Dissociation between delayed-type hypersensitivity and resistance to pathogenic mycobacteria demonstrated by T-cell clones.

One Lyt-2+ clone and fourteen T-helper clones (Lyt-1+ L3T4+ Lyt-2-) were isolated from Mycobacterium lepraemurium-infected BALB/c and C57BL/6 mice. All the clones were tested for their ability to transfer delayed-type hypersensitivity adoptively, and six clones were tested for their ability to transfer resistance. It was found that four L3T4+ clones that transferred delayed-type hypersensitivity responses locally and one L3T4+ clone that did not had no effect on resistance. The Lyt-2+ clone transferred increased resistance locally (77% reduction in the numbers of organisms recovered from the infection site) but did not transfer delayed footpad responses.     

Lack of correlation between delayed-type hypersensitivity and host resistance to Plasmodium chabaudi infection.

C57B1/6J mice immunized with Plasmodium chabaudi antigen plus saponin exhibited strong delayed-type hypersensitivity (DTH) reactions to footpad injections of P. chabaudi antigen. However, when immunized mice were challenged with P. chabaudi parasites, DTH was significantly depressed after 3 days of infection. This DTH depression coincided with a steep rise in the titre of malarial antibody in these immunized challenged mice when compared to unchallenged immunized mice. Serum from mice recovering from P. chabaudi infection depressed DTH levels of immunized mice significantly, but spleen cells from convalescent mice only slightly reduced the DTH levels. These results are discussed with respect to the role of cell-mediated immunity in malaria infections.     

Suppression of delayed-type hypersensitivity to histocompatibility antigens by ultraviolet radiation.

Delayed-type hypersensitivity (DTH) reactions to allogeneic histocompatibility antigens in mice could be systemically suppressed by a single exposure to UV-B irradiation. The extent of suppression reached its maximum 4 days after irradiation, gradually waned thereafter, and disappeared at Day 21. Re-exposure of these mice to UV-B after waning reinduced the state of suppression. The suppression could be transferred to naive mice by means of splenic T lymphocytes. The suppressor T (Ts) cells suppressed the proliferative activity in the lymph nodes draining the site of immunization, but not the activity of already activated DTH-reactive T cells. Phenotypical analysis of these Ts cells revealed that two subpopulations of T cells are involved: one with the Lyt-1+, 2- phenotype, the other with the Lyt-1-, 2+ phenotype.     

Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Failure of Mycobacterium leprae soluble antigens to suppress delayed-type hypersensitivity reaction to tuberculin.

In order to test a published claim that the inclusion of Mycobacterium leprae antigens with a tuberculin skin test reagent can suppress delayed-type hypersensitivity (DTH) to tuberculin in both paucibacillary and multibacillary leprosy cases, 109 leprosy cases and 104 non-leprosy controls were skin-tested simultaneously with tuberculin with and without M. leprae soluble antigens. Tests were randomized between arms and carried out double-blind. There was a clear tendency for larger DTH responses with the combined tuberculin plus M. leprae antigen than with tuberculin alone in paucibacillary leprosy cases and in non-leprosy controls. No evidence for M. leprae antigen-mediated suppression of DTH was observed in any group. It is unclear whether the difference between the results reported here, which were obtained in Malawi, and those in the published literature which were obtained in India, is attributable to geographic differences in important biological variables or to differences in the experimental protocols. The need for methodological rigour in skin-test studies is stressed.     

Half-body x-ray suppresses the acquisition of delayed-type hypersensitivity in the mouse.

800 R half-body x-ray suppressed the acquisition of allergic contact dermatitis in mice (1) if the x-ray field included the lymph nodes regional to the sensitization site, and (2) when x-ray was given during, but not immediately before, induction of the sensitivity.     

