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.     

Antigen-specific augmentation of delayed-type hypersensitivity by a humoral factor in the culture supernatant of immune spleen cells

Supernatants were harvested from a 24-hr culture of immune mouse spleen cells and erythrocyte antigens. Delayed footpad reactions to such heterologous erythrocytes were augmented antigen specifically when the supernatants were transferred a few hours before immunization of recipients. The augmentation factor(s) contained in the supernatants may exert its effect on the induction phase of delayed-type hypersensitivity.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. I. Nature of augmentation factor

A humoral factor capable of augmenting antigen-specific DTH has been found in the culture supernatant of immune spleen cells and erythrocyte antigen. In this study, a similar factor was identified in the sera of mice sensitized and elicited with heterologous erythrocytes, and the nature of this factor was investigated. Elicitation with antigen was essentially required for the production of the augmentation factor in sensitized mice. The factor showed antigen specificity and antigen-binding capacity. The activity was not assigned to immunoglobulins, as demonstrated by an absorption test with rabbit anti-mouse immunoglobulin-conjugated Sepharose. The activity was stable to heating at 56 degrees C for 30 min, to changes of pH from 3 to 10, and to treatment with trypsin or neuraminidase. The molecular weight of this factor was about 200,000 to 450,000.     

Augmentation of delayed-type hypersensitivity to serum proteins by vesicular stomatitis virus infection in mice: virus-suppressor cell interactions

The effects of infection with vesicular stomatitis virus (VSV) on delayed-type hypersensitivity (DTH) to heterologous serum proteins were investigated in mice. DTH was induced by a subcutaneous injection of antigen in complete Freund's adjuvant. Infection with VSV at the time of immunization did not affect the level of DTH elicited 3 wk later. Marked augmentation of DTH was observed only when previously immunized mice were infected with VSV simultaneously with restimulation by soluble antigen; either soluble antigen or the virus infection alone was ineffective. The augmentation was specific to the antigen used for the restimulation; in the mouse previously immunized with both bovine serum albumin (BSA) and human alpha-globulin (HGG), DTH to BSA but not to HGG was augmented by injecting soluble BSA and VSV, and vice versa. These results strongly suggest that cells involved in the suppression of DTH manifestation became susceptible to the virus after specific antigenic restimulation and were then eliminated.     

Ultraviolet radiation-induced immunosuppression of delayed-type hypersensitivity in mice

Ultraviolet (UV) radiation present in sunlight plays a critical role in the initiation and promotion of nonmelanoma skin carcinogenesis and immune suppression. The immune suppressive effects of UV have been identified as a risk factor for skin cancer induction. For these reasons, scientists have focused on elucidating the mechanisms of UV-induced immune suppression to better understand the pathogenesis of skin cancer induction. A hallmark of UV-induced immune suppression is the generation of antigen-specific suppressor T cells. These suppressor cells have been shown to suppress antitumor immunity as well as other cell-mediated responses such as delayed-type hypersensitivity (DTH) reactions. Due to the excessive cost and time involved in traditional UV carcinogenic experiments, scientists have opted to use UV-induced suppression of DTH reactions as a surrogate model. DTH has been, and continues to be, a widely used assay system to measure in vivo immune function. Although somewhat unsophisticated by today's standards, this assay has great advantages because it presents a fast, inexpensive, and reliable model system to help dissect the mechanisms involved in UV-induced immune suppression. Furthermore, the murine model of DTH enables scientists to perform additional procedures, such as adoptive transfer studies with suppressor T cells, which are currently unavailable with human subjects.     

