Studies on the induction and time course of repression of delayed hypersensitivity in the mouse by low and high doses of antigen

CAF₁ and CF-1 female mice pretreated with small or large quantities of the protein antigens chicken egg albumin or human serum albumin had their capacities to develop delayed hypersensitivity and some humoral antibodies in response to injection of these antigens in Freund's incomplete adjuvant specifically repressed. Repression following a single intraperitoneal injection of 10 mg of human serum albumin in saline was maximal at 2 weeks. Two appropriately spaced pretreatments were approximately 100-fold more effectual than one in eliciting repression, perhaps because of a secondary antibody response. Pretreatment with antigen in an inefficient variety of Freund's incomplete adjuvant also induced immunologic repression both at high (100 and 10 mg) and very low (optimal = 0·01 γg) doses; but this repression was weaker than that following pretreatment with antigen in saline. One experiment comparing four injection routes for relative capacity to induce repression showed no one clearly superior to the others, although the intravenous route gave some indication of being so. Repression affecting induction of delayed hypersensitivity lasted for 12 weeks in CF-1 and 30 weeks in CAF₁ mice, receded slowly thereafter, and was not followed by spontaneous sensitization. Among humoral antibody responses, pretreatment-induced repression affected Arthus sensitization, and passive haemagglutinin, precipitin and passive cutaneous anaphylaxis antibody production, roughly in descending order. Data from these experiments seem most compatible with the idea that the repression studied is active rather than passive and due, perhaps, to a humoral antibody which represses sensitization.     

Transfer of delayed Hypersensitivity by Lymph-Node Cells in Testis Autosensitization

Guinea pigs sensitized by an extract of homologous testis and Freund's adjuvant developed delayed skin hypersensitivity towards a purified testis antigen. When lymph-node cells from sensitized animals were transferred into normal guinea pigs by intravenous, intraperitoneal or intracutaneous injection the recipients also developed delayed skin hypersensitivity.

Maximum reactions in recipients were obtainable after the transfer of cells from donors which had been sensitized for only 6 days. The recipients became sensitized immediately after cell transfer but their sensitization lasted only a few days.

It can be concluded from this and earlier work that sensitized cells as well as free circulating antibody play a part in testis autosensitization.

     

The passive transfer of delayed hypersensitivity in the guinea-pig: II. The ability of passively transferred antibody to cause local inflammation and retention of antigen and the role of these phenomena in the passive transfer of delayed hypersensitivity

It is known that serum increases the passive transfer of delayed hypersensitivity to certain antigens, such as bovine γ-globulin, in the guinea-pig. This synergic effect of serum in the passive transfer of delayed hypersensitivity to bovine γ-globulin was partly, but not completely, produced by serum to haemocyanin when a mixture of bovine γ-globulin and haemocyanin was used for skin testing. It was concluded that part of the synergic action of serum was due to a local inflammatory reaction.

The ability of serum to cause local retention of antigen in the skin was studied using antigens labelled with radioactive iodine. Immune serum favoured the local retention of antigen. The passive transfer of antiserum to bovine γ-globulin, egg albumin and haemocyanin specifically increased the retention of antigen two- to twelve-fold. This ability of serum to cause the local retention of antigen at the site of intradermal injection was present in serum taken 3 weeks after immunization with bovine γ-globulin in Freund's complete adjuvant but absent in serum taken at 1 week. Antiserum also altered the distribution of antigen at the skin site. Autoradiography showed that it increased the area over which an appreciable concentration of antigen occurred.

Active immunization with bovine γ-globulin had a slight effect on the total amount of antigen retained in the skin after intradermal injection. It had a greater effect on the distribution of antigen. In control guinea-pigs 87 per cent of the total amount of bovine γ-globulin retained at 20 hours was found within a radius of 6.5 mm of the centre of injection. In contrast in guinea-pigs immunized with bovine γ-globulin in Freund's complete adjuvant 43 per cent was found beyond this radius. A similar change in the distribution of human serum albumin was seen in guinea-pigs immunized with bovine γ-globulin when the albumin was mixed with bovine γ-globulin. This indicated that factors other than the formation of immune precipitates were sometimes responsible for the local retention of antigen. The total amount of purified protein derivative of tuberculin (PPD) retained and its distribution in the skin was uninfluenced by immunization.

It was concluded that the synergic effect of immune serum on the passive transfer of delayed hypersensitivity was due in part to some aspect of the inflammation caused by antibody antigen reaction and in part to the local retention of antigen caused by antibody.

     

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.     

