Enhanced proliferation,survival, and yield of cultured myelin basic protein (MBP)-reactive Lewis rat lymph node cells following dual activation with concanavalin A and MBP

Lymph node cells (LNC) from Lewis rats sensitized 7 days previously to myelin basic protein (MBP) exhibit greatly increased survival and undergo much greater proliferation when cultured in the presence of both concanavalin A (Con A) and MBP. Dual activation, in contrast to culture with either the mitogen or the neural antigen, results in a substantially greater yield of lymphoblasts with the capacity to transfer experimental allergic encephalomyelitis (EAE) to normal syngeneic recipients. Dual activation provides a practical advantage over other methods reported to date for securing larger numbers of functional MBP-reactive lymphoid cells for immunologic studies.     

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freund's adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.     

Cellular transfer of experimental allergic encephalomyelitis in Lewis rats: Effects of different sensitization regimens on in vitro reactivity of donor spleen cells to concanavalin A

Splenocytes from Lewis rats sensitized to guinea pig spinal cord (GPSC) and complete Freund's adjuvant (CFA) or to myelin basic protein (MBP)-CFA plus pertussis vaccine were less effective than spleen cells from MBP-CFA sensitized donors in transferring EAE to syngeneic recipients following culture with concanavalin A (Con A). Moreover, splenocytes from rats sensitized to GPSC-CFA plus pertussis vaccine showed no EAE transfer activity following culture with Con A. Diminished EAE transfer activity occurred in parallel with decreased proliferative responses of primed splenocytes to Con A. These effects were due, at least in part, to the use of pertussis vaccine and to Con A activation of a suppressive adherent cell subpopulation in sensitized donor spleens. Proliferative responses and EAE transfer activity were restored upon removal of plastic-adherent cells from splenocytes of rats sensitized to MBP-CFA plus pertussis vaccine prior to Con A activation of the non adherent lymphoid cells. Deletion of plastic-adherent cells from splenocytes of donors sensitized to GPSC-CFA plus pertussis vaccine prior to activation with Con A, however, had no effect on proliferative responses or EAE transfer activity. Furthermore, EAE transfer activity of Con A-activated splenocytes from MBP-CFA-sensitized donors was lost when such cells were cultured with splenocytes from donors sensitized to GPSC-CFA plus pertussis vaccine.     

The role of myelin lipids in experimental allergic encephalomyelitis. III. Transfer of suppression from guinea pigs recovering from EAE, induced by myelin basic protein--galactocerebroside complexes

Juvenile strain 13 guinea pigs were immunized with myelin basic protein (MBP) combined with galactocerebrosides (MBP + GC) or with total myelin lipids without GC [MBP + (TL-GC)] in CFA. Control animals received dinitrophenylated-ovalbumin (DNP-OA) in CFA, CFA or IFA alone. The animals injected with MBP + GC showed a higher rate of recovery from the first EAE episode (83%) than those treated with MBP + (TL-GC) (50%). With the exception of the group treated with IFA alone, all animals were refractory to EAE following rechallenge with MBP in CFA 90 days after the first exposure. The in vitro proliferative response to MBP, of peripheral blood lymphocytes (PBLs) derived from guinea pigs freshly sensitized to MBP in CFA, was drastically suppressed in the presence of PBLs from animals injected with MBP + GC. Upon transfer to normal syngeneic recipients, spleen cells from MBP + GC-treated animals completely suppressed the clinical and histological manifestations of EAE following recipient challenge with MBP in CFA. Cell-free supernatants from PBLs and spleen cells of strain 13 guinea pigs treated with MBP + GC inhibited lymphocyte proliferation to MBP, of allogeneic responder cells, and spleen cell supernatants completely suppressed the induction of EAE upon transfer to allogeneic recipients. Suppression could not be transferred with cells from other treated groups. These results suggest that animals immunized with MBP + galactocerebrosides in CFA develop suppressor cells that may be in part responsible for the recovery from the first EAE episode and for protection against rechallenge with MBP in CFA. Their cell-free supernatants act in an MHC-nonrestricted fashion. These results do not rule out an additional protective mechanism since all animals exposed to CFA were refractory to rechallenge despite lack of demonstrable suppressor cell activity.     

