Dissociated development of T cells mediating delayed-type hypersensitivity and protective T cells against Listeria monocytogenes and their functional difference in lymphokine production.

CD4+ T cells mediating both delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR) were generated in mice after immunization with viable Listeria monocytogenes. In contrast, CD4+ T cells from mice immunized with killed L. monocytogenes in complete Freund's adjuvant were capable of mediating only DTH but not ACR. To determine the functional difference between T cells mediating DTH and T cells mediating ACR, we examined two different populations of T cells for profiles of lymphokine production after stimulation with a specific antigen in vitro. The production of interleukin-2 (IL-2) and IL-3 but not IL-4 was observed in both T cells mediating only DTH and those mediating DTH and ACR. In this respect, both types of T cells could be categorized into the TH1 population, and they produced macrophage chemotactic factor equally well. However, the production of gamma interferon (IFN-gamma) was observed only in T cells capable of mediating both DTH and ACR. This result was confirmed not only by an enzyme immunoassay specific for murine IFN-gamma but also by Northern (RNA) analysis for the detection of IFN-gamma mRNA. These results suggested that the TH1 population may be subdivided further into two distinct subsets and that the ineffectiveness of the killed bacterial vaccine may be partly explained by the dissociated development of T cell function.     

Induction of nonspecific acquired resistance and delayed-type hypersensitivity, but not specific acquired resistance in mice inoculated with killed mycobacterial vaccines.

A number of nonliving mycobacterial preparations were tested in vivo for their capacity to generate various relevant parameters of cellular immunity. All preparations tested had some detectable activity in raising resistance to challenge with Mycobacterium tuberculosis or with Listeria monocytogenes and in conferring the ability to mount a delayed-type hypersensitivity response to tuberculin. This report presents the first evidence, however, that none of these preparations were able to generate protective T cells capable of adoptive immunization against virulent tuberculosis. These data are discussed in terms of the use of these preparations in generating M. tuberculosis-reactive T-cell lines and the application of these lines in the continuing search for an improved vaccine against tuberculosis.     

Generation of Listeria monocytogenes-specific T cells mediating delayed footpad reaction and protection in neonatally thymectomized mice but not in nude mice

Neonatally thymectomized (NTx) mice, sham-operated control mice and congenitally athymic nude mice were immunized with viable Listeria monocytogenes and their spleen cells examined for the capacity to transfer both delayed footbad reaction and protection against challenge at the site of local transfer. Cells from immune NTx mice conferred significant degrees of delayed footpad reaction and protection comparable to sham mice, while cells from immune nude (nu/nu) mice did not. This abilty was completely eliminated by the treatment of cells with anti-Thy1, anti-Lytl or anti-L3T4 antibody plus complement but not with anti-Lyt2 antibody plus complement. These results indicated that NTx mice can normally mount the immunity to L. monocytogenes by generating Lyt1⁺2^–, L3T4⁺ T cells. Immune competence of NTx mice and thymus dependency of various immune responses are discussed.     

Expression of systemic protection and delayed-type hypersensitivity to Listeria monocytogenes is mediated by different T-cell subsets.

The relationship between acquired cellular resistance and delayed-type hypersensitivity (DTH) during the immune response to Listeria monocytogenes was investigated. Treatment of concanavalin A-stimulated Listeria-immune spleen cells with anti-CD8 antibody plus complement abrogated the adoptive transfer of systemic antilisterial immunity but had no effect on the transfer of DTH. In contrast, in vitro depletion of the CD4+ T-cell subset eliminated the ability of culture-activated cells to transfer DTH reactivity but did not interfere with the adoptive transfer of protection. In vivo, the infusion of anti-CD8 antibody inhibited the expression of both actively and adoptively transferred protection but did not influence the development of DTH skin test reactivity to L. monocytogenes antigens. In vivo depletion of the CD4+ T-cell subset eradicated the DTH response, with only minor influence of the protective anti-Listeria response. The apparent functional dissociation of the CD4+ (DTH) and CD8+ (protection) T-cell populations was further emphasized by our findings that the adoptive transfer of protection was dependent on a cyclophosphamide-sensitive cell population, whereas DTH reactivity was mediated by a cyclophosphamide-resistant population.     

Killed Listeria-induced suppressor T cells involved in suppression of delayed-type hypersensitivity and protection against Listeria infection.