A footpad weight assay method to evaluate delayed-type hypersensitivity in the mouse

Delayed-type hypersensitivity lesions have been difficult to evaluate objectively in the mouse. They are usually evaluated by measurement of footpad swelling or increased ear thickness after challenge with antigen. I have developed a footpad weight assay method which reduces the observer's error and the instrumental error. This method is reliable for the assessment of delayed-type hypersensitivity reactions when results are expressed as weight increase or weight ratio calculated on challenged and unchallenged footpad.     

The in vitro induction of delayed-type hypersensitivity to allo-antigens of the mouse

An in vitro procedure has been developed which allows the induction of delayed-type hypersensitivity (DTH) to major allo-antigens of the mouse. Murine spleen cells cultured with irradiated allogeneic cells develop allo-specific DTH reactivity (DTHR). The variables assessed to provide optimal induction of DTHR were (i) the culture system, (ii) the density of responders and the number of stimulator cells and (iii) the kinetics of induction. The reactivity of the allo-specific cells was assayed in 3 different ways in order to select the most sensitive: (i) by local footpad transfer into a mouse syngeneic with the responder cells together with the eliciting (irradiated) antigen-bearing cells; (ii) by local transfer into the footpad of a mouse syngeneic with the stimulator cells, in which the allo-antigens present in the subcutaneous tissues elicit the response, and (iii) by intravenous transfer into syngeneic or allogeneic mice which are challenged 24 h later in the footpad with spleen cells bearing the haplotype of the stimulator. The second assay is clearly the most sensitive (2X that of the first and greater than 20X that of the last). Observations are reported demonstrating the specificity of the swelling reaction. The kinetics of swelling and its T-cell dependence provide strong grounds for believing that the reaction is due to classical delayed-type hypersensitivity. Furthermore the T cells mediating the swelling are of the phenotype Lyt1+ Lyt2(+/-) and Ia- and are radiation resistant, whereas the ability to produce a swelling reaction is sensitive to 1000 rads. whole body irradiation. The system has been applied to determine both the specificity of T cells mediating DTH to major and minor allo-antigens and whether cytotoxic T cells (CTL) and DTH-active T cells are always induced under the same conditions.     

Regulation of delayed-type hypersensitivity. II. Specific suppressor factor for delayed-type hypersensitivity to sheep erythrocytes in mice.

An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x 10(9) SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH to SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 56 degrees C for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.     

Macrophages and resistance to tumours: I. Inhibition of delayed-type hypersensitivity reactions by tumour cells and by soluble products affecting macrophages

Mice were injected in the footpads with mixtures of an antigen (sheep red blood cells) to which they had developed delayed-type hypersensitivity (DTH) and syngeneic fibrosarcoma cells. DTH reactions were associated with depression of subsequent tumour growth compared with that in control animals. The degree of depression was proportional to the intensity of the reactions. Conversely, the presence of fibrosarcoma cells was associated with depression of the DTH reactions. Serum-free culture supernatants of all tumours tested (mouse, human and rat) depressed DTH reactions in mice, depressive activity being apparent even at dilutions greater than 1:100. Supernatants from cultures of nonmalignant cells had little or no depressive activity. Protein synthesis, but not DNA synthesis, was required for the production of the factor(s) responsible. in vitro, the active supernatants markedly inhibited macrophage migration, either spontaneously or in response to a chemotactic factor, but had much less effect on mitogen-stimulated or unstimulated lymphocyte cultures. The activity was decreased or lost after treatment with proteolytic enzymes, ribonuclease, neuraminidase or hyaluronidase. The supernatants were separated by membrane filtration into fractions of M.W. greater than, or less than 10,000. The fractions differed from each other in their effects in vivo and in vitro. Injection of concentrated supernatants from tumours together with fibrosarcoma cells led to more rapid initial tumour growth.     

Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

Mice injected with 1 X 10(8) sheep red blood cells (SRBC) into the footpad showed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after the injection. In contrast, mice injected intravenously with 1 X 10(9) SRBC were unresponsive to DTH induction through 1 X 10(8) SRBC injected into the footpad. This suppression of DTH was maintained for at least 6 weeks and was transferable spleen, lymph node and thymus cells to normal syngeneic recipients. Bone marrow cells, on the other hand, did not contain the suppressor cells. The suppression of DTH was antigen-specific in that DTH to chicken red blood cells and contact sensitivity to 2,4-dinitrofluorobenzene was not affected. The suppressor cells were theta-positive and Ig-negative. They appeared in the spleen in optimum number 3-4 days after induction. The suppressor cells affected both the induction and manifestation of DTH. The presence of suppressor and effector cells for DTH inducible by different routes of antigenic presentation reflects the dynamic balance in the regulation of DTH.     

Cross-reactivity between human adenoviruses in delayed-type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed-type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient-purified human Ad serotype 6 (Ad6) induced DTH in a dose-dependent manner. In Ad6-sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross-reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross-reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.     

Regulation of delayed-type hypersensitivity. VII. The role of I-J subregion gene products in the inhibition of delayed-type hypersensitivity to major histocompatibility antigens by specific suppressor T cells

Suppressor T cells for delayed-type hypersensitivity (DTH) to alloantigens are induced by injecting mice intravenously with a high dose of X-irradiated allogeneic cells. Using the same protocol, suppression of DTH directed against H-2 subregion products could also be induced, provided that the H-2 incompatibility between the cells used for the induction of suppression and the recipients includes the I-J subregion genes. Thus, the I-J subregion difference is both necessary and sufficient for the induction of suppression of DTH to the whole or part of the H-2 gene products. The suppression appears to be mediated by antigen-specific suppressor T cells which recognize the allo-I-J molecules and are able to suppress the DTH response to other H-2 subregion gene products in an associative recognition manner. T cells from (B10 × BALB/c)F₁ mice suppressively primed against B10.A(5R) cells (directed against J^kE^k antigens), when adoptively transferred to normal syngeneic recipients, were capable of suppressing the hosts' DTH response to B10.A(4R) cells (K^kA^k antigens) when the recipients were challenged with [B10.A(4R) × B10.A(5R)]F₁ cells (K^kA^k × J^kE^k). The recipients express normal DTH reactivity to B10.A(4R) cells when challenged with a mixture of B10.A(4R) and B10.A(5R) cells (K^kA^k + J^kE^k). These results provide direct evidence that when functioning as alloantigens, the I-J determinants preferentially induce suppressor T cells which specifically impair the immune response to the I-J molecules as well as other H-2 gene products if they are physically associated with the I-J determinants. The role of I-J subregions as the suppressor genes is discussed in terms of the possible application in transplantation immunity and the host's defence against infectious diseases.     

Strong delayed-type hypersensitivity induced against house dust mite antigens in the mice

The aim of this study was to assess the antigenicity of house dust mite antigens using delayed-type hypersensitivity (DTH) responses in mice. The crude extracts of Dermatophagoides pteronyssinus and Dermatophagoides farinae induced strong DTH responses and was transferred with T cells. Assessment of DTH responses to the house dust revealed that substantial cross-reactivity at T-cell level was observed between these two mite extracts. Almost all the fractions of the extract of D. pteronyssinus, which has a molecular weight of between 10 and 100 kilodaltons, elicited strong DTH responses. It was found that antigenicity of the mite extract was reduced by chemical denaturation with tannic acid.     

Distinction between suppressors of the delayed-type hypersensitivity and the humoral response to sheep erythrocytes.