Augmentation of delayed-type hypersensitivity by vesicular stomatitis virus infection in mice

Effects of the infection with vesicular stomatitis virus (VSV) on delayed-type hypersensitivity (DTH) to heterologous erythrocytes were investigated in mice. Infection at the time of immunization resulted in production of high levels of DTH that were specific to the antigen used for immunization. The high level of DTH produced in VSV-infected mice could not be attributed to the nonspecific enhancement of the footpad swelling with the infection. Augmentation of DTH was observed in all strains of the mouse (CBA, BALB/c, C3H/He, and C57BL/6) used. The augmenting effect of VSV infection was not as apparent in adult thymectomized mice in which the level of VSV-replicating T cells was reduced. These results strongly suggest that DTH-mediating T cells are resistant to infection by VSV, and also that there are VSV-sensitive cells that may be engaged in the suppression of DTH. It seems improbable, however, that the cells sensitive to VSV infection represent the suppressor cells themselves, since the enhancing effect was not observed in mice in which the suppressor cells were induced by the administration of high doses of the antigen.     

Inhibition of delayed-type hypersensitivity by heparin depleted of anticoagulant activity

The intravenous or intraperitoneal injection of heparin fractions depleted of anticoagulant activity (HFDA) into mice, either at the time of immunization or challenge, inhibited hapten-specific delayed-type hypersensitivity (DTH) reactions. The loss was not due to functional elimination of sensitized lymphocytes, since mice sensitized with the contactant and then treated with HFDA retained their ability to transfer reactivity into normal syngeneic recipients. In contrast, lymphocytes from sensitized mice were unable to produce DTH reactivity in recipient mice pretreated with HFDA. The intravenous injection of HFDA resulted in a rapid, but transient increase in the number of circulating leukocytes. The intravenous injection of HFDA also reduced the footpad swelling that resulted from a local injection of concanavalin A. It is postulated that HFDA exercise their inhibitory effects on the DTH response by interfering with the migration of cells into the challenge site.     

Induction of delayed-type hypersensitivity to measles virus in mice

Intracutaneous injection of inactivated measles virus (MV) into hind footpads of BALB/c mice infected 5 to 11 days previously with MV produces a strong delayed-type hypersensitivity (DTH) response. Pretreatment of mice with cyclophosphamide (CP) results in a significantly stronger response. In CP-pretreated mice, the optimal infecting dose of live MV and the restimulating amount of inactivated MV are approximately 10(7) plaque-forming units and 2 micrograms/mouse, respectively. The optimal time after infection for measuring DTH to MV is 7 days, while the optimal CP-pretreatment concentration is 200 mg/kg. The DTH response generated by MV is specific and not caused by fetal calf serum or Vero cell antigens. MV DTH is transferable to uninfected mice with lymph node cells. Transfer of DTH is sensitive to treatment with anti-Thy 1.2 serum plus complement, indicating the response is T cell dependent. With this sensitive assay for measuring cell-mediated immunity to MV, it will now be possible to analyze T cell cross-reactivity among paramyxoviruses and assess viral cell-mediated immunity in mice infected with neuroadapted MV.     

Genetic restriction of delayed-type hypersensitivity to tuberculin in mice

An adoptive local transfer method has been used to study the immunological features and genetic restriction of cell interaction during the development of the delayed-type hypersensitivity (DTH) to tuberculin in mice. Peritoneal cells from the BCG-infected mice transfer the DTH to intact animals (into hind footpad) in both syngeneic and allogeneic donor-recipient combinations. Nonadherent cells (macrophage-deleted) transfer the reaction in syngeneic but not allogeneic combination. The use of H-2 recombinant mouse strains demonstrated that successful transfer of the DTH requires I-A subregion compatibility. Treatment of CBA cells with anti-Thy-1.2 antiserum abrogates the reaction transfer. These results indicate that antigen presentation to immune T-cells proliferating during DTH to tuberculin is mediated through the molecular products of the I-A subregion.     

