Expression Of Delayed-type Hypersensitivity Research Articles

Immune Amplification of Murine CD8+ Suppressor T Cells Induced via An Immune-Privileged Site: Quantifying Suppressor T Cells Functionally

BackgroundCD8+ suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8+ suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8+ suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8+ suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells.MethodsSuppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen.ResultsSuppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8+ AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH.ConclusionsThe results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8+ suppressor T cells are specifically amplified by antigen during an immune response.

Mycobacterial trehalose 6,6′-dimycolate preferentially induces type 1 helper T cell responses through signal transducer and activator of transcription 4 protein

Mycobacterium tuberculosis is an intracellular pathogen of tuberculosis and its pathogenicity is related to the ability to escape killing by ingested macrophages and induce delayed-type hypersensitivity (DTH). A major component of the cell wall of M. tuberculosis is trehalose 6,6′-dimycolate (TDM), which has been implicated as a pathogenetic factor. The expression of DTH and cell-mediated immunity is dependent on the macrophage-cytokine-type 1 helper T (Th1) lymphocyte axis. Cytokines, interleukin-12 (IL-12) and interferon-γ (IFN-γ), play a critical role in the process and IL-12-activated signal transducer and activator of transcription (STAT) 4 is required for the development of fully functional Th1 cells. To clarify host responses to mycobacterial TDM, we have analyzed footpad reaction, histopathology and cytokine profile of experimental granulomatous lesions using STAT4-deficient mice. In the present study, we have demonstrated that mycobacterial TDM selectively induces the Th1 response through the STAT4 signaling pathway, because mice lacking STAT4 protein significantly reduced to develop DTH, hypersensitivity granulomas, and Th1 cytokine responses, when compared to BALB/c mice. These results shed light on the molecular pathogenesis of mycobacterial disease. Taken together with previous studies, TDM is a pleiotropic molecule against the host and participates in the pathogenesis.

A Deviant Immune Response to Viral Proteins and Transgene Product Is Generated on Subretinal Administration of Adenovirus and Adeno-associated Virus

The immune response after ocular exposure to foreign antigens varies substantially from that of a typical systemic response. Anterior chamber associated immune deviation (ACAID) has been well documented. The immune response of the subretinal space has not been studied in as much detail. Here, we characterized the immune response of the subretinal space when it encounters the antigens AdV-GFP and AAV-GFP (recombinant adenovirus or adeno-associated virus, respectively), each delivering the reporter gene encoding green fluorescent protein (GFP). Results indicate that the subretinal space possesses an immune-deviant property similar to ACAID. AdV-elicited immune responses following subretinal injections are significantly reduced compared with systemic responses elicited by intradermal injections of the same virus. Furthermore, subretinal AdV administration results in transduction of retinal pigment epithelial cells (RPE), which are the potential antigen presenting cells of the retina. This subsequently generates a population of immunosuppressive Th2-type, cytokine-secreting, splenic T cells. This response may be advantageous to the development of ocular gene therapy.

Neutralizing anti-IL-10 antibody upregulates the induction and elicitation of contact hypersensitivity.

Various cytokines have been shown to modulate the acquisition and expression of delayed-type hypersensitivity. In a mouse model, we tested the notion that neutralization of interleukin-10 (IL-10), a cytokine that inhibits T cell-mediated reactions, would upregulate delayed-type hypersensitivity. We used two different monoclonal antibodies with specificity for murine IL-10 and used allergic contact dermatitis as a prototypical example of delayed-type hypersensitivity. When anti-IL-10 antibody was given at the time of sensitization to a contact allergen, there was a substantial increase in the induced contact hypersensitivity (CHS). In other experiments, the challenge reactions to contact allergen in routinely sensitized mice were increased when anti-IL-10 antibody was given at the time of challenge. Primary irritant reactions to croton oil were increased but only if anti-IL-10 antibody was given at the time of challenge and not when it was given a week previously. It appears that anti-IL-10 antibody can potentiate CHS reactivity by inactivating otherwise downregulating endogenous IL-10.

