In vivo cyclophosphamide-mediated augmentation and aqueous HGG-induced suppression of murine delayed hypersensitivity to HGG are reflected by in vitro HGG-stimulated proliferation of lymph node cells from equivalent mice

The question addressed in this report was whether immunological processes which culminate in in vivo expression and augmentation as well as suppression of delayed effector cell activity are mirrored by events that can be quantified in vitro. For this purpose the previously characterized murine model of delayed hypersensitivity (DHS), which employs SJL/J mice immunized with aggregated human γ-globulin (AHGG) in complete Freund's adjuvant (CFA) was employed. The results indicated that lymph node cells (LNC) from cyclophosphamide (CY)-pretreated, AHGG-CFA immunized mice expressed increased proliferation in the presence of HGG and concanavalin A (Con A) but decreased LPS responsiveness compared with LNC from equivalently immunized but non-CY-treated animals. It was also found that LNC from CY-treated, AHGG-CFA immunized mice that were pretreated with aqueous HGG (aqHGG), but not aqueous bovine serum albumin (aqBSA), evidenced a markedly decreased capacity to proliferate in the presence of HGG compared with LNC from equivalent animals that were not pretreated with aqHGG. This suppressive effect was not attributable to antibody production. These findings support the conclusion that in vitro quantitation of antigen-induced proliferation by LNC from HGG-DHS mice appears to correlate with modulatory effects which are observed in in vivo expression of DHS responses.     

Editorial comment

A single injection of Cyclophosphamide (CY) in a dose of 300 mg of CY/kg of mice resulted in enhanced delayed hypersensitivity (DH) when administered between at least 7 days prior to and 15 days after intracutaneous (ic) immunization of sheep red blood cells in Freund's complete adjuvant. The maximal enhancement occurred when CY was applied 8 hr before the antigen. Using the latter interval, the effect of varying the dose of CY before ic or intraperitoneal (ip) injections of antigen was studied. Combined with ic immunization, increasing doses of CY resulted in increasing DH. The ip route of immunization needed CY in amounts of at least 100 mg/kg to augment DH to a detectable level. The enhancing effect of lower doses of CY was more pronounced when the interval between immunizing and eliciting injections was reduced. Administration of 300 or 200 mg of CY/kg before ip immunization suppressed the antibody formation, when measured S and 7 days later. A dose of 100 mg of CY/kg caused a suppression of antibody formation on Day 5, but an enhancement on Day 7. With this dose, a maximal enhancement of DH was found on both days. The results suggest that CY interferes with more than one regulatory mechanism of the immune response.     

Hymenolepis nana: adoptive transfer of protective immunity and delayed type hypersensitivity response with mesenteric lymph node cells in mice

A marked degree of footpad swelling was observed in BALB/c mice infected with Hymenolepis nana eggs, when soluble egg antigen was injected into their footpads 4 to 21 days after the egg infection, indicating delayed type hypersensitivity responses in infected mice. Adoptive transfer with mesenteric lymph node cells from donor mice (BALB/c strain; +/+) infected with eggs 4 days before cell collection could confer this hypersensitivity to recipient nude mice (BALB/c strain; nu/nu). These mesenteric lymph node cells were then divided into two fractions, blast-enriched and blast-depleted cells, by density gradient centrifugation with Percoll. The recipients intravenously injected with the blast-depleted cell fraction showed a marked increase in footpad thickness, whereas the intravenous transfer of the blast-enriched cell fraction resulted in an insignificant increase in footpad thickness. The transfer of the blast-enriched cell fraction, but not of the blast-depleted cell fraction, conferred a strong adoptive immunity on syngeneic recipient nude mice, when the immunity transferred was assessed by examining cysticercoids developed in the intestinal villi on Day 4 of challenge infection. The lack of delayed type hypersensitivity response in mice that received the blast-enriched cell population was not due to a lack of the capacity of the cells to induce the response, because the cells were capable of inducing a significant increase in thickness of footpads of normal mice when these cells were locally injected into the footpad together with soluble egg antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of adaptive immunity by antigen-carrying biodegradable nanoparticles

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying γ-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8⁺ T cells. Unlike complete Freund’s adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, γ-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice

The serum from C3H/He mice immunized with chicken erythrocytes (CRBC) in complete Freund's adjuvant contained a factor able to augment delayed-type hypersensitivity (DTH) antigen specifically, when transferred into naive syngeneic recipient mice before their sensitization with CRBC. This activity in immune serum appeared on Day 4 and reached a peak on Day 8 after immunization, and was enhanced when donor mice were treated with cyclophosphamide (CY) 2 days before immunization. The ability of recipient mice to respond to this factor was enhanced by CY treatment of these mice 4 days before being transferred. This factor could be discriminated from conventional antibodies. Production of this factor in the serum donor and the expression of its activity in transferred recipient was mediated by a T-cell subset which showed a low degree of thymus dependency in ontogenic development.     