Adoptive transfer of experimental allergic encephalomyelitis: requirement for macrophages in activation of spleen cells in vitro by concanavalin A or myelin basic protein

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in Lewis rats is markedly enhanced when sensitized spleen cells are incubated in vitro with either concanavalin A (Con A) or myelin basic protein (MBP). This phenomenon permits more detailed analysis of the inductive phase of EAE than has heretofore been possible. We have now demonstrated that macrophages are essential for the process to occur, and that they probably act by different means depending on whether activation is carried out with the mitogen or the specific antigen. Depletion of macrophages by passage through G-10 Sephadex columns prevented activation of spleen cells by either Con A or MBP. The effect of Con A could be partially restored with 2-mercaptoethanol, while activation by MBP could not. Reconstitution of macrophage-depleted cultures with peritoneal exudate cells from either immune or normal rats fully restored activation by both Con A and MBP. Supernatants taken from spleen cell cultures were unable to restore the transfer activity of macrophage-depleted cells, indicating that activation probably is not mediated by a soluble factor released by macrophages. Depletion of macrophages after incubation with Con A slightly reduced transfer activity, but depletion after incubation with MBP abolished it. Thus the mechanisms of activation appear to differ in the two systems, and in the case of MBP the macrophage-mediated portion of the process may be incomplete prior to adoptive transfer.     

Suppression of experimental allergic encephalomyelitis in Lewis rats treated with myelin basic protein-liposome complexes: clinical, histopathological, and cell-mediated immunity correlates

Guinea pig myelin basic protein (MBP) was inserted into phosphatidylserine liposomes and Lewis rats were injected by the intracardiac (ic) route with 75 microgram doses of MBP-liposomes according to various schedules. After challenge with 75 microgram guinea pig MBP in complete Freund's adjuvant, the rats were followed for clinical signs, were tested for delayed hypersensitivity (DTH) and lymphocyte transformation (LT) to MBP. The animals were sacrificed 30 days after challenge and the central nervous system tissue was examined for histological modifications. Rats treated with two injections of MBP-liposomes, 7 days before and 7 days after challenge, showed the highest degree of protection from clinical manifestations. Histological lesions were not significantly reduced. DTH reactions to MBP were all positive, regardless of treatment. LT assays were positive overall in only 50% of the animals tested. The response to rat MBP was significantly lower than to guinea pig MBP, especially in the groups treated with MBP-liposomes. Adoptive transfer of spleen cells from MBP-liposome-treated donors reduced the clinical scores of actively induced EAE in syngeneic recipients by 40-50%. These results suggest that at least one mechanism responsible for antigen-specific protection in EAE by MBP-liposomes operates through active suppression transferable by spleen cells.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Studies on the mechanism of suppression of experimental allergic encephalomyelitis induced by myelin basic protein-cell conjugates

The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the disease-inducing injection of MBP in Freund's complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells.     

Regulation of experimental allergic encephalomyelitis. II. Appearance of suppressor cells during the remission phase of the disease

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE). Most rats recover from paralysis and are subsequently resistant to the disease. In an adoptive transfer system, we found that lymph node cells (LNC) from rats that had recovered from EAE protect syngeneic recipients from the disease when the latter are challenged with encephalitogenic myelin basic protein and adjuvant after receiving donor cells. Suppression is antigen-specific and requires viable LNC. In contrast to the suppressor cells we previously studied in tolerized rats, which were nonadherent T lymphocytes, the suppressor cells found in rats that have recovered from EAE adhere to glass wool. However, they are not retained on Sephadex G-10 columns to which macrophages adhere. Suppressor activity is enriched in the nylon wool-adherent LNC population (which consists of approximately 80% Ig+ cells). Our findings suggest that activation of adherent suppressor cells may be implicated in recovery from EAE. These may be adherent T cells, or B cells that produce anti-BP blocking antibodies.     

T cell requirement for experimental allergic encephalomyelitis induction in the rat.

