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.     

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. 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.     

Antigen-specific suppressor factors produced by T cell hybridomas for delayed-type hypersensitivity.

This study describes the generation of T hybridoma lines which secret factors specifically suppressing delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC). AKR strain-derived T lymphoma BW 5147 cells were fused with spleen cells from mice primed with SRBC and containing antigen-specific T suppressor cells for DTH. Supernants from the derived hybridomas were tested for suppression of either expression of induction of DTH to SRBC. Six lines produced specific suppressor activity for the expression of DTH; 4 lines produced suppressor activity for the induction of DTH of which only one line was antigen-specific. These lines were passaged in normal AKR mice, and the serum obtained had activity up to 10(-4) dilution. The factor was effective across the H-2 barrier.     

Major histocompatibility gene complex (MHC)-coded determinants on antigen-specific suppressor factor for delayed-type hypersensitivity and surface phenotypes of cells producing the factor

Antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) was obtained by incubating in vitro spleen cells from CBA mice (H-2k) injected intravenously 3 days previously with 1 x 10(9) SRBC. The suppressor factor was characterized for major histocompatibility gene complex (MHC)-coded antigenic determinants by passing the factor through immunosorbents coupled with appropriate alloantisera. The suppressor factor was absorbed by anti-H-2k, anti-Iak and anti-I-Jk immunosorbents but was not retained by anti-Ias, anti-I-Js, anti-I-Ak, anti-I-E/Ck or anti-H-2Kk immunosorbents. In addition, the factor bound to an immunosorbent coupled with rabbit antibodies against carbohydrate-defined Ia antigens. Furthermore, the suppressive activity that was absorbed was quantitatively recovered in the acid eluates from the immunosorbents. Treatment of the spleen cells with anti-Lyt-1.1 antiserum and complement completely abrogated their ability to elaborate the suppressor factor in vitro. In contrast, treatment with anti-Lyt-2.1 or anti-Iak antiserum and complement had no effect. Thus, it appears that the suppressor factor for DTH to SRBC bears I-J subregion-coded determinants, and its production is dependent on cells which have the Lyt-1+,2- and Ia- phenotype.     

Cyclosporin A prevents suppression of delayed-type hypersensitivity in mice immunized with high-dose sheep erythrocytes.

When administered intraperitoneally to mice 2 days before immunization with a tolerogenic dose (10(9)) of sheep red blood cells (SRBC), cyclosporin A (CsA; 200 mg/kg) strikingly augmented 4-day delayed-type hypersensitivity (DTH) footpad reactions. These enhanced responses were similar in magnitude to those seen in animals sensitized with an immunogenic, low-dose (10(6)) SRBC. The stimulatory effect of CsA was observed over the dose range of 5-200 mg/kg and was obtained in animals given the drug in one injection, up to 7 days before sensitization. The augmentation of DTH was characterized by footpad swelling, intense mononuclear cell infiltration and increased deposition of 125I-fibrinogen within the challenge site. In addition, increased expression of procoagulant activity by spleen cells in response to antigen was observed. Cell transfer experiments showed that the CsA-enhanced DTH could be adoptively transferred to naive recipients. Additional transfers conducted at the time of antigen challenge suggested that, under the conditions described, CsA inhibited the action of a population of suppressor cells normally effective during DTH reactions.     

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.     

Suppressor T cells for delayed-type hypersensitivity to Japanese encephalitis virus.

The delayed-type hypersensitivity (DTH) to Japanese encephalitis virus (JEV) and the suppressor cells controlling it and the antibody-forming cells in inbred Swiss mice have been studied. JEV induces DTH, with a peak response at day 7 following infection which persists at low levels at least up to 119 days. Suppressor activity appeared on day 18. It was transferable by immune spleen cells. Treatment of spleen cells with anti-Thy-1.2 antisera and complement abrogated the suppressor activity. The homogenate of the spleen was equally effective in mediating suppression of DTH and the humoral response as measured by direct antibody plaque-forming cell (IgM-PFC) assay. The suppressor activity was antigen-specific both on DTH and T helper for antibody response as the immune responses against SRBC or Coxsackie B4 virus were not suppressed. The suppressor cells were sensitive to cyclophosphamide treatment when the drug was given 48 hr before their appearance. It is, therefore, concluded that in JEV infection of mice, antigen-specific suppressor T cells are generated, both for DTH and IgM antibody, which are cyclophosphamide-sensitive and mediate suppression through soluble product(s).     