Effective adjuvants for the induction of antigen-specific delayed-type hypersensitivity

Vaccines utilizing poorly immunogenic subunit antigens are dependent upon adjuvants to drive the appropriate T cell responses. In an effort to determine the ability of several adjuvants to promote cell-mediated immunity (CMI), we assessed delayed-type hypersensitivity (DTH) in mice inoculated with heat-killed Listeria monocytogenes (HKLM) vaccines. The vaccines were formulated as oil-in-water emulsions containing one or more of the following bacterial-derived immunostimulators: MPL ® immunostimulant, a monophosphoryl lipid A preparation, synthetic trehalose dicorynomycolate (TDCM) and Mycobacterium phlei cell wall skeleton (CWS). Oil-in-water emulsions containing HKLM without adjuvants did not induce DTH responsiveness in mice. The incorporation of TDCM, or MPL ® plus TDCM and/or CWS to the formulation enabled the HKLM vaccine to stimulate CMI characterized by DTH responsiveness. Following antigen challenge the resulting increases in footpad thickness ranged from 15–20% and were comparable to the DTH driven by complete Freund's adjuvant. Adjuvants composed of MPL/TDCM and MPL/TDCM/CWS induced responses equivalent to those measured in mice immunized with viable L. monocytogenes, and the responses remained at these levels for at least 2 months. Furthermore, in vivo of CD4+ T cells, but not CD8+ T cells, abrogated the induction and expression of DTH, indicating that the response is mediated by CD4+ T cells.

Immunoregulation of intraocular tumours.

Intraocular tumours reside within an organ that provides sanctuary from many immunological defence mechanisms. Antigens displayed on many intraocular tumours can elicit an aberrant systemic immune response in which systemic antigen-specific delayed-type hypersensitivity (DTH) is actively downregulated, thus denying the host one potential effector mechanism for controlling its intraocular tumour. Constituents within the aqueous humour inhibit the expression of DTH and natural killer cell effector mechanisms within the eye and thus protect intraocular tumours from immune-mediated rejection. Some experimental intraocular tumours in mice express potent tumour-specific antigens that stimulate the expansion of tumour-specific robust cytotoxic T lymphocyte (CTL) populations which enter the eye and mediate tumour rejection by piecemeal tumour necrosis. However, the presence of tumour-infiltrating lymphocytes (TIL) within an intraocular tumour does not inevitably lead to tumour resolution. In some cases CTL precursors infiltrate the intraocular tumour but fail to differentiate into mature cytolytic effector cells. Although uveal melanomas express melanoma-specific and melanoma-associated antigens that are capable of eliciting both humoral and cellular immunity, formidable barriers prevent the expression of tumour immunity. These barriers include: (a) anterior chamber-associated immune deviation; (b) in situ suppression of DTH effector cells; (c) suppression of natural killer cell activity in oculi; and (d) inactivation of the complement cascade by regulatory proteins expressed on uveal melanoma cells.

Mechanisms of anergy in tuberculosis.

Immunological hyporesponsiveness (anergy) is a concomitant of diseases due to intracellular pathogens and well-demonstrated in tuberculosis. The expression of anergy in tuberculosis encompasses depression of both delayed-type hypersensitivity (DTH), manifest as depressed tuberculin skin test reaction, and in vitro lymphocyte responses to antigens of Mycobacterium tuberculosis. Evidence for immunosuppression, which appears to be mostly limited to mycobacterial antigens, has been documented in different populations with active tuberculosis worldwide, regardless of prior BCG vaccination status. Whether attenuation of the host responses to antigens of M. tuberculosis that stimulate or maintain protective immunity is a factor in the development of tuberculosis or contributes to the chronicity of the disease is not known. However, it appears that anergy in active tuberculosis is mediated largely through monocytes that display signs of activation, possibly by exposure to mycobacterial antigens or cytokines in situ. M. tuberculosis and its constituents are potent in stimulation of monocytes to produce an array of cytokines, notably transforming growth factor β (TGF-(β), which is suppressive to lymphocytes, deactivates macrophage effector functions, and promotes fibrosis (Wahl 1992). Immunosuppressive circuits are activated in situ as newly recruited monocytes encounter M. tuberculosis and its products; at the site of infection these circuits may contribute to immunopathogenesis, while at local sites of exposure to antigens (skin test) they may modulate the expression of DTH.

