Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.     

Regulation of cytokine expression in mice immunized with cryptococcal polysaccharide, a glucuronoxylomannan (GXM), associated with peritoneal antigen-presenting cells (APC): requirements for GXM, APC activation, and interleukin-12

Mice immunized with peritoneal exudate cells (PEC; used as antigen-presenting cells [APC]) that are pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM), exhibit increased survival times and delayed-type hypersensitivity reactions when they are infected with Cryptococcus neoformans. These responses are GXM specific. The present study revealed that GXM-APC immunization enhanced development of anticryptococcal type-1 cytokine responses (interleukin-2 [IL-2] and gamma interferon) in mice infected with C. neoformans. The enhancement was not GXM specific, because immunization with GXM-APC and immunization with APC alone had similar effects. GXM-APC (or APC) immunization caused small increases in the expression of type-2 cytokines (IL-4 and IL-5), but the increases were not always statistically significant. IL-10 levels were not regulated by immunization with GXM-APC or APC. GXM-APC prepared with PEC harvested from mice injected with complete Freund's adjuvant (CFA) enhanced type-1 cytokine responses, while GXM-APC prepared with PEC induced with incomplete Freund's adjuvant were ineffective. The CFA-induced PEC had an activated phenotype characterized by increased numbers of F4/80(+) cells that expressed CD40, B7-1, and B7-2 on their membranes. The immunomodulatory activity of the CFA-induced APC population was not attributed to their production of IL-12 because GXM-APC prepared with peritoneal cells harvested from IL-12 knockout mice or their wild-type counterparts were equally effective in augmenting the type-1 response. Blocking of IL-12 in the recipients of GXM-APC early after APC infusion revealed that early induction of IL-12 secretion was not responsible for the immunomodulatory response elicited by GXM-APC. These data, considered together with previously reported data, reveal that the protective activity of GXM-APC immunization involves both antigen-specific and nonspecific activities of GXM-APC.     

The role of antigen-presenting cells in the regulation of delayed-type hypersensitivity. I. Spleen dendritic cells.

A previously unreported mechanism for the induction of delayed-type hypersensitivity (DTH) was studied in detail. The subcutaneous injection (without adjuvant) of spleen dendritic cells (DC) pulsed with keyhole limpet haemocyanin (KLH) into syngeneic BALB/c mice caused DTH when the ear was later injected with the same antigen. When KLH-pulsed DC were transferred intravenously, DTH was not induced, although the titre of anti-KLH antibodies rose after such transfer. The intravenous transfer of KLH-pulsed DC into mice immunized subcutaneously with KLH in complete Freund's adjuvant (CFA) at the same time (in the sensitization phase), but not when the ear was challenged with KLH (in the effector phase), had a suppressive effect on DTH, in an H-2-restricted way. When radiolabelled DC were transferred intravenously, they migrated into the spleen, but when transferred by subcutaneous injection, they stayed in the skin or migrated into the lymph nodes. In splenectomized mice immunized with KLH, the intravenous transfer of KLH-pulsed DC did not cause production of anti-KLH antibodies and did not suppress DTH. These findings suggest that the anatomical sites in which antigens are presented (i.e. the spleen or lymph nodes) rather than the type of cell that first presents antigens to the immune system governs whether DTH or antibody production is induced. Antibody production was induced when antigens were presented in the spleen.     

