Migration inhibition of lymph node lymphocytes as an in vitro assay for cell-mediated immunity in the draining lymph nodes of parenterally immunized mice

This paper describes a method for in vitro measurement of specific cell-mediated immunity in the mouse. Animals were immunized parenterally with ovalbumin in Freund's incomplete or complete adjuvant, and a direct migration inhibition assay was performed, using lymphoid cells from the draining lymph nodes. Migration inhibition was found to be antigen specific, correlated with systemic delayed-type hypersensitivity measured in vivo by skin testing, and had a high degree of sensitivity for ovalbumin. The migrating cells were identified as lymphocytes. Lymph node lymphocyte migration inhibition provides a reliable in vitro assay for regional CMI in the mouse.     

Lithium potentiates antigen-dependent stimulation of lymphocytes only under suboptimal conditions

Immunization of hamsters with DNP-BSA in either Freund's complete or incomplete adjuvant led to the induction of antigen reactive lymph node cells. As assessed by in vitro lymphocyte stimulation assays, antigen in complete adjuvant was more effective than antigen in incomplete adjuvant in inducing immunity. Supplementing antigen-stimulated cultures from animals 14 days post-immunization with LiCl led to no enhancement of tritiated thymidine incorporation into cells from animals immunized with antigen in complete adjuvant, but did enhance antigen-dependent stimulation of cells from animals immunized with antigen + incomplete adjuvant. LiCl was, however, able to enhance stimulation of cells from animals immunized with antigen + complete adjuvant at 22 and 29 days post-immunization, when in vitro responsiveness was declining. Lymph node cells from animals optimally immunized antigen + complete adjuvant were fractionated by passage over Sephadex G-10 columns. Sephadex G-10 non-adherent cells, deficient in cells such as macrophages, exhibited a depressed responsiveness to antigen, compared to unfractionated cells, and responsiveness was not restored by LiCl. Stimulation of cells by antigen was found to be inhibited by supplementing the cultures with theophylline or dibutyryl cyclic AMP and this inhibition could be reversed by LiCl. Lithium would, therefore, appear to be able to influence lymphocyte adenylate cyclase. Thus, LiCl can exert an immunopharmacologic effect on in vitro antigen stimulation primarily when conditions are suboptimal, possibly through an influence on cyclic AMP metabolism.     

Studies on mechanisms of immobilization of mononuclear cells in the delayed hypersensitivity (DH) reaction

Migration inhibitory (MI) activity in exudates, and migration and inhibition of migration of exudate cells was investigated in the delayed hypersensitivity (DH) reaction induced by intrapleural injection of PPD into complete Freund's adjuvant(CFA)-sensitized guinea-pigs. During the initial reaction (6-hour), two types of antigen-dependent MI activity were detected in serum and cell free exudate. One was a high molecular weight material associated with immunoglobulin, and the other was a low molecular weight material and appeared to be so-called antigen-dependent migration inhibitory factor (MIF). As the reaction progressed (i.e. 12–24-hour), two types of antigen-independent MI activity were revealed in exudate, but not in serum. One of these was a high molecular weight material, and the other was a low molecular weight material and thought to be socalled antigen-independent MIF.Similar experiments were performed on the reversed passive Arthus (RPA) reaction in the pleural cavity of guinea-pigs. A high molecular weight substance having MI activity was detected in 6-hour cell free exudate and was found to be antigen-independent. So-called MIF was not found in this reaction.Migration of unseparated exudate cells from the 18-hour DH reaction was less extensive than that of 6-hour unseparated cells. Addition of antigen caused further inhibition, the effect on 18-hour exudate cells being more pronounced. These results were further examined, using mononuclear cells separated on a Ficoll-Isopaque gradient. The migration area of the mononuclear cells was reduced as the DH reaction progressed. Mononuclear cells from the DH reaction gave a smaller migration area throughout the reaction in comparison with blood mononuclear cells. The migration area of the RPA exudate mononuclear cells was also reduced as the reaction progressed. However 6-hour RPA exudate mononuclear cells gave a larger migration area than blood mononuclear cells.The migration of mononuclear cells from DH exudate was inhibited by addition of antigen. A greater degree of inhibition of migration was induced by addition of antigen to mononuclear cells from 18- and 24-hour exudate cells in comparison with 6- and 12-hour exudates. The migration of mononuclear cells from normal blood (i.e. unsensitized animals) and RPA exudate (6- and 18-hour) was unaffected by addition of antigen. Similar results were obtained for blood of complete Freund's adjuvant sensitized animals from 0- to 18-hour.Adherent (A) and nonadherent (NA) mononuclear cells from 18-hour DH exudate were separated through a glass bead column. The migration of adherent cells from 6-hour exudates was not significantly inhibited whereas that of 18-hour mononuclear cells was markedly inhibited. The effect on migration of each mononuclear cell fraction was detected by mixing with peritoneal exudate cells (used as indicator cells). The effect of the A cells from 18-hour mononuclear cell exudates on peritoneal exudate cells was strong, whereas that of 6-hour exudates was less marked. A similar effect was observed with NA cells, from 6- and 18-hour exudate, on the migration of peritoneal mononuclear cells.     

