Cell-mediated and humoral immune responses in mice. III. Dynamic balance between delayed-type hypersensitivity and antibody response.

Delayed-type hypersensitivity in mice to bovine serum albumin (BSA) which would be induced by a s.c. injection of BSA in Freund's complete adjuvant was interrupted by an i.v. injection of alum-precipitated BSA plus bacterial endotoxin (AP-BSA plus ET). In contrast, s.c. injection of BSA restrained the antibody response in the spleen of the mice receiving AP-BSA plus ET. Subcutaneous injection of either the adjuvant alone or an unrelated antigen in the adjuvant did not affect the anti-BSA antibody response in the spleen. The antibody response to other antigens in the spleen was not affected by the s.c. injection of BSA. The reduction of the antibody response in the spleen does not seem to be attributable to suppressor cells, since the suppressor cell activity against the antibody response of normal mice and that of irradiated recipients of primed spleen cells was not observed in the spleen cells of mice given s.c. immunization. The results indicated that the reduced antibody response in the spleen may be caused by the migration of some part of the antigen reactive cells or their precursors from the spleen to the s.c. region.     

Cell-mediated and humoral immune responses in mice. I. Necessary conditions for the detection of delayed-type hypersensitivity.

Footpad assay of delayed-type hypersensitivity to bovine serum albumin (BSA) was studied. Injection of saline solution of BSA (S-BSA) into footpads of sensitized mice resulted in the development of only the local Arthus-type reaction. Injection of alumprecipitated BSA (AP-BSA), however, was effective in generating both Arthus- and delayed-type reactions. The difference between S-BSA and AP-BSA in the ability of elicitation of delayed-type reaction may be attributable to the difference in the retention of these two forms of antigen in the footpad, since S-BSA was found to be eliminated much more rapidly from footpads than AP-BSA.     

Differential sensitivity to cyclophosphamide of helper T cells for humoral responses and suppressor T cells for delayed-type hypersensitivity

The relative sensitivity to cyclophosphamide (CY) of helper T cells in antibody responses and of suppressor T cells in delayed-type hypersensitivity (DTH) responses against sheep red blood cells (SRBC) was measured by pretreating cell suspensions in vitro with the microsomally activated form of CY and evaluating their subsequent function in transfer experiments. It was found that suppressor T cells involved in DTH responses were sensitive to much lower concentrations of activated CY than were helper T cells for anti-SRBC antibody responses. Since the cytotoxic activity of CY is thought to be dependent on its ability to alkylate nuclear DNA, the differential sensitivity observed may be more easily explicable in terms of additional secondary enzyme-mediated degradation or DNA repair mechanisms.     

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.     

Regulation of the immune response. I. Suppression of delayed-type hypersensitivity by T cells from mice expressing humoral immunity.

The ability of horse red blood cell (HRBC)-specific T cells from mice expressing humoral immunity to suppress the induction of HRBC-specific delayed-type hypersensitivity (DTH) was investigated. The transfer of Ig-negative spleen cells, from mice injected 4 days previously with HRBC, completely suppressed the development of DTH in mice treated with cyclophosphamide and sensitized with HRBC. The suppressor cell was found to be lysed by treatment with anti-theta serum and complement. Furthermore, hemocyanin-specific immune T cells were able to suppress the DTH induced to HRBC, provided these two antigens were coupled together. These studies suggest that T cells present under conditions were humoral immunity is induced can suppress DTH and that such cells play an important role in the regulation of the immune response.     

Cell-mediated immune responses in Staphylococcus aureus infections in mice.

Delayed hypersensitivity to staphylococcal antigens was shown in mice repeatedly infected with Staphylococcus aureus. It was characterized by footpad swelling at 48 hours with a mononuclear cell infiltrate and could be transferred to non-infected recipients by T lymphocytes from infected animals, but not by serum. Recipients of immune T cells produced very severe necrotic lesions when challenged with staphylococci. This was in contrast to the protection against necrosis in recipients afforded by serum from infected donors. When both serum and cells were transferred into the same mouse the humoral effects overshadowed or perhaps inhibited those mediated by cells with resultant protection against staphylococcal dermonecrosis.     

