The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

Altered IFN-gamma and IL-4 pattern lymphokine secretion in mice partially depleted of CD4 T cells by anti-CD4 monoclonal antibody.

Complete elimination of CD4 cells by in vivo treatment with anti-CD4 mAb may result in B cell polyclonal activation. Additionally, mice treated with doses of anti-CD4 that eliminate half the CD4 cells produced higher anti-SRBC antibody responses than controls. This suggests that partial CD4 depletion enhances Th2-like function. To test this hypothesis we examined Th1 and Th2 lymphokine potential in mice partially depleted of CD4 cells. We measured IL-4 and IFN-gamma secretion by stimulated unfractionated spleen cells and analyzed activated, purified CD4 cells by RNA in situ hybridization to determine the percentage of IFN-gamma- or IL-4-producing cells. Unfractionated splenocytes from partially CD4-depleted mice secreted more IL-4 and less IFN-gamma than splenocytes from control mice. In situ hybridization proved that CD4 cells from partially depleted mice contained a higher percentage of IL-4 and a lower percentage of IFN-gamma-producing cells than controls. These results indicate that treatment with a dose of mAb resulting in partial CD4 depletion may permit increased Th2-like lymphokine expression. This study also provides evidence that cells committed to Th2-like function exist in vivo in mice.     

In vivo stimulatory effect of macrophage colony-stimulating factor on the number of stroma-initiating cells

The in vivo effect of human macrophage colony-stimulating factor (M-CSF) on the number of cells that formed stromal colonies in an in vitro culture system (stroma-initiating cells; SICs) was investigated. We found that the number of SICs in the femurs of C57BL/6 mice was significantly increased by the treatment with M-CSF. We also found that the SICs were resistant to at least three different chemotherapeutic reagents, 5-fluorouracil (5-FU), cytarabine, and cyclophosphamide, because the femoral cells of mice treated with these reagents contained higher numbers of SICs than those of untreated mice. M-CSF treatment also increased the number of SICs of the reagent-pretreated mice. The SICs detected in our culture system were present only in Mac-1(-)CD45(-) cells, and the M-CSF treatment of 5-FU-pretreated mice actually increased the number of Mac-1(-)CD45(-) SICs. The Mac-1(-)CD45(-) SICs collected from mice that were pretreated with 5-FU and then treated with M-CSF formed stromal colonies under in vitro culture conditions that did not contain M-CSF but did contain a high concentration of fetal calf serum. This result suggested that SICs collected following the treatment procedure did not necessarily require the presence of M-CSF for their in vitro proliferation. Our study indicated that M-CSF has the ability to increase the number of progenitor or precursor cells for bone marrow stromal cells in vivo system.     

In vitro studies of the natural humoral antitumor reactivity of BALB/c and C57BL/6J mice

Summary Antitumor antibodies were produced in vitro by spleen cells harvested from 12- to 18-month-old C57BL/6J and BALB/c mice with a naturally occurring complement-dependent serum cytotoxicity for tumor cells. The antibodies were of the IgM class and had a complement-dependent cytotoxic reactivity on EL4 cells, which was not absorbed by normal thymus cells. No natural antibodies were produced by untreated spleen cells from 1- or 3-month-old mice of the same two strains, which had no natural serum reactivity. However, after a treatment with an anti-Thy-1 serum and complement before culturing, spleen cells from 3-month-old mice, but not 1-month-old mice, produced the natural antitumor cytotoxic antibodies in vitro. When antibody-producing spleen cells from 12-month-old mice were cultured in vitro together with spleen cells from the unreactive 3-month-old syngeneic mice, inhibition of the antibody production occurred. This inhibiting capacity increased between 1 and 6 months of age and was abrogated by a treatment with an anti-Thy-1 serum and complement or with an anti-Ly-2 serum, whereas the passage of the inhibiting spleen cells on an anti-IgG column did not modify the inhibiting capacity. The in vitro data confirm previous in vivo findings on the nature, specificity and systems of regulation of the natural antitumor cytotoxic antibodies.     

NKT cells are critical for the initiation of an inflammatory bowel response against Toxoplasma gondii

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-gamma in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jalpha281(-/-)) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jalpha281(-/-) mice failed to overexpress IFN-gamma in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with alpha-galactosylceramide, which prevents death in C57BL/6, but not in Jalpha281(-/-), mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of alpha-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice.

MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse. CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice. Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment. The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice. The latter may reflect increased B cells in relative number in the peripheral lymphoid organs. Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group. Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT. Production of IFN gamma was suppressed by CT treatment in both strains of mice. This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J. Exp. Med. 172: 95-103, 1990). However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.     

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4(+)CD25(+) regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4(+)CD25(+) T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3(+) cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4(+)CD25(+) T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4(+)CD25(+) Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.     

Production of hapten-specific T cell hybridomas and their use to study the effect of ultraviolet B irradiation on the development of contact hypersensitivity

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.     