Enhancement of delayed hypersensitivity inflammatory reactions in guinea pig skin by 12(R)-hydroxy-5,8,14-eicosatrienoic acid.

Delayed-type hypersensitivity (DTH) reactions are initiated by sensitized T cells. Their progression is dependent upon the local release of various autacoids, including cytokines and eicosanoids, by T cells, infiltrating inflammatory cells, and resident tissue cells. 12(R)-hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE], an eicosanoid produced by skin and cornea, possesses potent proinflammatory properties at picomolar concentrations including vasodilation, increase in membrane permeability, neutrophil chemotaxis, and angiogenesis. Because DTH reactions are associated with many of these same phenomena, we examined the effect of 12(R)-HETrE and related 12-hydroxyeicosanoids on the expression of DTH to purified protein derivative of tuberculin in sensitized guinea pigs. In the absence of purified protein derivative of tuberculin, none of the eicosanoids evoked erythema or edema after intradermal injection at doses up to 100 pmol. When injected together with purified protein derivative of tuberculin, 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], but not its enantiomer 12(S)-HETE, significantly inhibited macroscopic expression of delayed reactivity (erythema) only at the highest dose tested, 10 pmol. In contrast, 12(R)-HETrE significantly enhanced expression of DTH at doses between 1 fmol and 1 pmol (50% and 30% increases above control, respectively). Its stereoisomer, 12(S)-HETrE, did not enhance DTH at any tested dose, but was able to block the activity of 12(R)-HETrE when injected simultaneously. Enhancement or inhibition of visible skin responses was not associated with qualitative or quantitative changes in cellular infiltrates at the reaction site. 12(R)-HETrE had no effect on the nonimmunologic inflammatory skin reaction induced by phorbol myristate acetate, suggesting selectivity toward DTH. We conclude that 12(R)-HETrE enhances DTH via a yet to be determined mechanism and that its stereoisomer, 12(S)-HETrE, may be a useful antagonist for studying the inflammatory actions of this eicosanoid.

CD8 cells play a critical role in delayed type hypersensitivity to intact Cryptococcus neoformans.

Although cell-mediated immunity is critical for optimal host defense to C. neoformans, the role of T lymphocyte subsets is complex and poorly understood. CD8 cells are important both for optimal host defense against C. neoformans, and for expression of delayed type hypersensitivity (DTH). Because host defense correlates with the ability to mount a DTH response to C. neoformans, the current studies were performed to determine the mechanism by which CD8 cells participate in DTH. Mice were immunized by the intratracheal route with live C. neoformans, or by the subcutaneous route with heat-killed C. neoformans. Mice were depleted of CD8 cells in vivo by administration of mAb. After challenge with soluble cryptococcal Ag, the DTH response was quantified as footpad swelling. We found that mice depleted of CD8 cells before immunization were unable to express DTH. Mice depleted of CD8 cells after immunization but before challenge also were unable to express DTH. Splenocytes of mice depleted of CD8 cells in vivo, before immunization, failed to transfer DTH to naive, undepleted mice. Immune splenocytes depleted of CD8 cells in vitro also failed to transfer DTH to naive, undepleted mice. These data indicate that CD8 cells were necessary during the challenge and immunizing phases of DTH, and were necessary for expression of DTH. However, CD8 cell depletion did not abrogate DTH in mice immunized with either soluble cryptococcal Ag in complete Freund's adjuvant, or sheep red blood cells, which are mediated by CD4 cells. These data suggest that CD8 cells play a critical role in the cell-mediated immune response to C. neoformans. Based on this information, it may be possible to protect hosts with deficiencies of CD4 cells, such as in AIDS, by designing immunizing strategies for stimulating CD8 cells.

Structural nature and functions of transfer factors.