Selective and specific inhibition of 24 hour skin reactions in the guinea-pig: III. Depression of cytophilic and haemolytic antibodies by pretreatment with antigen and the effect of irradiation

Dvorak and Flax (1966) and others reported that adult guinea-pigs pretreated with soluble antigens showed depressed immune responses following immunization with the same antigens in Freund's complete adjuvant. These depressed immune responses included haemolytic and cytophilic antibody and delayed hypersensitivity as well as antibody measured by passive cutaneous anaphylaxis and haemagglutination. This communication describes further studies on the effect of pretreatment with alum precipitated bovine γ-globulin rather than soluble antigen. Guinea-pigs given 1 mg of alum precipitated bovine γ-globulin prior to immunization with 50 μg bovine γ-globulin (BGG) in Freund's complete adjuvant show depressed haemolytic and cytophilic antibody and delayed hypersensitivity. This depression is immunologically specific as pretreatment with alum precipitated egg albumin does not depress immune responses to bovine γ-globulin. In contrast, antibody measured by passive cutaneous anaphylaxis and haemagglutination is either unaffected or raised.

Similarly, pretreatment with sheep red cells reduces the cytophilic antibody response to sheep red cells in Freund's complete adjuvant.

This depression is long lasting and 1 mg alum precipitated BGG depressed the haemolytic antibody response to BGG in adjuvant given 6 months later and the delayed hypersensitivity response to BGG in adjuvant given 9 months later. Alum precipitated BGG given up to 6 days after immunization with BGG in adjuvant caused some depression of haemolytic antibody. However, this depression was transient and much less than the long lasting depression caused by pretreatment with alum precipitated BGG 7 days before immunization with BGG in adjuvant.

The effect of irradiation followed by alum precipitated BGG on the immune responses to BGG in Freund's complete adjuvant was also studied. 300 r depressed haemolytic and haemagglutinating antibody but had no effect on cytophilic antibody or delayed hypersensitivity. There was no synergy between irradiation and pretreatment with BGG; the depression of immune responses caused by irradiation followed by alum precipitated BGG was no greater than the depression caused by the more effective agent when given alone.

     

A simple and effective method to assess murine delayed type hypersensitivity to proteins

Mice sensitized with various protein antigens emulsified in complete Freund's adjuvant by a subcutaneous injection at the base of the tail exhibited both immediate and delayed type hypersensitivities 7-8 days following sensitization. Hypersensitivity was assessed by the degree of footpad swelling elicited by challenge with a heat-aggregated form of the antigen. The swelling response was specific for the antigen used for sensitization and often resulted in a near doubling of footpad thickness. Histological examination is conjunction with studies having passive transfer of serum and adoptive transfer of either T cell-enriched or T cell-depleted preparations indicated that whereas the immediate swelling response was achieved with serum, the delayed swelling response was mediated by T lymphocytes. Adoptive transfer of specific delayed type hypersensitivity was also accomplished with small numbers of lymph node T cells obtained following in vitro enrichment and propagation.     

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.     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. II. Mechanism of the phenomenon

Recipient guinea-pigs pretreated with Freund's complete adjuvant alone show depressed 4 hr (Arthus) reactions following passive transfer of antiserum to bovine γ-globulin and human serum albumin. Recipient outbred guinea-pigs and inbred rats also show depressed 24 hr delayed reactions following passive transfer of immune peritoneal exudate cells and serum. This indicates that Freund's complete adjuvant depresses certain inflammatory responses.

Donor guinea-pigs and inbred rats pretreated with FCA alone and then immunized with BGG in FCA transfer smaller 24 hr reactions to normal recipients than comparable donor animals which have not been pretreated with FCA. This suggests that pretreatment with FCA depresses the central state of delayed hypersensitivity which normally follows immunization with BGG in FCA.

These two findings may explain why pretreatment with FCA depresses the 4 and 24 hr skin reactions which otherwise follow immunization with antigen in FCA.

     

The dual necessity for delayed hypersensitivity and circulating antibody in the pathogenesis of experimental allergic orchitis in guinea-pigs

1. Guinea-pigs injected with testis autoclavate (WT) in incomplete adjuvant develop antibody but no delayed hypersensitivity or testicular lesions. These animals fail to develop delayed hypersensitivity or a lesion when subsequently injected with antigen in complete adjuvant. Animals similarly treated, and then given repeated injection of immune cells from animals treated with whole testis and complete adjuvant, develop both delayed hypersensitivity and the characteristic orchitis.

2. Guinea-pigs injected with a purified fraction (designated H) with incomplete adjuvant fail to develop either circulating antibody, delayed hypersensitivity or a testicular lesion. Animals subsequently injected with this antigen and complete adjuvant, develop delayed hypersensitivity but no circulating antibody or testicular lesion. Three of the five animals similarly treated and given repeated injections of serum containing anti-testis antibody also develop orchitis.