Immunohistochemical localization of capsular polysaccharide antigen in the central nervous system cells in cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CME) is caused by the encapsulated fungus Cryptococcus neoformans (CN) and is a major cause of mortality and morbidity in patients with AIDS. The polysaccharide capsule of CN is important for virulence, and soluble polysaccharide has the potential to cause immune modulation. To better understand the interactions of central nervous system cells and cryptococcal capsular polysaccharide (CNPS) in the pathogenesis of human CME, postmortem brain tissue from 21 patients with CME (13 AIDS and 8 non-AIDS patients) was analyzed. Histopathology and distribution of tissue CNPS antigen were analyzed using monoclonal antibodies against CNPS in combination with cell type-specific markers (glial fibrillary acidic protein for astrocytes, Ricinus communis agglutinin (RCA)-l for macrophage/microglia and endothelial cells; UCHL-1 for T cells, L26 for B cells). The CN cells showed discrete capsular immunoreactivity as expected; however, diffuse and particulate cellular and tissue staining for CNPS was detected in the brain parenchyma and the meninges in all cases. By quantitative analysis, the CNPS immunoreactive area ranged from 0.1 to 88 percent of tissue cross sectional area, and tended to be higher in brains of AIDS (median values from two sections ranged from 1 to 57 percent; mean, 26 percent) than in non-AIDS (0.1 to 40 percent; mean, 9.6 percent) patients. The proportion of CNPS immunoreactive area was positively correlated with the estimated number of CN. None (0/13) of the AIDS patients displayed significant inflammatory responses to CN, whereas most (7/8) non-AIDS patients showed granulomatous inflammatory responses. The phenotype of infiltrating lymphocytes was UCHL-1+/L26-/RC4-, thus consistent with activated T cells, both in AIDS and non-AIDS patients. Double immunolabeling studies revealed that tissue CNPS immunoreactivity was most often localized in macrophages and microglia, less frequently in reactive astrocytes and endothelial cells, but not in lymphocytes. This study demonstrates that CNPS can be detected not only in the serum and cerebrospinal fluid (CSF) of patients, but also in the affected tissue, most often localized in cells of mononuclear phagocyte system. Potential implications of these findings for the pathogenesis of CME are discussed.     

Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.     

Regulation of delayed-type hypersensitivity. III. Effect of cyclophosphamide on the suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

A previous study (Eur. J. Immunol. 1977. 7: 714) has shown that mice injected intravenously (i.v.) with 4 x10(9) sheep red blood cells (SRBC) produce cells which suppress delayed-type hypersensitivity (DTH). These suppressor cells are theta-positive, antigen-specific and act via a soluble factor which does not bear immunoglobulin determinants (Eur. J. Immunol. 1978. 8: 168). The present paper demonstrates that these suppressor cells are inhibitable by cyclophosphamide (CY). Mice injected with graded amounts of CY two days prior to SRBC injection, showed maximum augmentation of DTH at 200 mg/kg body weight, a dose which completely suppressed the appearance of splenic plaque-forming cells (PFC) to SRBC. In contrast, lower doses of CY enhanced both DTH and PFC responses. Time course studies showed that CY inhibited the precursors of suppressor cells and had little or no effect on suppressor cells which have already encountered antigens. This was further confirmed by passive transfer studies which showed tha- suppressor cells were inhibited if CY was administered at the same time or 2 days before SRBC injection, but were not affected if CY was given after antigen stimulation. Direct evidence for the effect of CY on suppressor cells was obtained by cell fractination with a Ficoll density gradient. The denser suppressor cell population was absent from the spleens of mice treated with 200 mg/kg of CY 2 days before i.v. injection with 1 x 10(9) SRBC.     

Regulation of delayed-type hypersensitivity (DTH) response to hen egg-white lysozyme (HEL)

Delayed-type hypersensitivity (DTH) can be demonstrated in A/J mice immunized with hen egg-white lysozyme (HEL) in complete Freund's adjuvant (CFA) by challenging primed animals in the ear with aqueous HEL. Normal A/J mice receiving soluble HEL derivative peptide, P-Ib, sequence 29-54 and 109-123 linked by a single S-S bond, 7 days prior to immunization with HEL showed much lower DTH response specific to the protein. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T-cells (Ts). Thus, the suppression induced with a single i.v. injection of P-Ib solution was able to be transferred into syngeneic recipients by the spleen cells from mice pretreated with P-Ib. These suppressor cells are T-cells, since their ability to suppress DTH is completely abrogated by treatment wit anti-Thy 1.2 and complement. Amongst HEL derivative peptides tested in the present study, only P-Ib could induce the tolerance.     

