Delayed-Type Hypersensitivity Response in Mice to Treponema Pallidum

C3H/HeJ mice which had been primed with either virulent or killed T. pailidum were studied for in vivo delayed-type hypersensitivity (DTH) responses to T. pallidum following local footpad challenge. Mice sustaining a chronic infection of 5 months duration failed to develop a DTH to treponemal antigens, whereas priming by a single intravenous injection with killed organisms resulted in a significant DTH response in mice when challenged 5 days later. Treatment of mice prior to priming with a single sublethal dose of cyclophosphamide (100 mg/kg body weight) not only failed to potentiate T. pallidum-DTH, but abrogated the response observed in untreated primed animals.     

Delayed-Type Hypersensitivity to Ovalbumin in Bone Marrow Chimeric Mice

The site of action of the H-2 linked gene regulating the ability of mice to be sensitized to transfer delayed-type hypersensitivity was studied using radiation bone marrow chimeras. Lethally irradiated (non-responder × responder) F₁ were reconstituted with bone marrow cells of both parents. Cells of chimeras immunized with limiting doses of ovalbumin responded as did the parental strains. These results suggest that the gene regulating delayed type hypersensitivity is expressed at the level of the delayed-type hypersensitivity effector cells or that restrictions exits on the collaboration of the helper cells for DTH effectors (which differ at several loci including the major histocompatibility complex).     

The Effect of an Unrelated Delayed-Type Hypersensitivity Reaction on the Antibody Response to Diphtheria Toxoid

Four Lf fluid diphtheria toxoid were injected intracutaneously, together with tuberculin PPD, into guinea-pigs sensitized to tuberculin by a water-in-oil emulsion of killed tubercle bacilli 4–5 months previously. Antibody production, judged by antibody levels at 4 weeks, was two to four times greater than that in control guinea-pigs. Such an increase in antitoxin would correspond to a ten- to twelve-fold increase in the effective dose of toxoid, which is much greater than any likely non-specific retention at the injection site. It is suggested that the effect of a local tuberculin reaction in enhancing the immunogenic capacity of diphtheria toxoid is due to an increase in the number of immunologically competent cells to which the toxoid has access.     

Augmentation of Natural Killer Cell Activity by Anti-Host Delayed-Type Hypersensitivity during the Graft-versus-Host Reaction in Mice

During a graft-versus-host (GVH) reaction in unirradiated F1 hybrid mice there is a generalized activation of natural killer (NK) cells. We have examined whether the enhanced NK activity is due to an F1 resistance mechanism directed at the parental cells used to induce the GVH reaction. Spleen cells of C57BL/10 origin induce much more NK cell activation in B10F1 hybrids than the opposite parental type, despite a similar intensity of systemic GVH reactions. However, this does not correlate with in vivo resistance of mice with GVH reaction to a local challenge dose of B10 cells. NK cell activation in (CBA X BALB/c)F1 mice with GVH reaction involves both host and donor cells and is preceded by an anti-host delayed-type hypersensitivity (DTH) response. B10 cells have a greater ability to induce DTH in B10F1 mice than cells from the opposite parent. We propose that NK cells are one group of non-specific effector cells recruited by DTH in a GVH reaction and may contribute to the tissue pathology.     

Staphylococcidal Capability of Rabbit Peritoneal Macrophages in Relation to Infection and Elicitation: Delayed-Type Hypersensitivity Without Increased Resistance

The staphylococcidal capability of populations of peritoneal macrophages in rabbits has been measured before and after repeated infections with Staphylococcus aureus. Such rabbits after infection showed delayed-type hypersensitivity to S. aureus antigens, but the staphylococcidal capability of the peritoneal macrophages was not increased. This result at the cellular level is in agreement with previous assessment in vivo of the consequences of staphylococcal infection. Pathways to cell-mediated resistance, with and without delayed-type hypersensitivity, are presented.     

Two T-Cell Populations Mediate Delayed-Type Hypersensitivity to Murine Influenza Virus Infection

Two classes of T lymphocytes can mediate delayed-type hypersensitivity (DTH) to influenza virus in the mouse. If non-infectious virus preparations are used to sensitize for or to elicit a DTH response, the effector cells are found to be Ly-1-positive and are I-region-restricted. If infectious virus is used both to sensitize for and to elicit the reaction, a second set of effector cells is also detected, which are Ly-2,3-positive and are D- or K, D-region-restricted. The latter cells are cross-reactive within the A strains of influenza viruses, and pretreatment of the mice with high doses of cyclophosphamide markedly decreases their generation in the spleens of sensitized mice, suggesting that the cells that demonstrate DTH activity in vivo may also have cytotoxic activity in vitro.     

