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.     

The effects of cyclosporin A on the induction, expression and regulation of the immune response to herpes simplex virus.

Investigations were conducted to determine the effect of cyclosporin A (CsA) on the delayed type hypersensitivity (DTH) and antibody response of mice to Herpes simplex virus (HSV). Given only at the time of priming, the drug had little effect on the subsequent DTH response in mice receiving a 'DTH immunogenic' inoculation regime. However, CsA restored normal responsiveness in groups receiving a 'DTH tolerogenic' regime implying the abrogation of T suppressor (Ts) cells. Ts cell induction was insensitive to cyclophosphamide. Antibody responses were not suppressed after giving CsA with either of these regimes and enhancement was shown in some groups. DTH was substantially reduced by CsA when the drug was given repeatedly between the time of priming and challenge, or when previously primed mice received the drug shortly before challenge.     

Modulation of the immune system in the mouse 1. Drug administration prior to antien sensitization

A model has been developed in the mouse in which metylated bovine serum albumin (MBSA) and sheep red blood cells have been used to produced delayed type hypersensitivity (DTH) and humoral immunity (HI), respectively.The time coruse between antigen sensitization and challenge was chosen such that cyclophosphamide (CY), administered prior to sensitization, produced DTH enhancement and also produced suppression of HI when administered at high doses. A number of other drugs have been examined in this model but only CY-like drugs, viz. alkylating agents, produced similar DTH enhancement. DTH was suppressed in a few cases.The HI response was enhanced or suppressed by a number of unrelated drugs. CY was the only alkylatng agent to suppress the antibody titre.The mechanisms for these drug effects are uncertain. However, a number of drugs elicit effects on either DTH or HI which suggests there is selective removal of certain cell populations rather than non-specific cytotoxicity. Possible theories for the effects observed are discussed.These studies also suggest that CY possesses unique properties in this particular model.     

Modulation of the immune system in the mouse. 2. Drug administration following antigen sensitization

The effects of administering drugs 2 days after antigen sensitization in a combined model of delayed type hypersensitivity (DTH) and humoral immunity (HI) in the mouse are described and compared with a previous study in which drugs were administered prior to sensitization using the same model.Low doses of cyclophosphamide (CY) administered after antigen sensitization were found to enhance DTH to methylated bovine serum albumin (MBSA). In addition, high doses of this drug suppressed DTH for 3 days after challenge, and then enhanced the response for a further 5 days. The HI response to sheep red blood cells (SRBC) was simultaneously suppressed by a range of CY doses.A number of different drug types demonstrated a similar pattern of dose-dependent DTH effects. However, unlike the previous study DTH enhancement was not confined to the alkylating agents. Either DTH enhancement or suppression was also observed after treatment with a number of other drugs.HI was suppressed by several drugs, in particular by a number of alkylating agents and anti-metabolites. Selective effects on one or other limbs of the immune response were observed.The mode(s) of action of drugs active in the present study are uncertain but possible explanations are discussed in terms of the elimination and subsequent recovery of specific cell populations.     

Modulation of immune response in vitro and in vivo by human granulocyte factors

The adherence of granulocytes induces secretion of specific granule contents. The secreted proteins were termed granulocyte factors (GF). The experiments in vivo provide evidence that GF play an essential role in the stimulation of PFC in BALB/c mice immunized with SRBC when applied before challenge three times (5 micrograms per mouse), but 50 micrograms per mouse given in the same way diminishes the response. To elucidate this discrepancy, the effect of GF on the generation of suppressor cells (SC) and helper cells (HC) in vitro has been investigated. Antigen specific nonadherent SC or HC were induced in vitro using CBA mice spleen cells incubated with 100 micrograms/ml or 0.1 mg/ml of TNP-KLH, respectively, for 4 days. GF in concentrations of 0.1 to 1 microgram/ml abolish antigen specific SC generation. SC and HC activity was tested in cooperative cultures. Antigen specific SC in delayed hypersensitivity (DTH) to BCG were induced in an in vitro system as above using normal BALB/c spleen cells and 100 micrograms/ml PPD. Nonadherent suppressor cells were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer. DTH was measured by foot-pad reaction. This reaction was positive to PPD in CY treated mice immunized to BCG, while it was suppressed by the transfer of in vitro induced SC. When the SC were induced in the presence of 1 microgram/ml GF, the suppression was abrogated. The higher GF concentrations stimulated SC activities when they were measured in response to a nonrelated antigen and in specific anti-PPD response, but the HC inhibition could not be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro induction of suppressor T-cells in delayed-type hypersensitivity to BCG and an essential role of I-J positive accessory cells