Transfer factors are molecules that "educate" recipients to express cell-mediated immunity. This effect is antigen-specific. The most consistent effects of transfer factors on the immune system are expression of delayed-type hypersensitivity and production of lymphokines such as macrophage migration inhibitory factor (MIF), which is probably identical to gamma-interferon in response to exposure to antigen. Transfer factors bind to antigens in an immunologically specific manner. This discovery has enabled us to isolate individual transfer factors from mixtures that contain several transfer factors. This reactivity probably explains the specificity of individual transfer factors, and it has provided a method for purification of individual transfer factors to apparent homogeneity. The purified materials are immunologically active and antigen-specific. They have molecular weights of approximately 5,000 Da and appear to be composed entirely of amino acids. Transfer factors appear to offer a novel means of molecular immunotherapy for certain patients with defective cell-mediated immunity.

Biological properties of factors secreted by antigen-reactive suppressor cells in mice infected with Mycobacterium lepraemurium

Antigen-reactive cells were isolated from the spleens of Mycobacterium lepraemurium-infected C57BL/6 mice on petri dishes coated with mycobacterial antigens. When adoptively transferred to syngeneic mice, the mycobacterial antigen-reactive cells were found to depress the induction and expression of the delayed-type hypersensitivity (DTH) reaction to M. lepraemurium antigens. The adoptive transfer of soluble suppressor factors (SF) secreted by these cells inhibited only the expression of DTH. The cells depressing the induction of DTH mainly belonged to the L3T4+ (CD4+) T-lymphocyte subset, whereas those depressing its expression differed from the L3T4+ and Lyt-2+ (CD8+) subsets. Treatment of M. lepraemurium-infected mice with SF reduced their mean survival time and enhanced the multiplication of bacilli at the site of infection and their dissemination to the spleen and liver. In vitro at least, SF appeared to interfere at the level of mycobacterial antigen recognition by T lymphocytes rather than at the levels of antigen processing and presentation by macrophages.

A feverlike response inhibits expression of delayed-type hypersensitivity in rats

To determine the effect of fever on cell-mediated immunity, the preoptic area of male rats was cooled after either a sensitizing or a challenge injection of keyhole-limpet hemocyanin, and the delayed-type hypersensitivity inflammatory response was measured. Cooling the preoptic area for 5 days, starting shortly after the sensitizing injection of antigen, did not speed up the development of the capacity to respond to a challenge injection of antigen nor did it influence the magnitude of the resulting inflammatory response. In sensitized animals, however, cooling the preoptic area for 24 h, starting shortly after the challenge injection of antigen, raised core temperature to an average of 40 degrees C and decreased the magnitude of the inflammatory response by 25%, whereas heating the preoptic area caused a fall in core temperature to an average of 35 degrees C and increased the response by about 25%. There was, in fact, a moderate negative correlation (r = 0.85) between core temperature and the size of the inflammation. Furthermore, the inhibitory effect of cooling the preoptic area could be blocked neither by adrenalectomy nor by diazepam (0.5 mg/kg body wt), a drug that suppresses most endocrine responses to stress. It is therefore suggested that the expression of delayed-type hypersensitivity is inhibited at elevated body temperatures and that fever may thus weaken the defenses of the host against infection.

非定型抗酸菌に対する遅延型アレルギーのサプレッサー細胞についての研究

Mycobacterium intracellulare Mino strain (Mino) grows progressively in the organs of susceptible mice, such as C57BL/6, C57BL/10 or BALB/c. It is very difficult to induce acquired immunity against M. intracellulare in those susceptible mice. C57BL/6 (B6) mice show no or very weak delayed type hypersensitivity (DTH) to partially purified Mino antigens when sensitized with 10(7) living Mino subcutaneously. B6 mice pretreated with cyclophosphamide (CY) showed enhanced DTH to Mino, suggesting that suppressor mechanism exists in this system. Whether or not such a suppressor mechanism exists in the induction phase of DTH (A) and/or in the expression phase of DTH (B) was examined by cell transfer experiments. Recipients for testing (A) were B6 mice receiving CY 2 days before an immunization, and those for (B) were B6 mice receiving both CY and subcutaneous injections of Mino 3 weeks before the cell transfer. B6 mice which were intravenously injected with Mino a week or 6 weeks before preparing spleen cell suspension, were used as the donors. Single cell suspensions of the spleens were plated on tissue culture dishes and non-adherent cells were harvested after incubating for 60 min at 37 degrees C. These cells were used for cell transfer. In the induction phase experiment, recipient mice received the cells before sensitization, while they received the cells after sensitization in the expression phase experiment. DTH were markedly suppressed in the cell transferred groups as compared with the non-transferred groups. This activity disappeared by treatment of the transferred cells with antibody (alpha Thy-1 Ab) and complement.(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell subsets in delayed-type hypersensitivity, protection, and granuloma formation in primary and secondary Listeria infection in mice: superior role of Lyt-2+ cells in acquired immunity.