The Modulatory Effect of Exogenously Added Prostaglandin E1 Or E2 On the Delayed-Type Hypersensitivity Reaction of Normal or Tumored Mice

The effect of exogenously added prostaglandin (PG) E₁ or E₂ over concentration ranges of from 1 × 10^-4 to 1 × 10^-9 M were studied in order to determine their effect on the delayed-type hypersensitivity (DtH) reaction of normal or tumored mice. PGE or PGE₂. generally caused a stimulation over the control values of normal mice as detected by the footpad swelling assay. However, PGE¹ or PGE₂ at all concentrations tested were found to significantly inhibit the DtH reaction of CD₂F₁ tumored mice.     

Detection of Delayed Hypersensitivity in Mice Injected with Ribonucleic Acid-Protein Fractions of Salmonella typhimurium

Manifestations of delayed hypersensitivity (cell-mediated immunity) were found to be present in mice immunized with virulent Salmonella typhimurium SR-11 subfractions which contained appreciable concentrations of bacterial protein and in mice immunized with the living, attenuated RIA strain of S. typhimurium. Delayed hypersensitive responses were measured by footpad sensitivity in the immunized mice and in vitro by inhibition of spleen cell migration and by increase in (3)H-thymidine uptake by lymphoid cell populations when exposed to bacterial fractions rich in protein content.     

Local Administration of Various Cytostatic Drugs after Subcutaneous Immunization Enhances Delayed-Type Hypersensitivity Reaction to Sheep Red Blood Cells in Mice

Delayed hypersensitivity reaction in mice was enhanced with various anti-cancer drugs administered at the site of antigenic stimulation during 4 days following sensitization. The immunopotentiating effect of the presented local chemotherapy protocol is thought to result from impairment of a regulatory circuit, with activated suppressor T cells (Ts) as target rather than Ts precursors or Ts-inducing antigen-presenting cells.     

Delayed Hypersensitivity to Toxoplasma and Unrelated Antigens in Toxoplasma-Infected Mice: Induction and Elicitation of Delayed-Type Hypersensitivity by Antigen-Pulsed Macrophages

Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 10⁶ Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice.     

In Vivo Collaboration between Precursor T Cells and Helper T Cells in the Development of Delayed-Type Hypersensitivity Reaction to Influenza Virus in Mice

Nude, athymic mice do not mount a delayed-type hypersensitivity (DTH) response to influenza A virus. A single injection of T helper cells (gamma-irradiated, 2-day immune spleen cells) or three injections over 3 days of a concanavalin-A-activated spleen cell supernatant to virus-sensitized nude mice resulted in a 'normal' DTH response when the mice were challenged with the virus. It was previously shown that the cells responsible for the reaction were T cells and required I-region compatibility. Injection of T helper cells into normal mice did not affect the level of the subsequent DTH response. However, injection of such cells into mice pretreated with anti-thymocyte serum (ATS) restored the ability of the mice to mount a DTH response. The results show that (1) nude mice contain precursor T cells for influenza virus antigen; (2) an I region-restricted response can be generated in the absence of a thymus; and (3) in vivo collaboration between DTH T-cell precursors and helper T cells can be shown to occur in congenitally nude mice and ATS-treated mice.     

Adoptive Transfer of Delayed-Type Hypersensitivity to Sendai Virus

The intrathymic versus extrathymic influences on H-2-restriction specificity of K,D- and I-region-restricted Sendai virus-immune T lymphocytes, which mediate a delayed-type hypersensitivity response in vivo, were investigated. K,D- and I-region-restricted Td lymphocytes of P1 leads to F1 fetal liver chimeras showed preference but no absolute restriction for the P1 haplotype. T effector lymphocytes of thymic chimeras expressed a slightly higher degree of restriction for the haplotype of the thymus graft than F1 leads to P1 chimeras, which might reflect the activity of T lymphocytes of thymic origin. The lack of absolute restriction of both K,D- and I-region-restricted Td lymphocytes to the haplotype the thymus suggests an additional pathway of T-cell maturation.     

Experimental Salmonellosis: Hypersensitivity to Cell Wall Lipopolysaccharide and Anti-Infectious Resistance of Mice Infected with Salmonella

Mice infected with various strains of Salmonella enteritidis and S. typhimurium were found to be more sensitive to the cell wall lipopolysaccharide (LPS) extracted from certain strains of Salmonella than noninfected mice. This hypersensitivity was induced by those smooth or rough strains which possessed a polysaccharide chain longer than that of a glucoseless mutant. The major antigenic group participating in this hypersensitivity was presumed to be in rough core polysaccharide sequence because fractions containing either O side chain or LPS of a heptoseless mutant were ineffective in provoking a hypersensitivity reaction. Conditions for the induction and the phases of development of this hypersensitivity to LPS and for anti-infectious resistance were shown to be different. Present and previously obtained results suggest that the antigens participating in each of these two conditions were different.     

Host Defenses in Experimental Scrub Typhus: Delayed-Type Hypersensitivity Responses of Inbred Mice

Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.     

Cells Mediating Delayed-Type Hypersensitivity in the Lungs of Mice Infected with an Influenza A Virus

Effector cells that demonstrate delayed-type hypersensitivity (DTH) on transfer with antigen to naive mice can he recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5 × 10⁴EID₅₀) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly I phenotype and required I-region sharing between donor and recipient for activity to be elicited. They were cross-reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T-cell activity were shown to protect recipient infected mice from death.     