3. A sequential study of fourteen animals killed at daily intervals after they were injected with WT and complete adjuvant, showed that antibody was first detected in the testis on the same day as delayed hypersensitivity first appeared. Circulating antibody was not detected until 2 days later. The specific testicular antigen showed first signs of disappearing on the 10th day, by which time histological evidence of orchitis was first detected.

4. Antibody was not detected in the testes in any animal in the absence of delayed hypersensitivity.

     

Antibodies in delayed hypersensitive rats specifically desensitized by an intravenous injection of various antigen doses

Rats with delayed hypersensitivity to bovine serum albumin (BSA) and diphtheria toxoid were specifically desensitized by an intravenous injection of BSA. The degree of desensitization was positively related to the dose of antigen. The 4-hour skin reactions to BSA were weaker, and the passive haemagglutinin titres, antigen-binding capacity and average avidity of the antibodies were lower in the desensitized rats than in controls. The suppression of delayed hypersensitivity was apparently not caused by increased circulating antibody activity, but by a cellular defect or a humoral blocking factor.     

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.     

Adjuvant properties of Micropolyspora faeni.

The adjuvant properties of Micropolyspora faeni, an important source of antigenic material in the production of farmer's lung, were evaluated by comparing antibody- and cell-mediated immune responses of rabbits to bovine serum albumin (BSA) incorporated in complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and incomplete Freund's adjuvant with 5-10 mg/ml homogenized M. faeni (MFA). Rabbits immunized with BSA in CFA or MFA developed significantly increased antigen-induced macrophage migration inhibition, lymphocyte stimulation, and delayed skin reactivity when compared to those immunized with BSA in IFA. No similar adjuvant effect on specific antibody production was observed in rabbits immunized using BSA in MFA. These data suggest that M. faeni can act as a selective immunologic adjuvant for delayed hypersensitivity. This adjuvant property might be important in the induction of mononuclear cell infiltrates seen in human hypersensitivity pneumonitis.     

The immune response suppressed by specific antibody

Homologous or heterologous anti-sheep erythrocyte serum given passively to normal rats markedly suppressed their spleen plaque-forming cell and serum antibody response to sheep erythrocytes. Passive immunization against bovine γ-globulin prevented `sensitization' by a first injection of the antigen. The suppressive effect of passive antibody was prevented or partially prevented by adjuvants, B. pertussis vaccine, S. typhi endotoxin or Freund's complete adjuvant. Passive antibody or adjuvants had relatively little effect on the primary response when given more than 24 hours after antigen or on the secondary response when given with antigen.

The kinetics of the early spleen plaque-forming cell response were measured using sheep erythrocytes as antigen, homologous anti-sheep erythrocyte serum for passive immunization and B. pertussis vaccine as adjuvant. With a constant antigen dose, larger amounts of passive antibody caused increased suppression. Suppression apparently resulted from a decrease in the number of cells initially responding; the rate of proliferation of cells that did respond was not affected by passive antibody. If the amount of passive antibody was kept constant, an increase in antigen dose or addition of adjuvant to the antigen increased the rate of proliferation of the cells that did respond; an effect sufficient to completely mask suppression produced by smaller amounts of passive antibody. These findings can be accounted for by assuming that passive antibody and higher antigen doses or adjuvant affect different interactions required for the antibody response. Thus, the magnitude of the antibody response is dependent not only on the amounts of antigen and passively given antibody but also on the amount and activity of any intentionally or unintentionally introduced factor having adjuvant activity.

     

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.     

Inhibition of delayed hypersensitivity by pre-immunization without complete adjuvant

Guinea-pigs were immunized by injections of blood group substance with incomplete adjuvant, followed after an interval of approximately 2 weeks, by intracutaneous immunization with the same antigen and Freund's adjuvant containing M. tuberculosis. This treatment inhibited the appearance of delayed skin reactions, while circulating antibody production took place as in controls which had received complete adjuvant only with blood group substance, and had delayed skin reactions. The inhibition of the skin reaction was found to be antigen-specific with regard to unrelated antigens, but showed cross-inhibition for serologically different human blood group substances. The first immunization had to be given more than 2 days before the immunization with complete adjuvant. A similar phenomenon was seen with ovalbumin as antigen. In addition to inhibition of the delayed skin reaction, there appeared to be less γ₂-antibody to ovalbumin than in ovalbumin plus complete adjuvant-only controls. Passive administration of antibody did not affect the development of a delayed hypersensitivity state in complete adjuvant-immunized animals with blood group substance or ovalbumin as antigen. Present evidence favours an explanation of the phenomenon in terms of temporary paralysis on the part of some of the antibody-producing cells—viz. those concerned with delayed hypersensitivity and γ₂-antibody production.     