Immune Response to Sheep Red Blood Cells in Mice Pretreated with Mycobacterial Fractions

Ten micrograms of trehalose-6,6' -dimycolate (cord factor) injected into the footpads of mice increased the antibody response to sheep red blood cells (SRBC) subsequently injected into the same sites. There is a relationship between the antibody response and the cellular reaction induced locally and in the draining lymph nodes by cord factor, as judged by a much weaker response when antigen is injected into the contralateral footpads. The time intervals between injection of cord factor and antigen were from 5 to 20 days. A similar increased antibody response to SRBC was evident after preliminary administration of Freund's complete adjuvant or living BCG bacilli into the footpads. There was no increased antibody response in mice pretreated with living BCG or Freund's adjuvant to SRBC injected into the contralateral footpads. Administration of wax D from human strains Peurois and Test was without any effect. Administration of SRBC emulsified in incomplete Freund's adjuvant containing 5 mug of cord factor induced a very strong antibody response in the mice as compared to that after injection of the same amount of antigen in incomplete Freund's adjuvant containing wax D or mycobacteria. The results are discussed.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) in Freund's complete adjuvant (FCA) and boosted three times with DT in saline. Boosting increased anti-DT passive haemagglutinins, haemolysins, and cytophilic antibody on the surface of peritoneal exudate cells, but skin reactions to DT became negative and peritoneal cell migration was no longer inhibited by DT. When normal peritoneal cells were incubated in the sera of boosted animals, their migration was not inhibited by antigen, although they had cytophilic antibody on their surface as shown by rosette formation.     

Modulation of the immune response to transplantation antigens. I. The effects of immunization using adjuvants on the immune response of mice to a tumour allograft

Mice were immunized with an allogeneic tumour antigen using various adjuvants and were subsequently challenged with live tumour cells. Immunization using certain adjuvants led to accelerated destruction of the subsequent tumour allograft, whilst the use of Freund's complete and incomplete adjuvant (CFA and IFA) led to enhanced tumour growth and eventual death of the mice. Pretreatment with Freund's complete adjuvant without antigen also resulted in enhanced tumour growth. Enhancing antibody was not detected in the sera or on the tumour cells, though some of the sera were able to inhibit cell-mediated cytotolysis of tumour cells. The mice pretreated with CFA, with or without antigen, showed a depressed cell-mediated cytotoxicity against tumour cells when compared with untreated mice.     

In Vitro Target Cell Lysis Mediated by Normal Human Lymphocytes (K Cells) or Monocytes

Guinea pig IgG1 or IgG2 anti-dinitrophenyl (DNP) Antibody preparations were used for induction of target cell lysis by normal human lymphocytes (K cells) or monocytes. Target cells were DNP-coated 51Cr-labeled chicken erythrocytes. Antibody concentrations were assayed by an ammonium sulphate precipitation technique. When antiserum was obtained by immunization with the antigen in Freund's complete adjuvant (FCA), IgG2 antibodies predominated and were significantly more efficient inducers of K-cell-mediated lysis than IgG1 antibodies. The lowest activity in K-cell-mediated lysis was seen with IgG1 from antiserum obtained by immunization with antigen in Freund's incomplete adjuvant. When these antibody fractions were tested in monocyte-mediated erythrolysis, the two IgG1 fractions were as active as the IgG2 antibodies raised with antigen incorporated in FCA. The results suggest that the Fc receptors of mature human blood monocytes are different from those on the effector cells in the K-cell preparations.     