Immunity to Listeria monocytogenes was studied in mice treated with rat monoclonal antibodies (MAbs) specific for the Thy-1.2, L3T4, and Lyt-2 T-cell markers. Three characteristic T-cell-mediated phenomena were investigated. Delayed-type hypersensitivity (DTH) to listerial antigen was totally abolished in mice treated with anti-Thy-1.2 or anti-L3T4 MAbs, whereas anti-Lyt-2 MAb treatment had no effect, regardless of whether the MAb was given during the induction or the expression of DTH. On the other hand, the elimination of bacteria from the spleens of infected animals was inhibited only by the application of either anti-Thy-1.2 MAb or anti-Lyt-2 MAb. This could be shown most impressively during the secondary infection of immune mice with a normally lethal dose of listeriae. In this situation, treatment with anti-Lyt-2 MAb sufficed to completely abolish immunologic memory, whereas anti-L3T4 MAb had only a marginal effect on antibacterial protection. However, the accelerated development of mononuclear cell foci in the livers of immune mice was inhibited by the application of both anti-L3T4 MAb and anti-Lyt-2 MAb. It is concluded that in murine listeriosis, DTH and acquired immunity to reinfection are dissociable phenomena. Although DTH is a function of L3T4+ T lymphocytes, Lyt-2+ T cells are necessary and sufficient for the expression of acquired resistance to L. monocytogenes. The roles of the different T-cell subsets in granuloma formation warrant further investigation.

Assay for quantitation of mature monocytes in murine haemopoietic tissues

We propose a new assay which allows the extent of production of mature monocytes in murine haemopoietic tissues to be monitored with precision. To develop such a quantitative assay, we chose a T-cell function easily detectable in vivo, namely the ability of T lymphocytes to locally transfer a delayed-type hypersensitivity (DTH) reaction. T cells co-transferred with antigen into footpads of syngeneic mice are mediators of DTH through their ability to recruit phagocytes recently produced in haemopoietic tissues. Therefore, if recipients of DTH-mediating T cells (DTH-T cells) are depleted of phagocytes by lethal irradiation given 36 to 48 h before local transfer of these DTH-T cells mixed with antigen, they no longer respond by a DTH reaction, measured as an increase in footpad thickness, unless they receive i.v. bone marrow cells as a source of recruitable phagocytes. In such conditions, the footpad thickness increase is linearly related to the number of cells injected i.v. Pretreatment of bone marrow cells with the rat IgG2b F4/80 monoclonal antibody abolishes their ability to restore expression of DTH, indicating that monocytes (F4/80 + cells) are the phagocytes to be measured. This assay system thus provides a means of measuring levels of recruitable mature monocytes present in haemopoietic tissues. One illustration of the assay is given by the study of the recovery of recruitable phacogytes in bone marrow following a single treatment with cyclophosphamide.

Ferritin selectively suppresses delayed-type hypersensitivity responses at induction or effector phase

The effect of ferritin on the delayed-type hypersensitivity (DTH) response and Arthus-type reaction as assessed by footpad reaction using methylated human serum albumin, human serum albumin, or sheep red blood cells as antigens was investigated. Intraperitoneally administered ferritin was short acting and suppressed either induction or expression of DTH depending on the time of ferritin injection although it did not inhibit the antibody-mediated inflammatory response, the Arthus reaction. Investigation of ferritin's effect on the primary antibody response revealed that the number of IgG plaque-forming cells (PFC) was moderately decreased but IgM PFC were not. These results indicate that the afferent limb, ferritin selectively suppresses antigen presentation and/or clonal expansion of effector cells of cell-mediated immunity, but not that of the antibody response. Antigen presentation by Ia-positive cells and/or lymphokine-responsive inflammatory mononuclear cells at the efferent limb of DTH is suggested to be affected by ferritin. This conclusion is based upon the observations of successful T DTH effector cell transfer from sensitized but ferritin-treated donors and of successful reversal of ferritin-induced suppression of expression of DTH by supplementing normal bone marrow-derived cells containing Ia-positive ones. Thus our in vivo experimental system might be useful for the differential analysis of immunopathological lesions such as a T-cell-mediated, monocyte-dependent and an antibody-mediated inflammatory lesions.