Suppression of delayed hypersensitivity by antigen and antibody: Is a common precursor cell responsible for both delayed hypersensitivity and antibody formation?

Delayed-type hypersensitivity and antibody formation to sheep erythrocytes (SRBC) was studied in rats. Two immunologically specific suppressive effects on the primary induction of delayed hypersensitivity were found: one was short-lasting and due to a direct action of intravenous antigen; the other was mediated by anti-SRBC antibody and demonstrable both as a later-occurring and indirect effect of active immunization and as occurring consequent to passive immunization. Either of these two forms of suppression only partially prevented primary induction of delayed hypersensitivity, but used together their effects were synergistic and completely suppressed development of delayed hypersensitivity. The secondary delayed hypersensitivity response was insusceptible to antibody inhibition.

The data concerning delayed hypersensitivity to SRBC in the rat were in some ways analogous to previous findings concerning induction of haemolytic plaque-forming cells to SRBC in the same animal. The interpretation that delayed hypersensitivity and humoral antibody formation could represent alternative responses of potential antibody-forming cells to immunological induction was considered.

     

Mechanism of Intensification by Complete Freund's Adjuvant of the Acquisition of Delayed Hypersensitivity in the Guinea Pig

In the guinea pig, delayed administration of complete Freund's adjuvant (CFA) to skin sites previously primed with a cellular antigen leads to a marked intensification of the acquired sensitivity to that antigen. This effect has been demonstrated with diverse cellular antigens including foreign erythrocytes (both fresh and formaldehyde treated), HeLa cells and dead bacteria, Thus, when a borderline-sensitizing quantity of cells is given intradermally on Day 0, followed by adjuvant into the same site on Day 4, there results a marked intensification of the delayed hypersensitivity reaction to Day 6 testing with the priming cells in the adjuvant treated animals. It appears that CFA intensified acquisition of delayed hypersensitivity to cellular antigens, as well as to soluble protein antigens and contact allergens, involves two steps: 1) initial interaction of antigen with immunocompetent cell, 2) subsequent stimulation by adjuvant of immunocommitted cells (probably by enhancing their clonalization). It appears that the CFA effect takes place primarily in lymph nodes regional to the sensitization site.     

Immune deviation: demonstration of split tolerance in vitro by inhibition of macrophage migration

Migration of cells, taken from animals immunized with ovalbumin in Freund's complete adjuvant which gave normal delayed hypersensitivity skin responses, was found to be significantly inhibited in the presence of antigen. Migration of peritoneal exudate cells from guinea-pigs in which immune deviation had been induced by immunization with antigen in Freund's incomplete adjuvant was not inhibited.     

Desensitization in vitro—The specific inhibition, by antigen, of the passive transfer of delayed hypersensitivity by peritoneal exudate cells

A preliminary experiment showed that the injection of bovine γ-globulin into guinea-pigs with delayed hypersensitivity to bovine γ-globulin reduced the 24-hour skin reactions to bovine γ-globulin and (to a lesser extent) PPD. The peritoneal exudate cells from the desensitized donors had a reduced ability to transfer delayed hypersensitivity to bovine γ-globulin but a normal ability to transfer delayed hypersensitivity to PPD.

Likewise, it was possible to diminish the passive transfer of delayed hypersensitivity to bovine γ-globulin by peritoneal exudate cells, by exposure of the cells to bovine γ-globulin in vitro. The recipients were tested immediately after cell transfer. This in vitro desensitization was specific, in that the transfer of delayed hypersensitivity to PPD was unaffected.

Exposure of cells in vitro to hypotonic conditions and antibody to guinea-pig γ-globulin did not prevent the passive transfer of delayed hypersensitivity.

     

Cell-mediated and humoral immune responses in mice. III. Dynamic balance between delayed-type hypersensitivity and antibody response.

Delayed-type hypersensitivity in mice to bovine serum albumin (BSA) which would be induced by a s.c. injection of BSA in Freund's complete adjuvant was interrupted by an i.v. injection of alum-precipitated BSA plus bacterial endotoxin (AP-BSA plus ET). In contrast, s.c. injection of BSA restrained the antibody response in the spleen of the mice receiving AP-BSA plus ET. Subcutaneous injection of either the adjuvant alone or an unrelated antigen in the adjuvant did not affect the anti-BSA antibody response in the spleen. The antibody response to other antigens in the spleen was not affected by the s.c. injection of BSA. The reduction of the antibody response in the spleen does not seem to be attributable to suppressor cells, since the suppressor cell activity against the antibody response of normal mice and that of irradiated recipients of primed spleen cells was not observed in the spleen cells of mice given s.c. immunization. The results indicated that the reduced antibody response in the spleen may be caused by the migration of some part of the antigen reactive cells or their precursors from the spleen to the s.c. region.