Antigen-specific suppressor T-cells in delayed-type hypersensitivity (DTH) to BCG were induced in vitro. Normal spleen cells of C3H/He mice were incubated with 50 micrograms of PPD per ml for 4 days at 37 degrees C, and the non-adherent cells in the culture were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer, and DTH was measured by the footpad reaction to PPD two weeks later. Footpad reaction to PPD was positive in CY-treated C3H/He mice immunized to BCG, while it was suppressed by the transfer of the in vitro induced suppressor cells. When the suppressor cells were treated with anti-thy-1.2 antiserum and complement before transfer, the suppression was abrogated. Next, the spleen cells were separated into plastic adherent and non-adherent fractions. After treatment with anti-thy-1.2 and complement, the adherent cells were treated with either anti-I-Jk or anti-I-Ak antiserum and complement. Then, they were reconstituted with the non-adherent cells and cultured with PPD. Treatment of the adherent cells with anti-I-Jk antiserum and complement abrogated the suppressor cell induction, while the treatment with anti-I-Ak had no effect. These facts indicate that I-J positive non-T-adherent cells play an essential role in the induction of suppressor cells in DTH.     

Modulation of the immune responses against SRBC after oestriol treatment in mice.

Delayed-type hypersensitivity (DTH), primary direct and indirect PFC, memory antibody response and suppressor cell induction against sheep red blood cells (SRBC) have been examined in oestriol (E3) pretreated mice. The results showed that DTH and primary direct and indirect PFC responses were suppressed by E3 treatment. These suppressive effects could, however, be overcome when oestrogenized mice were given supra-optimal doses of SRBC for each response. On the other hand, the memory antibody response was markedly enhanced when E3 was given prior to the primary antigen stimulation. Induction of the suppressor cells for the antibody response seemed not to be affected by E3 treatment, but the characterization of the suppressor cells revealed that those obtained from E3 treated mice were surface immunoglobulin positive (sIg+) cells whereas those from control mice were not. These results were discussed in terms of the altered antigen distribution due to the activated phagocytic activity of the reticuloendothelial system (RES) after E3 treatment.     

Local administration of cytostatic drugs enhances delayed-type hypersensitivity to Sendai virus in mice

Cytostatic drugs known to exert immunomodulatory activity were studied in a local treatment protocol as to their effect on development of cell-mediated immunity to Sendai virus in mice. Strong enhancement of delayed-type hypersensitivity (DTH) was observed after treatment with the active cyclophosphamide (CY) derivative Z 7557 or the plant alkaloid VP-16 at the site of sensitization. Optimal enhancement of DTH was obtained after priming with subimmunogenic doses of Sendai virus and treatment with low, virtually nontoxic drug doses. Immunoenhancement is ascribed to elimination of CY-sensitive suppressor cells from the draining lymph nodes. The relevance of our findings with respect to protective immunity is discussed.     

Effect of cyclophosphamide on delayed hypersensitivity to Staphylococcus aureus in mice.

Mice given cyclophosphamide 2-3 days before a single subcutaneous infection with Staphylococcus aures on cotton dust develop much more delayed type hypersensitivity (DTH) than normal. The enhancement is due to removal of rapidly dividing cells from the spleen. Passive transfer experiments before infection or before challenge show that immune serum suppresses the induction but not the expression but do not prevent induction. There may therefore be 2 suppressor or regulating systems involved. Cell commitment to suppressor function may be self-limiting. The results explain why DTH to staphylococci is only fully established after repeated infections and support the view that the suppressor system may function as a check on the excessive and potentially harmful development of DTH.     