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. I. Preferential inhibition of the induction of delayed-type hypersensitivity.

Culture supernatants of spleen cells from susceptible CBA mice chronically infected with Trypanosoma cruzi were able to inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens as measured by 24-hr footpad swelling, bone marrow homing, and radioactivity accumulation assays. The suppressive activity, which was also present in the serum of these chronically infected mice, appears to be specific for the induction of DTH and had no effect on the 3-hr immediate-type hypersensitivity. It also failed to modify the expression of DTH in presensitized mice. Furthermore, it did not affect the synthesis in normal recipients of specific antibody or the induction of helper T cells or cytotoxic T cells. It also failed to induce DTH tolerance as recipient mice with markedly reduced DTH were able to develop a normal DTH response after secondary immunization. The suppressive activity was produced by an Ig- macrophage-depleted splenic T cell population, whose capacity to secrete the suppressive substance was completely abrogated by treatment in vitro with anti-L3T4 antibody and complement, but not with anti-Lyt-2 antibody and complement. These results therefore demonstrate that L3T4+ T cells from mice chronically infected with T. cruzi can produce substances which interfere with the induction of DTH. This finding may help to identify the differential antigenic stimulatory requirement for the activation of the various subsets of T cells.

Chagas' disease: Delayed type hypersensitivity to PPD in relation to seroreactivity to Trypanosoma cruzi in Ecuador.

In a population from an area endemic for Chagas' disease in Ecuador, cellmediated immune activity was assessed using delayed type hypersensitivity (DTH) reaction to PPD. Expression of DTH was significantly suppressed in individuals seropositive to Trypanosoma cruzi, and the suppression was markedly greater in the age group 10-39 years old than those over 40 years. The results show that cell-mediated immune response is apparently suppressed in patients with Chagas' disease, and tends to be restored in chronic stage. No defference was noted in the serum concentration of immunoglobulins between patients and controls.

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice.

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.

Delayed-type hypersensitivity and immunity to Salmonella typhimurium.

Studies were carried out to correlate immunity and expression of delayed-type hypersensitivity (DTH) in mice of the C3H lineage immunized with an avirulent strain of Salmonella typhimurium (strain SL3235). This strain belongs to a class of aroA- organisms which are being considered as vaccine strains for humans and veterinary use. In a systematic study, the relationship between the mouse strain and the immunizing dose of strain SL3235 on the development of protective immunity and DTH was examined. It was found that in hypersusceptible C3H/HeJ and C3HeB/FeJ mice, several doses of strain SL3235 afforded protection against intravenous challenge doses as high as 1,300 50% lethal doses. Despite these significant levels of immunity to challenge, mice of these two strains never mounted significant DTH responses following immunization with the doses of strain SL3235 tested, which spanned 3 orders of magnitude. Nonresponsiveness was not due to antigen overload, as all of the mouse strains were comparably colonized with strain SL3235 at the time of DTH elicitation. Further, it was found that the ability of responsive C3H/HeNCrlBR mice to display DTH was dependent on the immunizing dose of strain SL3235 and that a dosage could be found that resulted in increased resistance to challenge in these mice without a concomitant display of DTH. Thus, while both induction of protective immunity and DTH were vaccine dosage dependent in the responsive mouse strain (C3H/HeNCrlBR), DTH was a less sensitive measure of protective immunity than survival. Vaccine dosages ranging over three orders of magnitude failed to yield positive footpads to the Salmonella elicitin in the nonresponsive mice. The data suggest that caution should be observed in interpreting Salmonella DTH tests that are used as screens of immune status to typhoid fever in humans, as the extent of discordance between immunity and DTH in humans is unknown.