Susceptibility of Lewis Rats to Experimental Autoimmune Encephalomyelitis After Recovery from Passively Induced Disease

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by adoptive transfer of 5 × 10⁷ sensitized syngeneic lymphoid cells from donors challenged 12 to 43 days earlier with myelin basic protein and complete Freund's adjuvant. The donor cells were cultured for 72 hours with antigen prior to transfer. Following recovery, the recipients remained susceptible to EAE induced by challenge with antigen and adjuvant. The relevance of this finding to host recovery mechanisms is discussed.     

In vitro transfer of cellular immunity in experimental allergic orchitis by means of immune RNA.

Ribonucleic acid (RNA) extracts were obtained from lymph nodes of guinea-pigs that had previously been immunized with a purified testicular antigen emulsified in Freund's complete adjuvant. The RNA extracts were incubated with normal guinea-pig peritoneal exudate cells (NGP-PEC). After treatment, the NGP-PEC cells showed specific inhibition of migration when tested with the specific antigen. No inhibition of migration was observed when cells were tested with an unrelated antigen or when the cells were incubated with RNA obtained from animals immunized with adjuvant alone. Failure of inhibition of migration was also observed when the 'immune' RNA was degraded with RNAse. The appearance of this I-RNA in the immunized guinea-pigs correlates with the appearance of delayed hypersensitivity in vivo.     

In Vitro Target Cell Lysis Mediated by Normal Human Lymphocytes (K Cells) or Monocytes

Guinea pig IgG1 or IgG2 anti-dinitrophenyl (DNP) antibody preparations were used for induction of target cell lysis by normal human lymphocytes (K cells) or monocytes. Target cells were DNP-coated ⁵¹Cr-labeled chicken erythrocytes. Antibody concentrations were assayed by an ammonium sulphate precipitation technique. When antiserum was obtained by immunization with the antigen in Freund's incomplete adjuvant (FCA), IgG2 antibodies predominated and were significantly more efficient inducers of K-cell-mediated lysis than IgG1 antibodies. The lowest activity in K-cell-mediated lysis was teen with IgG1 from antiserum obtained by immunization with antigen in Freund's incomplete adjuvant. When these antibody fractions were tested in monocyte-mediated crythrotysis, the two IgG1 fractions were as active as the IgG2 antibodies raised with antigen incorporated in FCA. The results suggest that the Fc receptors of mature human blood monocytes axe different from those on the effector cells in the K-cell preparations.     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Immune responses to thyroglobulin in experimental allergic thyroiditis

Guinea-pigs immunized with homologous thyroglobulin in complete Freund's adjuvant were observed for lesions in the thyroid gland, and for development of specific delayed type skin hypersensitivity. Their blood leucocytes were examined for specific sensitivity in vitro by determining the inhibition of migration in the presence of thyroglobulin. Serum was tested for humoral antibodies by the passive haemagglutination technique. There was a significant correlation between the development of thyroiditis and the intensity of the skin reactions. No relationship was observed between the presence of thyroiditis and leucocyte sensitivity nor between thyroiditis and circulating antibodies. On the other hand serum antibody titres were correlated to the degree of leucocyte sensitivity. Thyroglobulin inhibited the migration of cells from normal animals in the presence of plasma from thyroglobulin-sensitized animals. The activity of sensitized plasma disappeared when diluted 1:10 in normal plasma irrespective of the serum antibody titre. Guinea-pigs immunized with thyroglobulin or kidney antigen in complete Freund's adjuvant and tested for skin or leucocyte hypersensitivity showed specific but no cross reactivity to the two preparations.     