The question of whether a cell-mediated or a humoral mechanism initiates EAE in rats sensitized with BP-CFA was investigated. The requirement of T cells for EAE induction was manifested when Tx, irradiated rats were reconstituted with normal lymphoid cells treated with ATS and then injected with BP-CFA. Neither EAE nor antibody was produced, indicating the T cell dependency of BP specific antibody production. More precise information regarding the role of the T cell in the production of EAE was obtained by means of passive transfer of EAE with sensitized lymphocytes. Thus, transfer of lymphoid cells from rats previously sensitized to BP-CFA into Tx, irradiated rats elicited EAE and antibodies to BP. However, no EAE followed when the transferred cells were first depleted of T cells by treatment with ATS. Nevertheless, ATP pretreatment did not depress the levels of antibody to BP produced in the transfer recipients. The latter finding indicates that the cells from animals sensitized 9 days previously were already committed to the production of antibodies to BP. Therefore, a) T cells are absolutely necessary for induction of EAE and b) antibody detected by antigen-binding is not responsible for the pathogenesis of this disease.     

Gammadelta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of gammadelta T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of gammadelta T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The gammadelta T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of gammadelta T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of gammadelta T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The gammadelta T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRalphabeta(+) cells, and CD40, CD80, and CD86 on CD19(+) and CD11b(+) cells. However, depletion of gammadelta T cells resulted in significant reduction in IL-12 production by LNC. That gammadelta T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the gammadelta T cell population restored IL-12 production, and that gammadelta T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for gammadelta T cells in the early effector phase of the immune response in EAE.     

Cellular transfer of experimental allergic encephalomyelitis: Altered disease pattern in irradiated recipient Lewis rats

Irradiation of recipient Lewis rats 6–24 hr prior to injection of sensitized lymph node cells (LNC) altered the pattern of transferred experimental allergic encephalomyelitis (EAE). Recipients subjected to total body irradiation in doses ranging from 500 to 1000 rads developed paralysis; nonirradiated control recipients did not do so. Histopathologic changes of EAE, in terms of number of descrete cellular infiltrates, were potentiated in the total body irradiated recipients. Among LNC recipients subjected to regional irradiation (850 rads) of the head or lower spinal column, paralysis was observed only in those animals where the irradiation impinged upon the spinal cord. Cellular infiltrates of EAE were numerically more common in the irradiated region of the neuraxis. The findings are discussed in terms of irradiation rendering the central nervous system of animals and man more vulnerable to autoimmune injury.     

Adoptive transfer of experimental allergic encephalomyelitis with activated spleen cells: Comparison of in vitro activation by concanavalin A and myelin basic protein

Adoptive transfer of experimental allergic encephalomyelitis (EAE) was carried out in Lewis rats using splenic lymphocytes incubated in vitro with either concanavalin A (Con A) or myelin basic protein (MBP). Requirements were established for sensitization of donors, culture conditions, numbers of transferred cells, and incubation period of EAE in recipients. These were strikingly similar whether Con A or MBP was used. In addition, cellular proliferation in vitro was not required in either system, but proliferation after transfer to the recipient was essential for the development of clinical signs and histological lesions. These methods have potential value for analyzing mechanisms of immune induction in this classic model of autoimmune disease.     

Effect of treatment with glutaraldehyde-fixed myelin basic protein-liposomes on active induction and passive transfer of experimental allergic encephalomyelitis in Lewis rats

Guinea pig myelin basic protein (MBP)-liposomes were prepared and fixed with 0.2% glutaraldehyde (GA). Lewis rats were treated with glutaraldehyde-fixed MBP-liposomes (MBP-L-GA) or with cytochrome-c-liposomes (CYC-L-GA), 7 days before and 7 days after challenge with MBP in CFA. Rats treated with MBP-L-GA, but not with CYC-L-GA, were very well protected against the clinical manifestations of EAE. The protection was better than that obtained after treatment with conventional MBP-liposomes (without glutaraldehyde). Furthermore, when grown in vitro for 72 hr in the presence of MBP, lymphocytes from rats treated with MBP-L-GA and challenged with MBP in CFA exhibited a marked decrease in their ability to transfer EAE to normal syngeneic recipients.     

Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2- T lymphocytes

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.     