Distinction between suppressors of the delayed-type hypersensitivity and the humoral response to sheep erythrocytes.

Suppressors for both delayed-type hypersensitivity (DTH) and the humoral immune response could be simultaneously induced in the spleens of mice by immunization with a high dose of SRBC. Normal recipient mice of the spleen cells from donors immunized 5 days previously elicited depressed DTH or humoral response when immunized with SRBC. The suppressive activity was found to reside in T not B enriched fraction. Four hundred rad irradiation of the primed spleen cells resulted in complete loss of DTH suppressor activity, but only in some reduction of the suppressor activity for the humoral response. In contrast, hydrocortisone treatment of the donor mice caused no loss of DTH suppressor activity while approximately half of the suppressive activity for anti-SRBC PFC response was lost. Adult thymectomy prevented completely the induction of the DTH suppressor in contrast to little loss of the suppressor activity for the humoral response. DTH suppression was antigen-specific for the induction, but nonspecific for the expression. However the suppression of the humoral response was antigen-specific not only for the induction but also for the expression. In addition, DTH suppressor was capable of suppressing both the induction and expression of DTH while the humoral response was suppressed only in the induction stage by the suppressor.     

Difference in antigen-presenting ability of macrophages between high- and low-responder mice in delayed-type hypersensitivity to Mycobacterium bovis BCG.

Purified protein derivative-pulsed spleen macrophages of Mycobacterium bovis BCG high-responder mice stimulated BCG-primed lymphocytes of F1 (low x high) mice well in vitro, but those of BCG low-responder mice did not.     

IgG antibody and delayed-type hypersensitivity responses in nude mice grafted with thymic epithelium.

This study compared the ability of foetal thymic epithelium depleted of lymphocytes and dendritic cells, by low temperature or deoxyguanosine (dGuo) treatment in organ culture, to reconstitute T-cell function in nude mice. It is shown that renal capsule grafts of either type could promote the development of functional T lymphocytes in the periphery, as judged by in vivo assays. Both syngeneic and allogeneic thymic epithelium endowed nude mice with the capacity to mount IgG antibody and delayed-type hypersensitivity (DTH) responses to the T-dependent antigen ovalbumin (OVA). Functional reconstitution was accompanied by the appearance of Thy-1-bearing cells in the spleens of thymic grafted nude mice. The results from allogeneically grafted recipients show that a substantial population of peripheral T cells was present that collaborated with B cells and other antigen-presenting cells (APC) which do not express major histocompatibility complex (MHC) molecules of the thymus donor haplotype.     

Cell-mediated and humoral immune responses of renal transplant recipients with cytomegalovirus infections

Cell-mediated and humoral immune responses were determined in immunosuppressed renal transplant recipients. A micro-phytohaemagglutinin (PHA) stimulation test utilizing whole heparinized blood and a macro-PHA test utilizing separated, washed lymphocytes were used to study cell-mediated immunity. Humoral immune status was estimated by determining quantitative immunoglobulins and complement fixing (CF) antibody titres to cytomegalovirus (CMV) infection. Micro-PHA responses were found to be markedly depressed in patients undergoing immunosuppressive therapy and in patients with chronic uraemia. Macro-PHA responses were normal, indicating that serum factors were responsible for the depressed micro-PHA responses. Antibody responses to CMV infections were found to be ten to a hundred times higher than in normal persons. An inverse relationship was demonstrated between micro-PHA responses and peak CF antibody titres to CMV infections. Humoral immune responses appeared to compensate for depressed cell-mediated immunity as measured by the micro-PHA test. Four patients had very low micro-PHA responses, did not respond to their CMV infections with CF antibody, and died of mixed bacterial and viral infections. Serum immunoglobulins of two were studied and were shown to be greater than two standard deviations below the normal mean. These patients appeared more suppressed than other patients receiving similar therapy and thus probably retained higher concentrations of suppressive drugs.     

Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity to Cryptococcus neoformans can be detected by delayed-type hypersensitivity (DTH) to a culture filtrate antigen of C. neoformans. Recently, we have identified a population of cells in spleens of mice immunized with cryptococcal antigen that, when transferred to recipient mice at the time of immunization, amplifies the anticryptococcal DTH response. If the cell donor mice are treated with cyclosporin A during induction of the anticryptococcal DTH response, the amplifier cells are not induced, whereas the cells which transfer DTH (TDH cells) are induced. The purpose of this study was to characterize the amplifier cells with respect to their surface and functional properties and, in so doing, determine whether or not the amplifier cells are analogous to long-lived memory cells. We demonstrated that the amplifier cells were nylon-wool-nonadherent, antigen-specific, CD4 (L3T4+ Lyt-2-) T lymphocytes which appear in the spleens of mice 5 days postimmunization with cryptococcal culture filtrate antigen in complete Freund adjuvant. The amplifier T (Tamp) cells are not considered to be memory cells because they are relatively short-lived, being present 14 but not 18 days after the stimulating immunization. Moreover, the amplified anticryptococcal DTH response does not fulfill the criteria of the typical secondary immune (anamnestic) response in that the amplified response does not appear early relative to the appearance of the primary anticryptococcal DTH response, and it does not persist longer than the primary DTH response. We speculate that Tamp cells are not long-lived memory cells but rather act in a T-helper cell capacity to amplify the anticryptococcal DTH response.     

Cell-mediated and humoral immune responses to herpes simplex virus and cytomegalovirus in renal transplant patients.

Cell-mediated immunity to herpes simplex virus and cytomegalovirus, using the lymphocyte transformation test and interferon induction in lymphocytes, was studied in 59 patients from 1 day to 7 years after allotransplantation and compared with the results in normal subjects. Both parameters were permanently depressed with regard to cytomegalovirus. With herpes simplex virus, interferon production was also permanently depressed, whereas the transformation reaction was normal during the first year after transplantation and only slightly depressed in patients more than 1 year after transplantation. In 6 patients the above-mentioned assays and the complement fixation reaction were performed serially and related to the clinical signs of herpes simplex virus and cytomegalovirus infection. The relationship between depression of the transformation reaction and interferon production in lymphocytes and the occurrence of clinically evident herpes simplex virus and cytomegalovirus infections was, however, equivocal. The humoral immune response to herpes simplex virus was measured by the complement fixation test and the more sensitive antibody-dependent, cell-mediated cytotoxicity reaction, and a good correlation was found between these two tests, although only a few persons were found to be negative in the antibody-dependent, cell-mediated cytotoxicity reaction. The suggestion is made that only a few adults are "true" herpes simplex virus seronegative.     

T-T cell interaction in the in vitro induction of delayed-type hypersensitivity (DTH) responses: demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of DTH responses

C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate virus-reactive helper T cell activity. 850R X-irradiated spleen cells from vaccinia virus-primed or unprimed mice as helper cells were stimulated in vitro with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP) in the presence of normal C3H/HeN spleen cells (responding cells). After 5 days of culture, effector cells were tested for anti-TNP delayed-type hypersensitivity (DTH) responses by adoptive transfer into footpads of syngeneic C3H/HeN recipient mice together with TNP-self. The results demonstrate that spleen cells from virus-primed mice failed to enhance anti-TNP DTH responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, spleen cells from vaccinia virus-primed mice, but not from unprimed mice, could augment anti-TNP DTH responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed mice was shown to be antigen-specific, and mediated by Lyt-1+2-T cells. DTH effector cells enhanced by helper cells were also antigen-specific and Lyt-1+2-T cells. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augmented induction of anti-tumor DTH responses by stimulation with virus-infected syngeneic fibrosarcoma tumor cells. Thus, these results provide evidence for the role of antigen-specific helper T cells in augmenting the development of DTH responses to cell surface antigens including tumor antigens.     

