. Cells from resistant mice proliferated only in response to foreign determinants on HTg and not to shared or mouse-specific epitopes of MTg, whereas susceptible mice had T cells reactive to shared determinants expressed on MTg and HTg as well as to foreign determinants on HTg.     

Suppression in murine experimental autoimmune thyroiditis: in vivo inhibition of CD4+ T cell-mediated resistance by a nondepleting rat CD4 monoclonal antibody.

Genetically susceptible mice become resistant to experimental autoimmune thyroiditis (EAT) induction with mouse thyroglobulin (MTg) and lipopolysaccharide after pretreatment with deaggregated MTg (dMTg). Recent work showed this suppression to be mediated by CD4+ suppressor T cells (Ts). To study Ts action in vivo, we used a rat IgG2a monoclonal antibody (mAb), YTS 177.9, which modulates CD4 antigen in vivo without depleting CD4+ cells. Initial studies showed that after two 1-mg doses of mAb 7 days apart, extensive CD4 antigen modulation of peripheral blood leukocytes occurred within 4 days. Mice given CD4 mAb 24 hr before dMTg (2 doses, 7 days apart) were resistant to EAT induction when immunized with MTg and LPS 20 days later. Also, anti-rat IgG2a titers were reduced following challenge with heat-aggregated rat IgG2a compared to controls. Subsequent analysis of serum in CD4 mAb-treated animals revealed that mAb was present in the circulation for 14 days. Moreover, mice given CD4 mAb and dMTg, then challenged after only 10 days, when CD4 mAb was still circulating, developed a significantly higher incidence of thyroid damage than controls. These findings suggest that modulation of CD4 antigen does not interfere with Ts activation, but the presence of CD4 mAb, at the time of autoantigenic challenge, can interfere with tolerance to EAT induction. Thus, the direct relationship between the presence of CD4 mAb and inhibition of EAT suppression implicates a role for CD4 molecules in the mediation of suppression.     

Induction of antitumor activities in spleen cells from mice and rats activated with lipopolysaccharide immobilized on beads

Summary Lipopolysaccharides (LPS) were coupled to polystyrene beads in order to apply the LPS without toxicity. The antitumor activity of the LPS-immobilizing beads was studied in experiments in vitro and in vivo. In vitro studies showed that spleen cells from C3H/HeN mice stimulated by beads immobilizing LPS fromEscherichia coli produced cytolytic activity as strong as that of lymphokine-activated killer (LAK) cells. Spleen cells from Sprague-Dawley rats stimulated by beads immobilizing LPS fromSalmonella minnesota produced cytolytic activity stronger than that of LAK cells. However, spleen cells stimulated by beads immobilizing each component of the LPS separately could not induce cytolysis. Contact stimulation, even for a brief period, sufficed for cytolytic activity, and was enhanced by culture for 48–72 h. Through in vivo studies, the suppression of tumor growth and a prolongation of the survival time were observed in tumorbearing mice injected with spleen cells activated by beads immobilizing LPS fromE.coli, and in mice injected with LAK cells. The effect of the activated spleen cells was stronger than that of the LAK cells. In rats bearing metastatic tumors, spleen cells activated by beads immobilizing LPS fromS.minnesota suppressed lung metastases more strongly than did LAK cells. These findings indicate that LPS immobilized by beads induced killer cells more strongly than interleukin-2. Ex vivo immunomodulation with LPS-immobilizing beads can be applied usefully as an anticancer treatment.     

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of epitope mimicry in potentiation but not initiation of autoimmune disease

We have examined whether the peptide (368-381) from the murine adenovirus type 1 E1B sequence, exhibiting a high degree of homology with the known pathogenic thyroglobulin (Tg) T cell epitope (2695-2706), can induce experimental autoimmune thyroiditis (EAT) in SJL/J mice. The viral peptide was a poor immunogen at the T or B cell level and did not elicit EAT either directly or by adoptive transfer assays. Surprisingly, however, the viral peptide was highly antigenic in vitro, activating a Tg2695-2706-specific T cell clone and reacting with serum IgG from mice primed with the Tg homologue. The viral peptide also induced strong recall responses in Tg2695-2706-primed lymph node cells, and subsequent adoptive transfer of these cells into naive mice led to development of highly significant EAT. These data demonstrate that nonimmunogenic viral peptides can act as agonists for preactivated autoreactive T cells and suggest that epitope mimicry may at times play a potentiating rather than a precipitating role in the pathogenesis of autoimmune disease.     