Pretreatment of mice by intravenous injection with killed Listeria provided neither delayed-type hypersensitivity to Listeria protoplasm nor protection against Listeria infection. Assuming that this suppression is due to suppressor cells, we attempted to clarify their induction and characterization. Pretreatment with killed BCG instead of killed Listeria suppressed the induction of DTH and protection in subsequent Listeria-immunized mice. Conversely, pretreatment with killed Listeria suppressed subsequent induction of DTH to PPD or protection from tuberculosis. Thus, these suppressions were induced antigen nonspecifically. Transfer of splenic non-adherent cells from killed Listeria-injected mice which had been treated with anti-BA theta serum plus complement, or had been passed through Sephadex G-10 columns, resulted in both afferent and efferent DTH suppression, suggesting that the DTH suppression is closely associated with suppressor T cells. Moreover, the splenic nonadherent cells from killed Listeria-injected mice showed suppression in vitro of listericidal activity of PEC from Listeria-immune mice in the presence of Listeria protoplasm.     

Decreased delayed-type hypersensitivity and increased protection to Listeria monocytogenes seen in mice infected with mucoid and nonmucoid Pseudomonas aeruginosa.

Infection of mice with Pseudomonas aeruginosa, washed and unwashed, mucoid and nonmucoid, altered subsequent immunity to Listeria monocytogenes. Mice were protected against lethal doses of L. monocytogenes yet exhibited decreased delayed-type hypersensitivity footpad swelling to sublethal doses. The mucoid coating of mucoid P. aeruginosa, an important pathogen in chronic bronchopulmonary disorders, imparted no additional immunomodulating capabilities to P. aeruginosa.     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.     

Delayed-type hypersensitivity and acquired cellular resistance in mice immunized with killed Listeria monocytogenes and adjuvants.

Delayed-type hypersensitivity (DH) and acquired cellular resistance (ARC) to Listeria monocytogenes in mice was studied following immunization with killed bacteria in combination with Freund's complete adjuvant or the adjuvant dimethyldioctadecylammonium bromide (DDA). Intracutaneous or intraperitoneal injections of killed listeria mixed with Freund's complete adjuvant did neither result in DH nor in ACR. Intracutaneous injections of killed listeria and DDA resulted in an antigen-dose dependent DH but not in ACR. Intraperitoneal injections of listeria and DDA, however, induced ACR but no DH. Optimal conditions for the induction of ACR were simultaneous intraperitoneal injection of 15 mg DDA/kg body weight and 10(7) or 10(8) listeria. The optimal interval between immunization and challenge was 7 days. No protection was found against challenge with a lethal dose of Salmonella enteritidis, suggesting that the protection is specific. Intraperitoneal injection of mice with DDA resulted in inhibition of phagosome-lysosome fusion in macrophages harvested 24 h later. Interference with macrophage activity is discussed as one of the possible mechanisms for the adjuvant effect of DDA.     

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.     

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.     

Effective protection against Listeria monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4+ and Lyt 2+ T cells.

Selected L3T4- and Lyt 2- T-cell subpopulations from Listeria monocytogenes-infected mice were transferred into syngenic recipients, and their capacity to adoptively mediate protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens was determined. Both functions were markedly reduced by pretreatment of cells with either anti-L3T4 or anti-Lyt 2.2 antibodies plus complement, but they could be restored by admixture of the two selected T-cell subsets. Thus, after systemic cell transfer effective protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4+ and Lyt 2+ T cells.     

Appearance of delayed-type hypersensitivity effector cells in murine gut mucosa.

Feeding of a protein antigen, human gamma globulin (HGG), to BALB/c mice prior to parenteral immunization resulted in the abrogation of a delayed-type hypersensitivity (DTH) response to challenge with that antigen. Unlike parenterally immunized mice, HGG-fed mice were unable to transfer DTH to naive syngeneic recipients using peripheral lymph node lymphocytes. Co-transfer experiments ruled out the possibility of a suppressor cell in the orally immunized mice operating on DTH effector cells. Intra-epithelial lymphocytes (IELs) from mice immunized either orally or parenterally were able to transfer a DTH reaction to unimmunized recipients, while mesenteric lymph node lymphocytes from orally, but not parenterally, immunized donors were capable of transferring DTH. The implications of these results for investigations of gastrointestinal disorders with a suspected immunological aetiology are discussed.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. II. Inflammatory helper T cells and effector T cells in mice with delayed-type hypersensitivity and in suppressed mice.

Peritoneal exudate cells were induced in mice 4 days after immunization with SRBC. A low dose of SRBC (10(6) i.v.) caused T lymphocytes to appear in inflammatory exudates. These cells, not only transferred DTH reactions, but also functioned as helper T cells in antibody production after transfer to syngeneic nu/nu recipient mice. After a high dose of SRBC (10(9) i.v.), very few helper T cells and no DTH transferring T cells were found in inflammatory exudates, although they were present in the spleen. It is postulated that T cells mediating DTH reactions and helper T cells behave similarly as far as those dose dependency of appearance in inflammatory exudates is concerned. A high dose of sensitizing antigen causes retention of helper and effector T cells in the spleen, in this way favouring antibody formation; low doses of antigen allow them to leave the spleen, thus favouring mediation of DTH reactions in the periphery.     