Maternal and postnatal dietary probiotic supplementation enhances splenic regulatory T helper cell population and reduces ovalbumin allergen-induced hypersensitivity responses in mice

Neonatal to early childhood is the critical period for establishing a balance of T helper 1 (Th1) versus T helper 2 (Th2) cellular immunity within the gut, which is strongly influenced by the source and establishment of gut microflora. Probiotic administration has been shown to attenuate Th2-biased cellular immunity and predisposition to food allergies. To test this hypothesis we provided ad libitum a probiotic-supplemented (Primalac 454 Feed Grade Microbials) or control diet to lactating dams with suckling pups and weaned pups until 10 weeks of age. Weaned mice were sensitized/challenged with egg allergen ovalbumin, saline or adjuvant at 6, 8 and 10 weeks of age. At 3, 6, 8 and 10 weeks, fecal samples were collected for microbial analysis, while blood samples were analyzed for ovalbumin-IgE and total plasma IgE levels. At termination, splenic T helper cell lymphocyte population subtypes were determined using FACS analysis and Th1/Th2/Th17 gene expression by PCR array. At 21 days of age, pups suckled by lactating dams fed the probiotic supplemented diet had significantly enhanced Lactobacillus acidophilus fecal counts compared to controls. Moreover, mice fed the probiotic supplemented diet had enhanced splenic naturally occurring and induced regulatory T cell populations, enhanced TGFβ gene expression and reduced expression of allergic mediator IL13 compared to controls. These results provide evidence that early probiotic supplementation may provide host protection from hypersensitivity reactions to food allergens by attenuating food allergen inflammatory responses.     

Maternal and postnatal dietary probiotic supplementation enhances splenic regulatory T helper cell population and reduces peanut allergen-induced hypersensitivity responses in mice

Neonatal to early childhood is the critical period for establishing a balance of T helper 1 (Th1) versus T helper 2 (Th2) cellular immunity within the gut, which is strongly influenced by the source and establishment of gut microflora. Probiotic administration has been shown to attenuate Th2-biased cellular immunity and predisposition to food allergies. To test this hypothesis we provided ad libitum a probiotic-supplemented (Primalac 454 Feed Grade Microbials) or control diet to lactating dams with suckling pups and weaned pups until 10 weeks of age. Weaned mice were sensitized/challenged with peanut extract, saline or adjuvant at 6, 8 and 10 weeks of age. At 3, 6, 8 and 10 weeks, fecal samples were collected for microbial analysis, while blood samples were analyzed for total plasma IgE levels. At termination (10 weeks of age), splenic T lymphocyte population subtypes were determined using FACS analysis and Th1/Th2/Th17 gene expression by PCR array. Mice given the probiotic-supplemented diet had significantly enhanced probiotic fecal counts compared to controls at 3, 6, 8 and 10 weeks. Moreover, mice fed the probiotic-supplemented diet had enhanced splenic naturally occurring T regulatory cell populations, and reduced splenic gene expression of allergic mediator IL-13 compared to controls. These results provide evidence that early probiotic supplementation may provide host protection to hypersensitivity reactions to food allergens by attenuating food allergen inflammatory responses.     

Regulation of delayed-type hypersensitivity. II. Specific suppressor factor for delayed-type hypersensitivity to sheep erythrocytes in mice.

An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x 10(9) SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH to SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 56 degrees C for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.     

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.     

Cellular and humoral immune responses in mice. II. Effect of intraperitoneal or subcutaneous injection of carrier of anti-hapten antibody and delayed hypersensitivity responses.

The subcutaneous (s.c.) injection of sheep red blood cells (SRBC) without adjuvant into mice preferentially induced delayed hypersensitivity (DH) reaction, as measured by footpad swelling, while intraperitoneal (i.p.) measured by the haemagglutinin test. Under these conditions, the properties of the helper activities of thymus-derived (T) cells for humoral responses were examined, in association with the features of the DH response, by measuring the anti-hapten and anticarrier antibody responses after a booster injection of trinitrophenylated (TNP) SRBC and by changing the combination of doses and injections routes of the carrier and the hapten-carrier conjugates. When mice were presensitized with i.p. injections of SRBC and boosted with i.p. injections of TNP-SRBC, the anti-TNP antibody production was maximally enhanced by presensitization with a low dose of SRBC, and gradually abolished with higher doses of SRBC for pre-sensitization. In the latter case, anti-SRBC antibody production was increases with increasing doses of SRBC..     

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.