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.     

Induction of TNP-specific cytotoxic T lymphocyte memory in vivo in the absence of T helper cell activity

The question of whether TH cells are required for the priming of CTL precursors (CTLp) in vivo was studied by using Txbm mice (Thymectomized, irradiated, and stem cell-reconstituted mice). In these mice, TNP-specific CTL could be induced in vitro with TNP-coupled spleen cells only if the cultures were supplemented with an IL 2-containing supernatant (ConAsup). In contrast to normal mice, TNP-specific Lyt-2-TH cells could not be induced by skin painting with trinitrochlorobenzene (TNCB) (as tested by the ability to help CTL formation from thymocyte or normal spleen precursors). These data confirm previous findings that Txbm mice possess CTLp but that their TH compartment is deficient. TNCB skin painting had, however, a clear priming effect on the CTLp population: spleen cells from TNCB-painted mice could give rise to specific CTL with a lower amount of ConAsup than spleen cells from unprimed mice. In addition to this, priming changed the CTLp so that stimulation with lightly coupled cells (0.1 mM trinitrobenzene sulfonic acid [TNBS] instead of 10 mM TNBS) became effective. These changes took place without a significant increase in the frequency of TNP-specific CTL precursors. The data obtained are consistent with the concept that at least with some antigens, CTLp proliferation (clonal expansion), which is probably caused by activated TH cells, is not required for the induction of immunologic memory in vivo.     

LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice

Recent studies have suggested that lipopolysaccharide (LPS) derived from gram-negative organisms such as Bacteroides, which are not members of the Enterobacteriaceae, stimulate B cells from the classic LPS-hyporesponsive C3H/HeJ mouse. In the present study, purified, phenol-water-extracted LPS from Bacteroides fragilis ATCC 25285 (B-LPS) was tested for its ability to induce in vivo and in vitro responses in classic LPS-responsive C3H/HeN, LPS-hyporesponsive C3H/HeJ, and (C3H/HeN X C3H/HeJ)F1 hybrid mice. B-LPS induced mitogenic responses in both C3H/HeN and C3H/HeJ spleen cell cultures when cells were cultured under standard conditions, i.e., 8 X 10(5) cells/well. Interestingly, when lower spleen cell numbers were tested with B-LPS, a typical responsive-nonresponsive pattern developed in which good mitogenic responses were induced by B-LPS in C3H/HeN cultures and in which low responses in C3H/HeJ spleen cell cultures were evident. In vivo immunization of mice with B-LPS resulted in high antibody responses in C3H/HeN, intermediate responses in F1, and low responses in C3H/HeJ mice. When purified splenic B cells were incubated with B-LPS, both mitogenic responses and polyclonal immunoglobulin M (IgM) synthesis occurred in C3H/HeN cultures, whereas intermediate responses were noted in F1 cultures and no response was seen in B cell cultures from C3H/HeJ mice. Furthermore, in vitro TNP-B-LPS responses were induced in C3H/HeN spleen cells or purified B cell cultures, and intermediate anti-TNP PFC responses occurred in F1 spleen cells or purified B cell cultures. The toxicity of B-LPS was tested in galactosamine-sensitized mice. The LD50 values for B-LPS in classic LPS-responsive C3H/HeN and C57BL/6J mice were 0.6 microgram and 1.1 microgram, respectively; F1 hybrid mice were approximately 15-fold more resistant, whereas C3H/HeJ mice gave an LD50 of 1650 micrograms. This study shows that phenol-water preparations of B-LPS are biologically active and induce responses in the classic LPS-responsive but not in the LPS-hyporesponsive C3H/HeJ mouse strain.     

Suppression of GvHR by in vitro allogeneically stimulated lymphocytes: the role of lymphoblasts in the regulation of a primary T-cell response

Graft versus host reaction (GvHR) induced in 10-day-old F1 mice by in vitro allogeneically or mitogenically stimulated spleen cells is lower than that induced by unstimulated fresh spleen cells. In vitro stimulated lymphoblasts are unable or only slightly able to induce a GvHR. An active suppression by the blasts is not involved. Since lymphoblasts after in vivo stimulation show an increased ability to elicit a GvHR it is concluded that in vitro and in vivo stimulated lymphoblasts have different properties. A different homing cannot be excluded after transfer to the mouse.