Antigen-specific inhibition of immune interferon production by suppressor cells of autoimmune encephalomyelitis

Previous work from this laboratory has revealed that spleen and/or lymph node cells from Lewis rats, that have recovered from an acute episode of experimental autoimmune encephalomyelitis (EAE), suppress the development of EAE when injected into syngeneic recipients subsequently challenged with myelin basic protein (MBP) in CFA. In an effort to understand the mechanism of this suppression, we measured the production of immune IFN-gamma, which may be required for the induction of an immune response, by EAE effector T cells (which transfer disease) and EAE suppressor cells when cultured in vitro with MBP. We now report that EAE effector T cells produce IFN-gamma when cultured in vitro with MBP. In contrast, spleen cells from recovered rats (which manifest suppressor activity in vivo) do not produce IFN-gamma. Moreover, in cell mixing experiments, these suppressor spleen cells inhibited the production of IFN-gamma by EAE effector cells. This inhibition was not eliminated by the removal of macrophages nor by the inhibition of PG synthesis by indomethacin. Furthermore, the inhibition was shown to be Ag-specific and mediated by nylon-adherent, radiation-sensitive splenic T cells. The findings suggest that suppressor cells regulate EAE by inhibiting IFN-gamma production by effector cells. This inhibition may result in the down-regulation of IFN-gamma-induced expression of class II major histocompatibility Ag on cells of the central nervous system, thus reducing the presentation of tissue-specific Ag (i.e., MBP) to autoreactive lymphocytes.     

Suppression of experimental allergic encephalomyelitis by MBP-coupled lymphoid cells and by MBP-liposomes: a comparison.

In previous experiments, we showed that administration of myelin basic protein (MBP) inserted into phosphatidyl-serine liposomes, to susceptible animals suppressed the clinical manifestations of both acute and chronic-relapsing EAE. In this report we compare the effectiveness of treatment with MBP-liposomes and with MBP-coupled syngeneic spleen cells in EAE protection. Lewis rats treated with 150 micrograms MBP-liposomes or with 160 micrograms (35 x 10(6] MBP-coupled spleen cells, given 7 days before and 7 days after encephalitogenic challenge were equally protected against clinical EAE, when compared to untreated controls. In addition to clinical protection, in vitro proliferative responses of lymphocytes from treated rats were significantly reduced, but delayed hypersensitivity (DTH) reactions remained unaffected. Proliferation of lymphocytes from MBP-sensitized donors was inhibited by the addition of spleen cells but not of lymph node cells from treated donors. The inhibitory effect was observed with spleen cells regardless of whether the donors were treated or not, was antigen nonspecific, and localized in a radio-resistant, adherent cell population. Adoptive transfers of spleen cells from treated donors, after a 48-hr in vitro incubation with concanavalin A, showed that the cells from donors treated with MBP-coupled spleen cells, but not with MBP-liposomes, suppressed the disease in recipients, following challenge with MBP-complete Freund's adjuvant (CFA). These results suggest that two distinct mechanisms operate in the protection by MBP-coupled cells and MBP-liposomes, respectively.     

Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis.

Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.     

Enhanced transfer of experimental allergic encephalomyelitis with strain 13 guinea pig lymph node cells: requirement for culture with specific antigen and allogeneic peritoneal exudate cells

Previously, we reported that transfer of experimental allergic encephalomyelitis (EAE) with sensitized peritoneal exudate cells (PEC) in strain 13 guinea pigs is markedly enhanced if the cells are first cultured with specific antigen, myelin basic protein (BP). These cells also undergo considerable antigen-specific proliferation. In contrast, the data reported here show that lymph node cells (LNC) from sensitized animals display neither enhanced transfer nor antigen-specific proliferation after culture with BP. Enhanced transfer is obtained, however, if a second nonspecific signal is available. This second signal is provided by the presence of normal allogeneic strain 2 PEC in culture. After culture with BP and strain 2 PEC, 2.5 to 5 x 10(7) strain 13 LNC transfer disease reproducibly, in contrast with approximately 1 x 10(9) previously required for successful transfer. Addition of allogeneic or syngeneic PEC without antigen does not lead to enhanced transfer by LNC. Culture with normal syngeneic PEC plus BP oly infrequently enhances transfer by LNC. The intense mixed lymphocyte reaction (MLR) induced by addition of strain 2 PEC to strain 13 LNC precludes the use of 3H-TdR incorporation for detection of proliferation by EAE effector cells. However, inhibition of transfer with low doses of mitomycin C (2 to 5 micrograms/ml) pluse the fact that EAE effector cells are found almost exclusively in the light fraction of BSA gradients after (but not before) culture suggests that the latter are induced to proliferate in culture.