Defective tumoricidal capacity of macrophages from A/J mice. I. Characterization of the macrophage cytotoxic defect after in vivo and in vitro activation stimuli.

Macrophages from A/J mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that activate cells from C3H/HeN mice. Peritoneal macrophages from A/J mice treated i.p. with viable Mycobacterium bovis, strain BCG, killed Corynebacterium parvum, or pyran copolymer fail to develop in vitro tumoricidal activity; varying the numbers of macrophages from treated mice added to target cells, or the dose and time of treatment, or the treatment schedule of these in vivo activation stimuli did not evoke cytotoxic activity. Moreover, cytotoxic activity by macrophages from A/J mice was not observed with any of four target cell lines derived from three different mouse strains. In vitro treatment of peritoneal exudate macrophages from A/J mice with lymphokine-rich supernatants, bacterial endotoxins, or T cell mitogens was also ineffective; varying the numbers of treated macrophages added to target cells, the dose of in vitro activation stimuli, or the time of treatment did not evoke cytotoxic activity. Thus, A/J mice exhibit a profound defect in macrophage tumoricidal capacity to both in vivo and in vitro activation stimuli over a wide range of experimental conditions.     

Transfer Agent of Immunity

Detection of the cells which contain antibody was accomplished by a method of immune adherence of human erythrocytes to a single cell, termed SCIA (single cell immune adherence) reaction. Peritoneal exudate cells were collected from mice immunized with flagella of either Salmonella enteritidis or S. tennessee. Serologically specific antibody was detectable in some of the peritoneal exudate cells of such mice. An immune ribonucleic acid (RNA) was extracted from the peritoneal exudate cells of mice immunized with salmonella flagella. When mice were injected intraperitoneally with this preparation, serologically specific antibody was found in some of their peritoneal exudate cells by the SCIA method. This preparation was inactivated by treatment with ribonuclease, but was resistant to proteinases, deoxyribonuclease and anti-flagella antibody, suggesting that this agent is of RNA nature and does not contain antigen or fragment thereof.     

Modulation of granulomatous inflammation in murine schistosomiasis mansoni by enteric exposure to schistosome ova: in vitro characterization of a regulatory mechanism within the granuloma

Enteric immunization with schistosome ova results in a diminished granulomatous response. This study explored a mechanism by which enteric immunization may decrease granuloma size. Granulomas from livers of acutely infected mice were dissociated and the dispersed cells were depleted of macrophages. As defined by a direct in vitro migration inhibition factor (MIF) assay, the macrophage-depleted cells, composed of lymphocytes and eosinophils, inhibited the migration of normal peritoneal exudate cells when exposed to soluble egg antigens. Anti-Thy 1.2 or -Lyt 1.1, but not -Lyt 2.1, treatment of these cells abrogated MIF activity. Next, mice were exposed enterically to eggs 4 weeks prior to sacrifice. Cells from granulomas isolated from these animals demonstrated no MIF activity unless treated with anti-Lyt 2.1. When granuloma cells from enterically immunized mice were mixed with those from unimmunized animals, MIF activity by the latter was abrogated. Treatment of cells from immunized mice with anti-Lyt 2.1 or -Thy 1.2, but not -Lyt 1.1 prior to mixing once again permitted MIF activity. These results suggest that the diminished granulomatous response induced by enteric immunization could be mediated by Lyt 2+ suppressor T cells. These suppressor cells may regulate the MIF activity of Lyt 1+ T lymphocytes residing within these lesions.     

Autoimmune CD4+ T cells interfere with immune tolerance to a thymic-independent antigen

The ability of autoimmune T cell subsets to interfere with tolerization of B cells can be studied by using thymic-independent Ag. We have defined an abnormality within the CD4+ T cell compartment in young NZB and MRL-lpr/lpr mice by studying tolerance of spleen and B cells to the thymic independent Ag, fluorescein-Brucella abortus. Tolerization of spleen cells is defective in MRL-lpr/lpr mice, but not MRL-+/+ or C3H.lpr mice, suggesting that the defect requires both the autosomal MRL background and the lpr gene to be present. T enriched cells from NZB mice and from MRL-lpr/lpr mice (but not MRL-+/+ or C3H.lpr mice) reverse tolerance in spleen cells from [NZB X DBA/2]F1 and C3H/HeJ mice, respectively. This interference is removed by treatment with anti-CD4 antibody and C. Supernatants from cultured T cells of NZB and MRL-lpr/lpr mice also prevent tolerance in spleen cells of [NZB X DBA/2]F1 and MRL-+/+ mice, respectively, unless CD4+ cells are removed prior to T cell culture. Removal of T cells from NZB and MRL-lpr/lpr spleen cells allows normal tolerization of B cells, which is abrogated by the addition of syngeneic T cells or cultured T cell supernatants. This effect also depends on the presence of CD4+ T cells. These studies show that in MRL-lpr/lpr mice, through interaction of the lpr and MRL background genes in a T cell subset, and in NZB mice, CD4+ T cells interfere with B cell tolerance to a thymic-independent Ag.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.