Evanescent delayed-type hypersensitivity: mediation by effector cells with a short life span.

Four days after i.v. immunization of mice with optimal low doses of heterologous erythrocytes (2 x 10(5) RBC), strong delayed-type hypersensitivity (DTH) responses can be elicited in the footpad. At later intervals after immunization, DTH responsiveness is progressively diminished and replaced by 4-hr antibody-dependent reactions. These evanescent T cell-mediated DTH responses, which are progressively replaced by antibody-dependent reactions, resemble Jones-Mote type delayed hypersensitivity responses of humans and guinea pigs. Since higher doses of immunizing antigen activate suppressor mechanisms that inhibit DTH responses, we examined the possibility that the evanescence of DTH in mice immunized with an optimal low dose of antigen might also be due to suppression. Using techniques that could clearly demonstrate the suppression produced by high antigen doses, we failed to find evidence for either humoral or cellular suppression in optimally immunized mice with declining of DTH responses. Thus, it appears that the evanescence of produced by optimal low dose immunization with RBC may be due to an intrinsic short life span of the effector cells rather than to the activation of an identifiable shut-off mechanism.     

Suppression of delayed-type hypersensitivity to syngeneic testicular cells in mice: involvement of suppressor T cells which act at the induction stage

Suppressor cells for delayed footpad reaction (DFR) against syngeneic testicular cells (TC) were detected in the spleen cells of donor mice immunized intravenously (iv) with viable syngeneic TC. Cyclophosphamide (CY)-pretreated recipients were given spleen cells from donors iv, immunized subcutaneously (sc) with syngeneic TC, and the footpad reaction at 24 hr was elicited with syngeneic TC 6 days after immunization. DFR in the recipients was suppressed by the transfer of spleen suppressor cells. The suppressor cells induced were Thy-1+, CY-sensitive, adult thymectomy (ATx)-resistant and act only at the induction stage. They directly suppress the generation of effector T cells for delayed-type hypersensitivity (DTH). When mice pretreated with CY were actively immunized with syngeneic TC, DFR could be provoked to a measurable level only when they were immunized sc. However, peritoneal exudate cells of those tolerant mice immunized sc without CY pretreatment or immunized iv with CY pretreatment also passively transferred DFR locally, suggesting the existence of effector T cells for DTH even in tolerant mice.     

Mechanisms of polyclonal tolerance induced by lipopolysaccharide and cyclophosphamide

The treatment of recipient mice with LPS from S. marcescens followed by the injection of CY 48 h later inhibited a subsequent antibody production against unrelated antigen (SRBC) and polyclonal mitogen (LPS from Br. abortus). Such a reactivity persisted for 2-3 weeks after treatment. It was shown that the number of Ig+ cells in the spleens of treated mice was decreased, while the population of spleen Thy-1.2+ cells remained unaltered. Cell-cooperative test revealed that the function of B cells, but not T cells, was inhibited by the treatment. There were no changes in DTH response to SRBC. Thus, a subsequent treatment of mice with LPS and CY led to B-cell deficiency. The nature of this phenomenon is presumably the same as the nature of CY-induced antigen-specific immunological tolerance.     

Immunologic effects of neonatal infection with mouse thymic virus.

Mouse thymic virus is a herpesvirus that causes extensive thymic necrosis when given to newborn mice. During the time of acute infection spleen cells have markedly diminished reactivity to T cell phytomitogens and to allogeneic cells and are incapable of effecting a primary in vitro response to a "T-dependent" antigen; responses to B cell mitogens and to a T-independent antigen are unimpaired. Spleens from acutely infected mice have low theta antigen normal numbers of immunoglobulin-bearing cells. Surprisingly, despite widespread necrosis and cellular depletion, thymic cell reactivity to mitogens is unimpaired. However, the ability to thymocytes to proliferate and to generate cytotoxic killer cells in response to allogeneic cells is diminished.     