Enhancing effects of locally administered cytostatic drugs on T effector cell functions in mice

It is well established that systemic treatment with cyclophosphamide (CY) can augment cell-mediated immunity by selective toxicity for suppressor cells. The present paper analyzes the effect of local administration of the cytostatic drugs Z 7557, an active CY-derivative, and VP-16 (etoposid) on the development of cell-mediated immunity to KLH in mice. Both drugs were found to induce a long-lasting state of enhanced DTH responsiveness. Induction of enhancement was antigen-specific but required a conjunction of local drug treatment and s.c. priming. This indicates that locally applied cytostatics interact with the sensitizing process in the draining lymph node. In support of this view, draining lymph node cells showed an enhanced capacity to transfer DTH reactivity to syngeneic recipients in vivo, and to proliferate and produce IL-2 and MIF in response to KLH in vitro. Kinetic studies showed a lag in appearance of antigen-specific T cell responsiveness in drug treated mice, which indicates that effector cells are also sensitive to the drugs. If local chemotherapy is properly scheduled, the effector cells recover quickly and, released from the putative inhibitory influence of suppressor cells, give rise to an augmented T cell response. As systemic treatment with cytostatic drugs is known to cause adverse side effects, restricting its use for immunopotentiating purposes, the present local chemotherapy protocol provides a new, versatile approach for enhancing cell-mediated immunity.     

Suppression of delayed hypersensitivity to tuberculin by antigenic competition. A positive immunoregulatory mechanism sensitive to cyclophosphamide.

Effector mechanisms that produce delayed hypersensitivity reactions to tuberculin are subject to positive immunoregulation. Two different immunoregulatory mechanisms can be demonstrated. One is specific and the other, antigenic competition, is non-specific; both are sensitive to cyclophosphamide (CY). Delayed hypersensitivity to purified protein derivative PPD in guinea-pigs can be enhanced by the administration of cyclophosphamide 3 days before but not after immunization. The enhanced response seems to result from the reduced influence on effector cells of CY-sensitive suppressor cells. Passive transfer of delayed hypersensitivity to PPD is facilitated by the use of cells from CY treated animals. The response to both immunization and skin testing with ovalbumin in animals immunized with this antigen in Freud's complete adjuvant (FCA) produces a marked, non-specific reduction in the delayed hypersensitivity response to PPD. CY given 3 days before or 1 day after immunization prevents this suppression of the PPD response by antigenic competition. The data suggests that in the generation of both the specific suppressor cells for tuberculin and the non-specific suppressor cells of antigenic competition, that can influence effector cells for tuberculin, a period of rapid cell proliferation occurs that renders both mechanisms sensitive to cyclophosphamide.     

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.     

Antigenic competition in IgE antibody production. II. Effect of cyclophosphamide.

The effects of cyclophosphamide (CY) on antigenic competition in IgE antibody production were studied in mice treated with the drug on different days and immunized with a mixture of two non-related antigens. Injection of 100 mg of CY/kg of body weight 3 days before or 6 days after immunization resulted in a partial or total recovery of the IgE, but not of the IgGl antibody response to the test antigen. In contrast, when the same dose was given together or 3 days after immunization both responses were much more suppressed than in untreated animals. This same effect was obtained when a higher concentration (200 mg/kg) of cyclophosphamide was injected on day--3. When a different antigenic system was tested, the suppressive effects of competition in IgGl antibody production were also abolished after CY treatment. These results seem to provide further evidence for an important role of suppressor T cells in the mechanism of antigenic competition.     