Regulation of delayed-type hypersensitivity V. Suppressor cell memory in antigen-specific suppression of delayed-type hypersensitivity to sheep erythroeytes

Mice primed i. v. with 10⁹ sheep red blood cells (SRBC) produce antigen-specific T suppressor (Ts) cells which inhibit both the induction and the expression of delayed-type hypersensitivity (DTH). These Ts cells are detectable in the spleen and lymph nodes 3–5 days after priming but are largely absent by 6 days. The transient detect-ability of the Ts cells contrasts sharply with the profound antigen-specific suppression which persists in primed donor mice for at least a year. Evidence is presented that this long-term impairment of DTH is maintained, at least in part, by memory Ts cells which are Thy-1⁺, cyclophosphamide-resistant and antigen-specific. Although they appear to be co-induced with the short-lived primary Ts cells and localize initially in the lymphoid organs, they are present in the long-lived circulating pool of T cells and can be adoptively transferred by celomic parabiosis. Memory Ts cells are readily reactivated by lower doses of SRBC which would induce T effector cells rather than Ts cells in naive animals. Reactivated memory Ts cells seem to generate a population of antigen-specific secondary Ts cells which again localizes in the lymphoid organs and can adoptively suppress the induction and expression of DTH to SRBC.     

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.     

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.     

Long-lasting enhancement of the delayed-type hypersensitivity response to heterologous erythrocytes in mice after a single injection of cyclophosphamide.

Previous reports have indicated that cyclophosphamide (CY) treatment can enhance delayed-type hypersensitivity (DTH) reactions by abrogating suppressor T cell functions. Such findings have suggested that cells in the suppressor lineage may be particularly sensitive to this alkylating agent. The experiments reported here demonstrate that a single injection of CY before sensitization can induce a long-lasting state of enhanced DTH responsiveness to sheep red blood cells (SRBC) in mice. This enhancement required concurrent antigenic stimulation and appeared to be antigen-specific. Additionally, CY treatment of sensitized mice before the first antigenic challenge for DTH resulted in suppressed responses to that challenge, followed by enhanced DTH to subsequent challenge with the same antigen. The suppressed response was achieved with a lower dose of CY than the subsequent enhancement and also required concurrent antigenic stimulation. These results indicate that the effects of CY on both effector and suppressor mechanisms are critically dependent upon antigenic stimulation, and suggest that apparent suppressor sensitivity to CY may be a function of differential ability to recover from CY treatment in a context of antigenic stimulation.     

Junin virus-induced delayed-type hypersensitivity suppression in adult mice

Junin virus (JV) infection of suckling mice leads to lethal meningoencephalitis consistent with a delayed-type hypersensitivity (DTH)-like immune response. In contrast, there are no central nervous system (CNS) alterations, and high antibody titers are induced in resistant adult mice. As a possible explanation, JV infection in adult mice may provoke DTH depression. Thus in this work we study the alterations induced by JV in the immune response of adult mice by using sheep red blood cells (SRBC) as an unrelated indicator antigen. JV infection was found to abrogate DTH significantly, regardless of SRBC priming time, virus strain attenuation, and viral route of inoculation. The effect proved viral dose-dependent and required live and infectious virus. However, the humoral response to SRBC, as determined by splenic "plaque-forming cell" count was found higher than controls. These results are consistent with adult mouse response to JV infection. In contrast to the guinea pig model, there is no destruction of immunocompetent cells. T-cell percentage in the spleen was high, suggesting involvement with DTH-suppressive action. We suggest that the immune response to SRBC in adult infected mice may be used for understanding the mechanisms involved in resistance.     