Selective and specific inhibition of 24-hour skin reactions in the guinea-pig: II. The mechanism of immune deviation

Guinea-pigs injected with antigen in Freund's complete adjuvant develop delayed hypersensitivity to the antigen. This is reduced by the prior injection of the same antigen precipitated with alum. This may be termed immune deviation and has been shown to differ from immune tolerance. Four possible mechanisms for this phenomenon were considered: (a) that there was a specific defect in the cell population responsible for the passive transfer of delayed hypersensitivity; (b) that there was an antibody blocking the detection of delayed hypersensitivity; (c) that there was an antibody blocking the induction of delayed hypersensitivity; and (d) that the cells responsible for delayed hypersensitivity were desensitized in vivo. Experiments on the passive transfer of delayed hypersensitivity from deviated and control guinea-pigs showed that there was a defect in the cell population involved in the passive transfer of delayed hypersensitivity. Serum transfer failed to reveal evidence of antibody blocking the induction or detection of delayed hypersensitivity. Transfer of cells into recipients which had been pretreated with alum-precipitated antigen gave no evidence of desensitization in the PPD system. The findings with bovine γ-globulin were equivocal. It was concluded that there was a defect in the cell population involved in the passive transfer of delayed hypersensitivity and, in the absence of evidence for other mechanisms, this was attributed to a direct effect of the first injection of antigen on the cells responsible for the state of delayed hypersensitivity after the injection of the same antigen in Freund's complete adjuvant.     

Heat-killed Listeria monocytogenes and L. monocytogenes soluble antigen induce clonable CD4+ T lymphocytes with protective and chemotactic activities in vivo.

In the present study we attempted to analyze the possibility to induce in mice a T-cell response using killed Listeria monocytogenes in adjuvant. Clearly, nonviable antigen is capable of inducing protective and granuloma-forming T cells in C57BL/6 mice when emulsified in complete Freund's adjuvant. These T cells were cloned in vitro by using antigen and irradiated splenocytes, as antigen-presenting cells, and the clones were characterized in vivo. Listeria-specific T-cell clones showed protective and chemotactic activity upon adoptive transfer, although some degree of functional heterogeneity among different clones was observed. The heterogeneous in vivo functions could not be correlated with the ability of the clones to produce gamma interferon or T-cell growth factor (interleukin-2 and/or interleukin-4). It was demonstrated that an in vivo relevant fraction of listeria-specific T lymphocytes can be induced by nonviable antigen in complete Freund's adjuvant. These results show that the low immunogenicity of heat-killed bacteria is not due to the expression of specific protective T-cell epitopes only by live bacteria.     

Immunization of virgin cows with surface antigen TF1.17 of Tritrichomonas foetus.

Protection by surface antigen TF1.17 of Tritrichomonas foetus was investigated because it reacted with a monoclonal antibody which immobilized and mediated complement killing of the organism and prevented adherence to vaginal epithelial cells. This monoclonal antibody was used to demonstrate conservation of the antigen in most strains and to immunoaffinity purify the 50- to 70-kDa glycoprotein antigen. In preparation for immunization studies, the appropriate challenge dose of parasites was determined by intravaginal inoculation of 23 virgin cows (heifers) with 10(2), 10(4), or 10(6) live organisms at the time of estrus. More animals became infected and vaginal infection was maintained at a higher rate (P < 0.005) over 10 weeks for the group that received 10(6) organisms than in the other two groups. Therefore, this dose was used for challenge of immunized animals. Animals immunized with immunoaffinity-purified TF1.17 antigen in incomplete Freund's adjuvant or incomplete Freund's adjuvant plus dextran sulfate cleared the infection more quickly than adjuvant controls (P < 0.005). Isotype-specific enzyme-linked immunosorbent assay with T. foetus antigen showed that serum immunoglobulin G1 (IgG1) and IgG2 antibody responses as well as cervicovaginal mucus IgG1 and IgA antibodies peaked at about the time of clearance of infection in vaccinated animals. Controls developed later cervicovaginal mucus IgA antibody responses as would be expected in a primary local immune response to infection. These results indicate that vaccination with this immunoaffinity-purified surface antigen of T. foetus enhances antibody responses as well as clearance of the parasite from the female reproductive tract.     