Auto-Delayed-Type Hypersensitivity Induced in Immunodeficient Mice with Syngeneic Modified Self-Antigens

The control of the autoimmune response to modified self-antigens was explored, using immunodeficient mice injected with syngeneic trinitrophenylated spleen cells (TNP-SC) as an experimental model system. X-irradiated (250 rad) A mice injected with TNP-SC and footpad-challenged 7 to 14 days later with syngeneic lymphoblasts generated a delayed-type hypersensitivity (DTH) response that was expressed by footpad swelling measured 24 h, 48 h and 72 h later. Histopathological examination showed massive inflammatory infiltration in the soft tissues of the limbs with extensive necrosis. This was not observed in X-irradiated mice that received the lymphoblast challenge only. The immunological activity was transferred from the X-irradiated TNP-SC-immunized mice to naive recipients by T cells (Lyt-1+) and not by serum, thus excluding the possibility that the inflammatory reaction is mediated by antibodies. We have previously presented evidence that the differentiation status of the lymphoblasts, and not contaminants from the incubation media, was the determinant factor eliciting the DTH response of immunodeficient mice injected with TNP-SC. Since only syngeneic lymphoblasts were able to elicit the DTH response of immunodeficient mice injected with syngeneic TNP-SC, we suggested that immunological activity was directed against self-antigens, thus expressing an autoimmune reactivity. The ability of immunodeficient mice to generate syngeneic DTH was not restricted to the TNP hapten or to inbred A-strain mice. X-irradiated BALB/c mice injected with syngeneic penicillinated spleen cells and challenged with syngeneic lymphoblasts generated a significant DTH response, in contrast to X-irradiated BALB/c mice exposed to the challenge dose only. X-irradiated A mice injected with syngeneic TNP-SC and simultaneously reconstituted with syngeneic splenocytes failed to generate a DTH response after the lymphoblast challenge, indicating that the syngeneic DTH response is controlled by normal suppressor cells. The suppressor cells were characterized as T cells carrying I-Jk, Lyt-1+, Lyt-2+ and Lyt-3+ antigenic markers. The suppressor cells abrogated the syngeneic DTH response of immunodeficient mice injected with TNP-SC, even when transferred a few days after the induction of immunological activity, but not when transferred 1 h before the lymphoblast challenge, indicating that even the established immunological activity can be restrained. Various immunological aspects of these observations and the significance of the findings in illuminating human autoimmune disorders are considered.     

Effect of cyclophosphamide pretreatment on defective delayed-type hypersensitivity in autoimmune-prone MRL mice

Autoimmune MRL mice develop a poor delayed-type hypersensitivity (DTH) after BCG cell wall (BCG CW) immunization. To test the possible participation of suppressor cells on DTH induction, MRL/MpJ-lpr/lpr (MRL-lpr) and MRL/MpJ-+/+ (MRL-+/+) mice were pretreated with cyclophosphamide (Cy) 4 days before the immunization. The footpad reaction to the purified protein derivative of tuberculin (PPD) was enhanced remarkably by Cy pretreatment in both MRL-lpr and MRL-+/+ mice. In addition, the lymph node cells obtained from nonpretreated MRL-lpr and MRL-+/+ mice at 7 days after BCG CW immunization suppressed the DTH induction of Cy-pretreated MRL-+/+ mice. On the other hand, a positive proliferative response to PPD in vitro was seen only in the cells from Cy-pretreated MRL-+/+ and younger MRL-lpr mice, but not in the cells of nonpretreated MRL-+/+ or both pretreated and nonpretreated older MRL-lpr mice. Removal of B220-positive cells from the lymph node cell populations of Cy-pretreated MRL-lpr mice restored PPD responsiveness, but the same treatment had no effect on the cells from nonpretreated MRL-lpr mice. The results suggest that DTH responses to PPD in MRL mice immunized with BCG CW are regulated by Cy-sensitive suppressor cells. These suppressor cells occur in MRL mice irrespective of whether they carry the lpr gene. And it is also demonstrated that MRL-lpr mice have TDTH precursor cells in a sufficient number for TDTH production even after expressing lymphadenopathy.     

The surface phenotype of a suppressor cell of delayed-type hypersensitivity in the mouse.