Interleukin 6 inhibits delayed-type hypersensitivity and the development of adjuvant arthritis

We studied the effect of interleukin 6 (IL 6) on the delayed-type hypersensitivity (DTH). In mice immunized with sheep red blood cells (SRBC), a DTH response was evoked by antigen challenge into the hind paw 5 days after immunization. The magnitude of the response was assessed by footpad swelling measured 24 h after antigen challenge. IL 6 significantly suppressed the DTH in its induction phase in a dose-dependent manner when administered s.c. into the back at a dose of greater than 2.5 micrograms twice a day (5 micrograms/day) for 5 consecutive days from the day of immunization (day 0) to 1 day before antigen challenge (day 4). Heat-inactivated IL 6 did not suppress the DTH response. Furthermore, the suppressive activity of IL 6 was completely abolished by affinity chromatography on an anti-IL 6 antibody. This suppression was also obtained when IL 6 was administered only on day 0 and day 1, but not on days 3 and 4. This indicates that IL 6 acts on the early part of the induction phase of DTH development. Furthermore, footpad swelling was suppressed even by the administration of IL 6 after antigen challenge. These results show that IL 6 suppresses both the induction and effector phases of DTH. To confirm further this inhibitory effect of IL 6, we examined its effect on the development of adjuvant arthritis in rats. Administration of IL 6 also significantly suppressed the development of adjuvant arthritis.     

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.     

Kinetics of murine delayed-type hypersensitivity response to Eimeria falciformis (Apicomplexa: Eimeriidae)

Mice recovering from a primary infection with an intestinal protozoan parasite, Eimeria falciformis (Apicomplexa: Eimeriidae), showed a classic delayed-type hypersensitivity (DTH) reaction to oocyst antigen challenge. This reaction was characterized by a biphasic pattern of footpad swelling. The first swelling peaked at 2 h after antigen challenge, whereas the second swelling peaked at 24 to 48 h after challenge. The DTH reaction was transferable with a T-cell-enriched spleen cell population from mice that had recovered from E. falciformis infection. Cytotoxic depletion of immune T cells with anti-L3T4 antibody and complement abrogated DTH transfer, indicating that L3T4-positive T cells were required. A T-cell-enriched spleen cell population from acutely infected mice suppressed the transfer of DTH with immune cells from recovered animals, implicating the existence of infection-induced immunoregulatory cells controlling the parasite-specific immune response during infection. Immune spleen cells also transferred resistance to infection as measured by oocyst production and death rate of recipients. Together, these results indicate that the DTH reaction, induced by infection with E. falciformis, is mediated by L3T4-positive T cells and is associated with resistance to infection.     

Cyclosporin A inhibits the delayed-type hypersensitivity reaction: impaired production of early pro-inflammatory mediator(s)

The effect of cyclosporin A (CsA) on the delayed-type hypersensitivity (DTH) elicited in mice by sheep red blood cells was investigated. Evidence is presented that a single injection of CsA adversely affects the inflammatory reaction. Sensitized T lymphocytes initiate the DTH reaction by their recruiting activity on phagocytic cells which infiltrate the cutaneous site of antigen deposition. CsA administration has no adverse effect on the recruitment of phagocytic cells at the site of the inflammatory reaction. The present studies show that CsA acts on specific T cells: (a) in adoptive transfer, T-DTH-mediating cells cannot elicit a response in mice treated with CsA 8 h before; (b) when collected 8 h after a single injection of CsA, T-DTH-mediating lymphocytes are no longer able to adoptively transfer the reaction. This conclusion is strengthened by in vitro studies: (a) the frequency of T-DTH-mediating lymphocytes is 50-fold decreased after a short in vitro incubation with CsA; (b) in vitro production by concanavalin A-activated lymphocytes of chemotactic pro-inflammatory mediator(s) is abolished in presence of CsA.     