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. I. Quantitation of suppressed antigen-specific T cell responses as a consequence of prior exposure to antigen in incomplete Freund's adjuvant

We have asked whether a correlation exists between T cell proliferation and the in vivo suppression of delayed type hypersensitivity observed after administration of antigen in incomplete Freund's adjuvant before exposure to antigen in complete Freund's adjuvant. We find that in vivo suppression is indeed paralleled by diminished in vitro responsiveness to the immunogen. Suppression of T cell proliferation is antigen-specific, dependent upon prior immunization of antigen in IFA, and can be transferred adaptively into unprimed but not primed animals by lymphoid cells from actively suppressed syngeneic donors.     

Effect of polysaccharides from Ganoderma applanatum on immune responses. I. Enhancing effect on the induction of delayed hypersensitivity in mice.

Prior intraperitoneal (i.p.) or oral administration of the polysaccharide preparation from a kind of mushroom, Ganoderma applanatum (Pers.) Pat. of Basidiomycetes, exerted an enhancing effect on the induction of delayed hypersensitivity (DH) to protein antigen as measured by the footpad reaction (FPR), and expanded the size of T cell memory for the IgG antibody response. One of the active principles was partially purified and found to be associated with a polysaccharide-rich fraction. The induction of DH was enhanced by treatment with an appropriate dose of the mushroom extract, whereas increasing the dose resulted in almost complete loss of the enhancing activity. The mechanism for the enhancing effect of the mushroom extract on the induction of DH was explored by the adoptive cell transfer technique. Although an i.p. injection of methylated bacterial α-amylase (M-BαA) in incomplete Freund's adjuvant (IFA) has been found to generate in the spleen the antigen-specific suppressor T cells capable of inhibiting the induction of DH 5 days after immunization, the same treatment of mice given prior injections of the mushroom extract did not raise the suppressor cell activity, but transfer of these spleen cells (6 × 10⁷) into syngeneic recipient mice which had been primed with a subcutaneous (s.c.) injection of M-BαA in complete Freund's adjuvant (CFA) resulted in substantial amplification of the expression of DH. The absence of effector T cells for DH in the transferred spleen cells was confirmed by the failure to transfer DH into cyclophosphamide (CY)-treated mice with the amplifying cells. The amplifying activity was antigen-nonspecific and mediated by cells sensitive to treatment with anti-θ antiserum plus complement. Therefore, the nonspecific enhancing effect of the mushroom extract could not be explained by the possibility that pretreatment with the extract eliminated the antigen-specific suppressor T cells. Other adoptive cell transfer experiments revealed that nylon wool-passed cells from mice unprimed but treated with the mushroom extract were able to exert an enhancing activity on the expression of effector T cells in DH. The results indicate that the treatment with an appropriate dose of the extract enhances the induction of DH by activation of the nonspecific amplifier T cells.     

Immune amplification of murine CD8 suppressor T cells induced via an immune-privileged site: quantifying suppressor T cells functionally

Background

CD8⁺ 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.

Methods

Suppressor 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.

Results

Suppression 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.

Conclusions

The 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.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. IV. Fractionation and antigenic properties of a soluble adult worm immunoprophylactic activity

An aqueous buffer-soluble, nonparticulate fraction of adult Schistosoma mansoni worms (SWAP) was separated by gel filtration on Ultragel AcA-34, and portions of the eluate were tested for their capacity to induce protective immunity against cercarial challenge when administered intradermally to mice in combination with the adjuvant BCG. All of the immunogenic activity was found in a single peak of protein excluded in the void volume of the column. This same fraction was determined by SDS-PAGE and Western immunoblotting to be unique in that it contained a component of Mr (X 10(-3) 97 (97,000) recognized monospecifically by antibodies from mice vaccinated with unseparated SWAP plus BCG. Similarly, the protective fraction was unique in possessing the capacity to elicit 24 hr delayed footpad swelling responses, as well as lymphokine production, in SWAP-BCG-immunized mice. These results suggest that the immunogenic activity of SWAP resides in a restricted population of molecules, and possibly in the 97,000 antigen detected with antibodies from vaccinated animals. Because both the protective capacity of unfractionated SWAP and the serologic reactivity of the 97,000 antigen are sensitive to digestion with protease, it is likely that the immunologic activity of these molecules is dependent on peptide-bonded structural elements.     