Anti-receptor antibody-induced H-Y-specific delayed-type hypersensitivity responses in nonresponder mice

The present study examines an antiserum prepared against antigen-reactive T cells that induces murine H-Y-specific delayed-type hypersensitivity (DTH) responses. This anti-H-Y receptor antibody (ARA) was raised in C57BL/6 male mice against splenic T lymphocytes from H-Y immune syngeneic females. Subcutaneous administration of ARA to cyclophosphamide-pretreated C57BL/6 females is able to induce H-Y-specific delayed-type footpad swelling responses. The DTH inducing capacity in ARA was selectively retained on rabbit anti-mouse immunoglobulin columns and was absorbed completely by H-Y immune lymphoid cells from C57BL/6 females. The induction of H-Y DTH reactivity was due at least in part to the activation of H-Y antigen-specific T lymphocytes that could adoptively transfer DTH-like responses to naive female mice. ARA induces DTH responses in strains with the same lgh regions, including selected strains of H-Y nonresponders. Therefore, MHC-linked lr genes do not appear to be as critical when responses are triggered by ARA instead of by antigen. Possible mechanisms for the induction of immune responses by ARA are discussed.     

Anti-tumor cytostatic mechanism and delayed-type hypersensitivity against a syngeneic murine tumor. Comparison between neonatally thymectomized mice and congenitally athymic nude mice

We studied the anti-tumor mechanism against a syngeneic tumor using a BALB/c-MA tumor system by cytolysis and cytostasis assays in vitro comparing mice neonatally thymectomized at 1 day or 7 days after birth (NTx-1, NTx-7), sham-operated (sham) mice, and congenitally athymic nude BALB/c mice. NTx-1 mice showed more rapid tumor growth and a slightly lower degree of strong cytostatic activity in peritoneal exudate cells (PEC) than NTx-7 or sham mice. Nude mice showed more rapid MA growth than NTx-1 mice and no cytostatic activity in PEC. After immunization with mitomycin C-treated MA (MMC-MA), NTx-1 mice acquired an immunoprophylactic capacity against MA and showed cytostatic activity and delayed footpad reaction (DFR) to MA, however, nude mice showed no acquisition of such an immunity, or cytostatic activity, or DFR to MA. These differences between NTx-1 and nude mice could be well-explained by less capacity of nude mice to produce a macrophage-activating factor, which activates macrophages to exert cytostasis and DFR. However, NTx-1 mice could not reject MA by immunization with MMC-MA in CFA (MMC-MA/CFA), although such immunized sham mice could eliminate MA completely. Both PEC and spleen cells from Sham mice immunized with MMC-MA/CFA showed cytostatic activity, whereas NTx-1 mice showed cytostatic activity of the same level in PEC and less in spleen cells compared to Sham mice. Cytolytic activity was never detected throughout this study in a BALB/c-MA system. These data suggest that cytostasis plays an important role in antitumor immunity against a syngeneic MA tumor and that two types of cytostasis is included from the standpoint of thymus-dependency of ontogenic development, relatively low and high.     

Leukocyte procoagulant activity in man: an in vitro correlate of delayed-type hypersensitivity

Human mononuclear leukocytes generate cell-bound procoagulant activity (LPCA) after incubation with an antigen (mumps or tuberculin) to which the donor was previously sensitized. An inhibitor of coagulation appears to be liberated into the extracellular culture fluid during incubation of leukocytes with the sensitizing antigen. Removal of this activity before measuring LPCA resulted in a reliable test that correlated directly with delayed skin reactivity. The assay was particularly sensitive in that cells from weakly sensitized donors who reacted only to high doses of tuberculin (100 TU) in the delayed skin tests produced detectable LPCA in vitro. By contrast cells from weakly sensitized donors did not react to PPD in the lymphocyte blast transformation test or the direct macrophage migration inhibition factor test. The LPCA assay correlated closely with the blast transformation and MIF tests in which cells were used from more strongly sensitized donors who reacted in skin tests with lower doses of tuberculin (1 or 10 TU). The assays were antigen-specific in that cells from donors sensitive to mumps antigen but not to tuberculin reacted only to mumps antigen in vitro. The assay was extremely reproducible; cells from individual donors reacted to the same extent over a period of 8 mo). We propose that the assay system reported here represents an improved method for the measurement of cell-mediated immunity in vitro because it requires fewer donor cells, is technically simpler, and is more sensitive than previously described methods.     