Suppression of the delayed-type hypersensitivity and cell-mediated immune responses to Listeria monocytogenes induced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa-mediated suppression of the immune response to Listeria monocytogenes was investigated in mice. Because delayed-type hypersensitivity (DTH) footpad swelling to L. monocytogenes was suppressed equally in lipopolysaccharide-responsive and -hyporesponsive mouse strains, the lipopolysaccharide component of P. aeruginosa could not have been the suppressive agent. Mucoid P. aeruginosa cells were no more suppressive than their nonmucoid revertants; therefore, mucoid coating was not an additional immunosuppressive element. Interleukin-1 and macrophage inhibitory factor production to L. monocytogenes and clearance of L. monocytogenes from mouse spleens were all decreased by prior Pseudomonas infection, indicating that cell-mediated immunity, as well as DTH, was decreased to a sublethal Listeria dose. The timing of Pseudomonas exposure relative to Listeria sensitization was varied. P. aeruginosa injected 24 or 6 h before or at the same time as L. monocytogenes depressed DTH to Listeria challenge 7 days later. Animals treated in this way could not respond to reinfection with L. monocytogenes at 13 days. P. aeruginosa administered to L. monocytogenes-sensitized mice at the time of footpad challenge was suppressive, but these mice responded normally upon reinfection. It appears that P. aeruginosa induced two types of suppression to L. monocytogenes: a transient suppression, affecting DTH challenge but not resensitization, and a longer lasting suppression that did not permit mice exposed to P. aeruginosa at the time of Listeria sensitization to respond to subsequent Listeria exposure.     

Induction and characterization of delayed-type hypersensitivity to influenza virus in mice.

Mice infected with an aerosol of influenza virus or immunized with purified UV- inactivated whole virus or viral subunits developed delayed-type hypersensitivity (DTH) to influenza virus. The level of DTH was greatly enhanced when mice were injected intraperitoneally 2 days prior to immunization with 200 mg/kg of cyclophosphamide. The reaction peaked at 24 h after elicitation, had the classical DTH histology, and was transferable by immune cells but not by immune serum. DTH induced in cyclophosphamide-pretreated mice persisted for at least 40 days after sensitization. DTH induced by deoxycholate-treated subviral particles (DC particles) or the matrix protein of the influenza virus was type-specific, i.e. DTH induced by DC particles or matrix protein of type A virus cross-reacted only with type A virus and not with type B virus. DTH induced by purified hemagglutinin (HA) subunits, on the other hand, showed subtype specificty. Thus, DTH induced by HA of X 31 virus (H3) did not cross-react with PR 8 virus (H 0). However, DTH induced by HA of one subtype cross-reacted significantly with the variants of the same subtype. Thus, it appears that the effector T cells of DTH do not discriminate between the antigenic variants of influenza virus that are distinguishable by serology. DTH induced by purified, UV- inactivated whole virus showed extensive cross-reactivity. This nonspecific reaction was shown to be due to host-derived material, the lipid bilayer. - The theoretical and practical implications of these findings are discussed.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice

We produced transgenic mice expressing Tcell receptor-αβ chain genes, derived from the chicken ovalbumin (OVA)-specific I-A^d-restricted CD4⁺CD8^? T helper cell clone 7–3–7. In transgenic mice with H-2^d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-γ, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α and MIP-1β, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.     

Specific suppressor T cells for delayed-type hypersensitivity in susceptible mice immunized against cutaneous leishmaniasis.

BALB/c mice injected intravenously with 10(6) or higher doses of formaldehyde-fixed promastigotes (ffp) of Leishmania major developed significantly lower levels of delayed-type hypersensitivity (DTH) compared with uninjected control mice when they were subsequently immunized intradermally with ffp. The suppression of DTH was antigen specific and was also inducible with lethally irradiated promastigotes or soluble parasite antigens. The suppressive effect was adoptively transferable with splenic T cells which express the Lyt-1+2+ and L3T4+ phenotypes. These specific suppressor T cells were active against both the inductive and expressive phases of DTH. They were sensitive to 200 rads of gamma-irradiation in vitro and appeared to manifest the suppressive activity via soluble factors. In spite of this profound suppression of DTH, BALB/c mice injected intravenously with 4 X 10(7) ffp were substantially protected against a challenge infection with L. major promastigotes. The possible relationship between the suppressor T cells for DTH and prophylactic immunization against fatal cutaneous leishmanial infection in susceptible BALB/c mice is discussed.     

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.