Suppressors for both delayed-type hypersensitivity (DTH) and the humoral immune response could be simultaneously induced in the spleens of mice by immunization with a high dose of SRBC. Normal recipient mice of the spleen cells from donors immunized 5 days previously elicited depressed DTH or humoral response when immunized with SRBC. The suppressive activity was found to reside in T not B enriched fraction. Four hundred rad irradiation of the primed spleen cells resulted in complete loss of DTH suppressor activity, but only in some reduction of the suppressor activity for the humoral response. In contrast, hydrocortisone treatment of the donor mice caused no loss of DTH suppressor activity while approximately half of the suppressive activity for anti-SRBC PFC response was lost. Adult thymectomy prevented completely the induction of the DTH suppressor in contrast to little loss of the suppressor activity for the humoral response. DTH suppression was antigen-specific for the induction, but nonspecific for the expression. However the suppression of the humoral response was antigen-specific not only for the induction but also for the expression. In addition, DTH suppressor was capable of suppressing both the induction and expression of DTH while the humoral response was suppressed only in the induction stage by the suppressor.     

Regulation of delayed-type hypersensitivity V. Suppressor cell memory in antigen-specific suppression of delayed-type hypersensitivity to sheep erythroeytes

Mice primed i. v. with 10⁹ sheep red blood cells (SRBC) produce antigen-specific T suppressor (Ts) cells which inhibit both the induction and the expression of delayed-type hypersensitivity (DTH). These Ts cells are detectable in the spleen and lymph nodes 3–5 days after priming but are largely absent by 6 days. The transient detect-ability of the Ts cells contrasts sharply with the profound antigen-specific suppression which persists in primed donor mice for at least a year. Evidence is presented that this long-term impairment of DTH is maintained, at least in part, by memory Ts cells which are Thy-1⁺, cyclophosphamide-resistant and antigen-specific. Although they appear to be co-induced with the short-lived primary Ts cells and localize initially in the lymphoid organs, they are present in the long-lived circulating pool of T cells and can be adoptively transferred by celomic parabiosis. Memory Ts cells are readily reactivated by lower doses of SRBC which would induce T effector cells rather than Ts cells in naive animals. Reactivated memory Ts cells seem to generate a population of antigen-specific secondary Ts cells which again localizes in the lymphoid organs and can adoptively suppress the induction and expression of DTH to SRBC.     

Increased resistance and depressed delayed-type hypersensitivity to Listeria monocytogenes induced by pretreatment with lipopolysaccharide.

Intravenous injection of a small dose of lipopolysaccharide 24 h before infection with Listeria monocytogenes enhanced the resistance of mice to this organism. This protective effect of lipopolysaccharide related to the ability of nonimmune macrophages to inhibit bacterial proliferation in livers and spleens. Surprisingly, lipopolysaccharide-treated mice exhibited inferior acquired immunity, as measured by adoptive transfer of immunity to normal mice, delayed-type hypersensitivity to Listeria antigens, and uptake of tritiated thymidine by lymphocytes in the spleen. These results support the view that lipopolysaccharide stimulates a highly effective anti-Listeria immunity via the macrophage component, despite interference with the lymphocyte component.     

Suppression of delayed-type hypersensitivity reactions and lymphokine production by cyclosporin A in the mouse.

Two consecutive daily i.m. injections of cyclosporin A (Cs A) (greater than 50 mg/kg) inhibited delayed type hypersensitivity (DTH) responses in mice immunized with SRBC. Maximal suppression was observed when Cs A was administered 24 and 48 h after sensitization. Culture of spleen cells from these animals with antigen, insoluble concanavalin A (iCon A) or PHA revealed inhibition of the production of two lymphokines: that inducing macrophage procoagulant activity (MPCA) and macrophage chemotactic factor (LDCF). The inhibitory effect on lymphokine production was not due to depletion of T cells. In vitro, 25 ng/ml Cs A suppressed T cell proliferative responses to antigen and mitogen but much higher doses were required to impair the response to LPS. Similar doses of Cs A also suppressed lymphokine production, but the responses of macrophages to these lymphokines was unaffected, even at doses which totally inhibited lymphokine production. Production of interleukin 1 by LPS stimulated macrophages was inhibited by Cs A only at concentrations much greater than those required to suppress lymphokine production.