Delayed-type hypersensitivity (DTH) to horse red blood cells was induced in cyclophosphamide-treated CBA/H mice. The DTH reaction, represented by an increase (0 x 8--1 x 0 mm) in footpad thickness 24 h after secondary challenge, could be suppressed by the adoptive transfer of 10(7) splenic lymphocytes from syngeneic mice primed with 10(9) HRBC. The surface antigenic phenotype of the suppressor cell was determined by the formation of EA, EAC, or Ig rosettes followed by depleting the rosetted populations on Isopaque-Ficoll. The suppressor cell was found to be Ig-, FcR- and CR-, although some suppression was observed with FcR+ cells. Cell depletions with cytotoxic alloantisera and rabbit complement further characterized the suppressor cell as being Thy-1+, Ly-1+, 2-, 3-, 4-, 5+, 6+, 7-, Ia- and IJ-. This cell surface phenotype if unique and differs from the Ly-1-, 2+, 3+, I-J+ suppressor cell of antibody formation and from the recently described Ly-+, 2+, 3+ feedback suppressor T cell.     

Effects of cyclophosphamide treatment and gamma irradiation of SJL mice on the IgE antibody response and the nature of IgE-binding factors

Immunization with alum-absorbed ovalbumin (OA) failed to induce IgE antibody responses in SJL mice, while mice pretreated with either cyclophosphamide (CY) or gamma-irradiation prior to immunization transiently formed IgE antibodies. Spleen cells of OA-primed SJL mice formed IgE-suppressive factors upon incubation with OA. In contrast, spleen cells of the CY-treated or irradiated mice formed IgE-potentiating factors. It was found that CY treatment diminished the formation of glycosylation inhibiting factor (GIF) and enhanced the formation of glycosylation enhancing factor (GEF). The results suggest that the enhancement of the IgE antibody response by CY treatment is due not only to elimination of suppressor T cells but also to the activation of GEF-forming lymphocytes.     

Studies on delayed-type hypersensitivity (DTH) reactions in the pleural cavity in mice: prolonged DTH reaction and its interruption by cyclophosphamide treatment

DTH reactions to sheep erythrocytes (SRBC) and purified protein derivative (PPD) antigens were produced in the pleural cavity in mice. The profiles of the DTH reactions with respect to time course and cellular exudate reactions differed greatly according to the strains of mice. In particular, HY mice that were established in our laboratory displayed prolonged DTH reactions, characterized by macrophage followed by lymphocyte reactions. HY X C3H F1 mice showed a similar tendency. On the other hand, strains such as C3H, BALB/c, DBA/2 and B6 mice showed short-lived and macrophage-predominant DTH reactions. BALB/c nu/nu mice showed no DTH reactions. Characteristic features of the prolonged DTH reactions in HY mice were transferred with sensitized T cells. However, DTH reactions in HY mice treated with cyclophosphamide (CY) terminated in a short period, and mainly consisted of macrophages and polymorphs, although they were greatly enhanced. Such profiles of the reactions could also be transferred with sensitized T cells from CY-treated and SRBC-sensitized mice. Spleen cells taken from CY-treated and SRBC-sensitized HY mice, when injected intravenously into SRBC-sensitized HY mice just prior to challenge, could not interrupt prolonged DTH reactions. These results thus indicated various phenotypes of the DTH reactions in terms of time course and exudate cellular component involved might be carried by the specific effector T cells in each phenotype of the DTH reactions and could be induced using strains of mice and CY.     

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

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

Reversal of antigen-induced resistance to collagen arthritis by cyclophosphamide

Treatment of rats with intravenous injection of 1 mg of soluble native type II collagen induced resistance against the subsequent induction of active arthritis by type II collagen immunization. This resistant state was accompanied by suppressed antibody response and delayed-type hypersensitivity (DTH) skin reaction to type II collagen. However, pretreatment of rats with 20 mg/kg of cyclophosphamide (CY), an agent reputed to damage suppressor T-cell function, 2 days before intravenous injection of soluble type II collagen abrogated the antigen-induced resistance against the subsequent induction of active arthritis. The DTH skin reaction to type II collagen was completely restored and the antibody response to type II collagen was significantly though not completely restored by CY pretreatment. These results provide evidence that antigen-induced resistance to collagen arthritis is mediated, at least in part, under the control of CY-sensitive events.