Adoptive transfer of suppression of experimental allergic orchitis with lymphoid cells from antigen-pretreated guinea pigs

The injection of lymph node cells or spleen cells, obtained from strain 13 guinea pigs rendered unresponsive to experimental allergic orchitis (EAO) by pretreatment with testicular antigen (TA) in incomplete Freund's adjuvant, into normal syngeneic recipients markedly prevented the development of EAO, especially of the interstitial inflammatory cell response, which was expected to occur 2 weeks following orchitogenic challenge with TA in complete Freund's adjuvant (CFA). The suppressive effect of thymus cells from the same donors was much less prominent. The inhibition of EAO by suppressor cells was specific for the relevant antigen TA. In such EAO-suppressed animals delayed skin reaction to TA was suppressed, whereas antisperm antibody formation was not impaired. The active suppressor cells residing in the lymph nodes had characteristics of T lymphocytes, in that they did not adhere to the plastic dish surface and nylon wool and in that they formed rosettes with rabbit erythrocytes. B lymphocytes from the same animals did not have detectable suppressive properties. Lymph node cells from protected donors that had been treated with a single dose of cyclophosphamide (CY) 3 days before cell transfer were unable to transfer unresponsiveness to EAO. The results suggest that the immune prevention against EAO is explainable at least in part by the generation of CY-sensitive suppressor T lymphocytes with the capacity of inhibiting development of effector T cells after antigenic stimulation and that suppressor cells that mediated unresponsiveness to EAO may also regulate the cellular hypersensitivity to TA.     

Ovalbumin (OVA) and Mycobacterium tuberculosis bacilli cooperatively polarize anti-OVA T-helper (Th) cells toward a Th1-dominant phenotype and ameliorate murine tracheal eosinophilia.

A recent increase in allergic disorders has coincided with a decrease in infections, including tuberculosis. Although an inverse association between tuberculin responses and atopic disorders was reported, it was not known how T-helper (Th)1-biased immune responses to Mycobacterium tuberculosis influenced Th2-dominant responses to allergens. We examined whether M. tuberculosis could modulate ovalbumin (OVA)-induced eosinophilic inflammation in the murine trachea in a manner that transcended the barrier of antigen specificity. We found that CD4(+) T cells primed with OVA in complete Freund's adjuvant (CFA) inhibited OVA-induced tracheal eosinophilia through interferon (IFN)-gamma secretion. Immunization with an irrelevant antigen in CFA or with OVA in incomplete Freund's adjuvant failed to induce suppressor cells. In vitro experiments confirmed that both M. tuberculosis and OVA (as opposed to either one alone) were necessary to evoke polarized development toward a Th1-like phenotype through interleukin-12 secretion. These results indicate that exposure to an allergen along with M. tuberculosis switches development of allergen-specific T cells toward a Th1 phenotype, which, in turn, downregulates allergic manifestations in an antigen-specific manner. The possible implications of these results are discussed in the context of the causal relationship between a decrease in tuberculosis and an increase in allergic disorders.     

Cross reactions among flaviviruses in macrophage migration inhibition assay.

Adult Swiss albino mice were immunized intravenously with flaviviviruses. Cellular immune response was studied by macrophage migration inhibition assay. Migration of cells from sensitized mice was significantly inhibited in the presence of homologous virus or antigen. Migration inhibition was also significant with serologically related viruses, thus establishing cross reaction in cellular immune response.     

The effect of lymphokines on the development of delayed hypersensitivity to an unrelated antigen

The development of delayed hypersensitivity to sheep red cells in rabbits was assessed by measuring the inhibition of migration of cells from spleen fragments in the presence of sheep red cell antigen. Supernatants containing migration inhibition factor were prepared by incubating lymph node cells from rabbits immunized with Freund's complete adjuvant with PPD. Sheep red cells injected together with these supernatants intravenously gave rise to delayed hypersensitivity. In contrast the injection of sheep red cells alone or with control supernatants did not give rise to delayed hypersensitivity.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) or with DT-anti-toxin precipitate, both in Freund's complete adjuvant (FCA). DT-induced inhibition of peritoneal cell migration was equally strong in both groups and increased with time after immunization. Some guinea-pigs immunized with DT-antitoxin precipitate were boosted with DT or DT-antitoxin precipitate, both in FCA. This increased the antibody titre but did not affect 24-hour skin reactivity or migration inhibition. Migration inhibition did not correlate with anti-DT passive haemagglutinins, haemolysins, or cytophilic antibody on peritoneal cells, but did correlate with 24-hour skin reactivity.