Junin virus-induced suppressor cells are effective only on the efferent limb of the delayed-type hypersensitivity response

Junin virus (JV), the etiological agent of Argentinian hemorrhagic fever, induces high mortality in the suckling BALB/c mouse, which correlates with delayed-type hypersensitivity (DTH)-like immune response. In contrast, the adult mouse is resistant to infection, and no DTH response can be detected. An antigen-nonspecific DTH suppressor T-cell activity induced by JV has been described that may be related to adult mouse survival. In this report, we present evidence supporting the inability of such cells, identified here as bearing the L3T4 marker, to affect the normal establishment of a DTH reaction. In the same form, virus-induced suppressor cells were unable to alter the expression of DTH effector cells in the absence of antigen-specific DTH suppressor cells. Besides, we found that a single high dose (200 mg/kg) of cyclophosphamide before or after JV inoculation was unable to alter the activity of virus-induced suppressor cells.     

Effect of irradiation on the precursor, activated and memory suppressor T cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

The relative radiosensitivities of precursor (Tsp), activated (Ts) and memory (Tsm) suppressor T cells for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were investigated in mice. Spleen cells from CBA mice, primed i.v. with 10(9) SRBC 3-4 days previously, contain specific Ts cells which substantially impair the induction of DTH to SRBC in normal syngeneic recipients. Exposure of mice to 400 rad irradiation 1 day before the priming completely eliminated the subsequent development of Ts cells. In contrast, 3 days after the priming injection, Ts cell activity in mice is resistant to doses higher than 600 rads. Mice primed 40 days previously with 10(9) SRBC contain Ts-cell memory which can be readily recalled by i.p. injection of 10(8) SRBC. The secondary Ts cells which specifically inhibit DTH induction can be demonstrated adoptively in normal recipients. Mice were exposed to various doses of irradiation 40 days after the priming and 1 day before the i.p. injection. Ts memory was significantly reduced by 300 rads and was completely abrogated by 400 rads. The relative radiosensitivities of the three subsets of suppressor T cells are in the order of Tsm = Tsp greater than Ts.     

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.     

Cellular cooperation in the expression of murine delayed-type hypersensitivity (DTH): dual roles of accessory cells.

A recently described murine local passive transfer (LPT) system was modified to investigate accessory cell function in the expression of in vivo delayed-type hypersensitivity (DTH). An enriched antigen-presenting accessory cell population was prepared by overnight culturing and antigen pulsing. These cells, when cotransferred locally with enriched populations of immune DTH effector T lymphocytes, induced antigen-specific DTH responses. A consistently positive relationship between DTH expression and quantity of pulsed cells transferred was demonstrated. The further addition of fresh accessory cells to the transfer mixture provided 'augmentation' of DTH responses. In contrast to antigen presentation, 'augmentation' appeared restricted to an optimal range of augmenting cell: T cell ratios. That antigen presentation and augmentation were separate accessory cell functions was further supported by differential selection during overnight culture and differential sensitivity to paraformaldehyde (pCHO) exposure. The demonstration of multiple accessory cell functions indicates considerable promise for this system in the investigation of in vivo immune expression.     

Effects of relaxation on the delayed-type hypersensitivity (DTH) reaction to diphenylcyclopropenone (DCP)

Delayed-type hypersensitivity (DTH) reactions to the experimental allergen diphenylcyclopropenone (DCP) were measured in four groups, which either trained (+) or did not train in relaxation (−) during the sensitization and/or the challenge phase. All groups consisted of high and low hypnotic susceptible subjects. While there were no differences in erythema, the mean induration of the group which trained in relaxation in both the sensitization and the challenge phase (+/+) was significantly greater than that of the group which trained in relaxation in the challenge phase only (−/+). Significant correlations were found between induration and hypnotic susceptibility scores, and between induration and degree of perceived relaxation during challenge. High hypnotic susceptible subjects experienced a higher degree of perceived relaxation and exhibited greater indurative and erythematous DTH reactions to DCP than low hypnotic susceptible subjects in all four experimental conditions. Though the mediating mechanisms remain unclear, our results suggest that relaxation may affect the DTH reaction, and support previous findings of higher psychophysiologic reactivity of high hypnotic susceptible subjects.     

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.