Absence of Correlation Between Delayed-Type Hypersensitivity and Protection in Experimental Systemic Candidiasis in Immunized Mice

We found that in mice which had been immunized intraperitoneally with 2 × 10⁸ heat-killed Candida albicans cells there was a striking temporal relationship between resistance to systemic challenge with 10⁶ living C. albicans cells and a number of measurable cellular parameters of the host response. These included the emergence of delayed-type hypersensitivity and the development of granulocytosis. Since it had been shown in previous work that granulocytosis was associated with an increase in resistance when nonspecific immunostimulation was used, we performed experiments to induce delayed-type hypersensitivity without any measurable modification of the granulocyte population. Adoptive transfer of delayed-type hypersensitivity with spleen cells from immune and resistant donor mice did not produce any increase in resistance in normal recipients. When separate groups of mice were immunized intraperitoneally or subcutaneously with varying doses of heat-killed C. albicans, we found that doses of less than 10⁸ cells did induce significant delayed-type hypersensitivity without any increase in granulocytosis. In such mice, as well as in animals pretreated with immunomodulators before immunization with heat-killed C. albicans, the presence of cell-mediated immunity, as measured by the delayed-type hypersensitivity test, was not associated with an increase in resistance against systemic candidiasis. On the contrary, the results suggested that cell-mediated immunity was associated with an increase in the susceptibility of these mice. The same effect on candidiasis susceptibility was observed when animals were immunized with heat-killed filamentous C. albicans.     

Interleukin 18 Contributes to Host Resistance and Gamma Interferon Production in Mice Infected with Virulent Salmonella typhimurium

Spleen and peritoneal macrophages obtained from innately resistant A/J mice released low levels of interleukin 18 (IL-18) upon infection with Salmonella typhimurium C5 RP4. Incubating the cells with recombinant gamma interferon (rIFN-γ) enhanced IL-18 production. A/J mice treated in vivo with anti-IL-18 antibodies showed impaired resistance to infection, with increased bacterial loads in the liver and spleen. Administration of rIL-18 could protect A/J mice from challenge with a lethal dose of virulent salmonellae, with a dramatic reduction in bacterial numbers in the tissues. rIL-18 administration did not ameliorate the disease in IFN-γ-R^−/− mice. IL-18 proved to be required for IFN-γ production by mouse splenocytes from conventional, scid, and rag-1^−/− mice; in vivo IL-18 neutralization caused a decrease in circulating IFN-γ levels. Thus, IL-18 is a key factor in early host resistance to Salmonella and probably acts via IFN-γ.     

At Least Four Percent of the Salmonella typhimurium Genome Is Required for Fatal Infection of Mice

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.     

Antigenic Modification, Rosette-Forming Cells, and Salmonella typhimurium Resistance in Outbred and Inbred Mice

To assess the separate contributions of host T cells and the physical state of the antigen in the development of effective. Salmonella resistance, glutaraldehyde-treated and untreated protein- and ribonucleic acid-rich extracts (E-RNA extracts) of virulent Salmonella typhimurium SR-11 or attenuated S. typhimurium RIA were used to immunize Salmonella-resistant Salmonella-susceptible strains of mice for the purpose of determining whether antigen-specific T-cell or B-cell responses were formed and, if so, which responses predominated. The resistance imparted to each mouse strain after vaccination with S. typhimurium RIA was used as the standard for comparison. The inbred mouse strains C57BL/6 and DBA/2 and their F₁ hybrid (strain BDF₁), outbred ICR Swiss mice, and endotoxin-resistant C3H/HeJ mice were examined for the capacity to develop resistance to lethal Salmonella infections, as well as the ability to generate antigen-reactive T cells. Only the BDF₁, C3H/HeJ, and ICR Swiss mice were able to develop resistance to challenge infections mediated by the virulent SR-11 strain of S. typhimurium after vaccination with the living, attenuated RIA strain of S. typhimurium or immunization with E-RNA extracts. We developed an assay to identify the antigen-reactive rosette-forming lymphocytes present in lymph nodes and spleens of immunized mice. Levels of 0.2% or higher of theta antigen-bearing, antigen-reactive rosette-forming cells were found in the lymph nodes or spleens or both of only the BDF₁, C3H/HeJ, and ICR Swiss mice (i.e., in the “Salmonella responder” strains). Mouse strains C57BL/6 and DBA/2, which failed to develop resistance to lethal infections after immunization with the S. typhimurium RIA vaccine or with the E-RNA extracts, lacked effective numbers of antitheta antigen-sensitive rosette-forming cells. Modification of the effective E-RNA extracts by polymerization with glutaraldehyde resulted in a marked diminution in their abilities to induce resistance to salmonellosis in the two responder mouse strains tested (BDF₁ and ICR Swiss), even though detectable levels of antibody were induced.