Delayed-Type Hypersensitivity (DTH) Response to Dengue Virus in Mice: Effect of Route of Sensitization and Splenectomy

This study was designed to determine the role of the sensitization route and the spleen in the development of delayed-type hypersensitivity (DTH) to dengue virus in mice. DTH was measured by footpad swelling response. Strong but transient DTH was produced in cyclophosphamide (CY) pretreated mice sensitized subcutaneously (s.c.) or intravenously (i.v.) with dengue virus type 4. Subcutaneous inoculation of virus in incomplete Freund's adjuvant (IFA) further enhanced the DTH elicited. The time course of DTH generated by s.c. and i.v. sensitization were similar with the peak reactivity seen on day 6 after sensitization. Poor DTH was observed in mice given an i.p. inoculation even when CY and/or IFA were used. Intracerebral (i.c.) inoculation also sensitized mice poorly. Splenectomized mice showed enhanced DTH response when compared to intact mice. In contrast to intact mice, pretreatment of splenectomized mice with CY did not alter the DTH level. Splenectomized mice inoculated s.c. with virus in IFA showed poorer DTH than mice sensitized with virus alone.     

Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

The involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

Immune sensitization to syngeneic organ-specific intestinal antigens in the Lewis rat

In human chronic inflammatory bowel disease involving mucosal epithelium, sera and lamina propria mononuclear cells are reactive with cell surface components isolated from gut epithelial cells. To define a model system in which the disease-inducing potential of such immune factors could be rigorously evaluated, we sought to immunologically sensitize inbred murine strains to syngeneic colonic epithelial cell-associated components (ECAC-C), to define precise in vivo and in vitro conditions to optimize ECAC-C reactivity, and to initially explore whether such cells could elicit tissue injury in epithelium after adoptive transfer to naive animals. Following footpad immunization, Day 42 lymph node cells but not splenocytes were reactive with syngeneic ECAC-C, as shown by a linear increase in [3H]thymidine incorporation over a wide range of antigen concentration (0.5 to 100 micrograms/ml). A subsequent 48-hr exposure to ECAC-C and/or interleukin 2 resulted in a more restricted responsiveness, proliferation occurring only in the presence of ECAC-C or mitogen and not to a coimmunogen (PPD). Further evidence that lymph node cells from ECAC-C/CFA immunized animals were indeed sensitized to syngeneic ECAC-C included ability of donor animals to mount highly significant earlobe DTH responses to ECAC-C, indicating the presence of antigen-specific T-DTH cells, and the failure of polymyxin B, in doses sufficient to inhibit LPS-induced mitogenesis, to reduce lymph node cell responsiveness to ECAC-C, known to be contaminated with LPS. ECAC-C-specific circulating antibody and T-cytotoxic cells were not detected. Adoptive transfer of Day 42 lymph node cells, sensitized in vivo and conditioned in vitro, was not associated with tissue injury in syngeneic recipients in preliminary experiments. This model system, with antigen-specific cells analogous to those present in diseased mucosa of human chronic inflammatory bowel disease, may be an important means to determine the pathophysiologic significance of anti-epithelial cell immune responses in these disorders.     

The enhancement of humoral and cellular immune responses by dimethyldioctadecylammonium bromide

Dimethyldioctadecylammonium bromide (DDA) produced marked enhancement of both cellular and humoral immune responses to SRBC when administered to mice intraperitoneally, or of cellular immunity when given subcutaneously. Stimulated cellular responses were seen as increased footpad swelling as a measure of delayed hypersensitivity and increased antigen-induced blastogenesis. Elevation of humoral response was reflected in increased numbers of splenic plaque-forming cells (PFC) and in circulating anti-SRBC antibody. Adjuvancy did not depend on addition of the lipid of DDA to antigen, as both humoral and cellular responses were enhanced whether DDA and SRBC were admixed or injected separately 4 hr apart intraperitoneally. DDA also enhanced the PFC response to the T-cell independent antigen TNP-LPS. The DDA effects are accompanied by macrophage activation, which may mediate at least in part the observed responses. DDA-activated macrophages exhibit fast spreading, are highly phagocytic, and elaborate significantly greater amounts of thymocyte mitogenic factor(s) than do normal resident peritoneal macrophages. This activation may effect the stimulation of antigen-specific primary lymphocyte responses by adjuvant and expansion of memory-cell populations which lead to the observed enhancement of secondary responses.