Suppression of delayed-type hypersensitivity to oxazolone in whole-body-irradiated mice and protection by WR-2721

The effect of whole-body irradiation on cellular immunity, as measured in vivo by delayed-type hypersensitivity (DTH) to oxazolone (4- ethoxymethylene -2-phenyl- oxazol -5-one), was determined in CD2F1 mice. DTH, determined by changes in ear swelling after challenge with oxazolone, was significantly depressed in irradiated mice (500-900 rad of 60Co) in a dose-dependent fashion when animals were irradiated after sensitization and before challenge with oxazolone. Administration of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] 30 min before irradiation (2 days after sensitization) resulted in protection against suppression of DTH, which was dependent on drug and radiation dose. An effective dose of WR-2721 (200 mg/kg body wt) provided an approximate dose-modifying factor of 1.3. The data suggest that WR-2721 interacts with cells involved in that DTH response (lymphocytes and/or macrophages) and that WR-2721 may be useful in protecting against radiation-induced decrements in cell-mediated immunity.     

Nude mice produce a T cell-derived antigen-binding factor that mediates the early component of delayed-type hypersensitivity

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is caused by the sequential action of two different T cells. An early-acting, DTH-initiating T cell produces an Ag-specific T cell factor, that is analogous to IgE antibody and initiates DTH by sensitizing the local tissues for release of the vasoactive amine serotonin. In picryl chloride or oxazolone contact sensitivity, this T cell factor is Ag-specific, but MHC unrestricted. We, therefore, hypothesized that DTH-initiating T cells are primitive T cells with Ag receptors that can bind Ag without MHC restriction. In order to characterize the origin of this DTH-initiating T cell and the conditions that are necessary for its development, we contact-sensitized various strains of immunodeficient mice. Surprisingly, we found that the early phase of DTH was present in athymic nude mice. In contrast, the early component of DTH was absent in mice with severe combined immunodeficiency. These mice lack T and B cells, but have NK cells. These findings suggested that the early component of DTH was not caused by NK cells, and was caused by cells belonging to a lineage from a rearranging gene family. The early component of DTH in nude mice was Ag specific, was caused by MHC unrestricted Thy-1+ T cells, and was mediated by Ag-binding, Ag-specific T cell factors. We found that DTH-initiating, T cell-derived, Ag-binding molecules from nude mice and normal CBA/J mice had the same functional properties. The early component of DTH was elicited in two different systems (contact sensitivity and SRBC-specific DTH) in two strains of nude mice (BALB/c athymic nudes and CByB6F1/J-nu) from two different suppliers, but not in BALB/c and athymic nudes from a third supplier. From these findings we concluded that DTH-initiating T cells, which produce IgE-like Ag-specific T cell factors, are present in some strains of athymic nude mice and thus are relatively thymic independent T cells.     

Compromised humoral and delayed-type hypersensitivity responses in IL-23-deficient mice

The heterodimeric cytokine IL-23 consists of a private cytokine-like p19 subunit and a cytokine receptor-like subunit, p40, which is shared with IL-12. Previously reported IL-12p40-deficient mice have profound immune defects resulting from combined deficiency in both IL-12 and IL-23. To address the effects of specific IL-23 deficiency, we generated mice lacking p19 by gene targeting. These mice display no overt abnormalities but mount severely compromised T-dependent humoral immune responses. IL-23p19(-/-) mice produce strongly reduced levels of Ag-specific Igs of all isotypes, but mount normal T-independent B cell responses. In addition, delayed type hypersensitivity responses are strongly impaired in the absence of IL-23, indicating a defect at the level of memory T cells. T cells stimulated with IL-23-deficient APCs secrete significantly reduced amounts of the proinflammatory cytokine IL-17, and IL-23-deficient mice phenotypically resemble IL-17-deficient animals. Thus, IL-23 plays a critical role in T cell-dependent immune responses, and our data provide further support for the existence of an IL-23/IL-17 axis of communication between the adaptive and innate parts of the immune system.     

Regulatory mechanism of delayed-type hypersensitivity in mice. II. Effect of suppressor cells on the development of memory cells for delayed-type hypersensitivity

Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr ⁵¹Cr-release cytotoxicity assay and an overnight ¹²⁵IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.