Secondary delayed-type hypersensitivity to sheep red blood cells and minor histocompatibility antigens in mice: transfer of memory by recirculating thoracic duct lymphocytes

Secondary delayed-type hypersensitivity (DTH) in mice to sheep red blood cells (SRBC) and minor histocompatibility (H) antigens is dependent on long-lived memory T cells. In this paper we investigated whether these memory T cells recirculate. It was shown that late phase "immune' thoracic duct lymphocytes (TDL) from mice which were immunized with SRBC or non-H-2-incompatible spleen cells several weeks previously could adoptively transfer secondary DTH to these antigens. Passing the immune TDL through intermediate recipients demonstrated that these SRBC- or minor H-antigen-reactive memory T cells recirculate from blood to lymph. In contrast to mice immunized with minor H antigens, no secondary type DTH reactivity could be demonstrated in mice immunized with H-2-incompatible spleen cells. Also, after adoptive transfer of TDL from mice immunized with H-2-alloantigens, it was impossible to demonstrate an accelerated DTH reactivity.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. IV. Fc receptors on specific peritoneal exudate lymphocytes and their role in delayed-type hypersensitivity reactions.

In mice, delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) is mediated by T cells. Peritoneal exudate T cells (PETLs) from mice optimally sensitized for DTH to SRBC form rosettes when interacted with sensitized sheep red blood cells (EA). The binding of EA to PETLs is mediated by a receptor specific for the Fc portion of the antibody (FcR). Biological activity (mediation of DTH) depends on the unreacted state of PETLs and is lost when the latter are either rosetted with EA or reacted with aggregated IgG. Transfer of EA or aggregated IgG-treated PETLs from mice with DTH to SRBC does not lead to adoptive sensitization of recipients. It is suggested that FcR found on the membrane of T cells mediating DTH play a role in the regulation of the cellular immune response to SRBC.     

Immunoregulatory effect of antibody on delayed-type hypersensitivity in mice

We analyzed the conditions for in vivo toleration of delayed-type hypersensitivity (DTH). The intravenous injection of a high dose of keyhole-limpet hemocyanin (KLH) into BALB/c mice did not induce DTH in vivo, but the serum titers of the anti-KLH antibody were significantly elevated. This lack of DTH response was antigen-specific, and the intravenous injection of the antigen induced effector-phase suppressor T cells. The findings suggested that the lack of a DTH response brought about by the intravenous injection of a high dose of antigen was not immunological tolerance. Treatment with a high dose (250 mg/kg) of cyclophosphamide - but not a low dose (50 mg/kg) - enhanced the DTH, but suppressed antibody production. These results indicate that humoral immune response participate in the regulation of DTH. In addition, the transfer of serum or immunoglobulin from mice that were injected intravenously with a high dose of the antigen suppressed the DTH. We concluded that the antibodies regulate DTH in the antigen-specific manner.     

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.     

Effect of thymectomy on formation of immunologic tolerance in delayed-type hypersensitivity effectors

The effect of thymectomy on the formation of tolerance of delayed-type hypersensitivity (DTH) to sheep's red blood cells was investigated. If tolerance was induced by combined injection of a massive dose of antigen and cyclophosphamide, thymectomy did not prevent this process and prolonged the state of tolerance. If area activity was induced by a massive dose of antigen alone, thymectomy restored the ability to form DTH and prevented the formation of suppressor cells. Thymetome weakened DTH formation somewhat in intact animals, but not in animals receiving cyclophosphamide. The results confirm views regarding the diversity of the mechanism of tolerance (clonal-deficiency and suppressor). It is also suggested that among DTH effectors and their precursors there are two subpopulations which differ in their sensitivity to cyclophosphamide and thymectomy.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 10, pp. 434–437, October, 1979.     

Factors influencing the enhancement of delayed-type hypersensitivity to ovalbumin by cyclosporin A in the guinea pig: possible role of suppressor cells

Enhancement of delayed-type hypersensitivity (DTH) reactions to ovalbumin (OVA) was demonstrated in guinea pigs given a single, high dose of cyclosporin A (CsA) intraperitoneally, 2 days before immunization. Courses of oral CsA, commencing at the time of immunization and lasting until day 4 or 13 also resulted in augmented DTH responses at days 14 and 28, respectively. However, the enhancing protocol (CsA; day 0-4) did not significantly affect circulating anti-OVA antibody titres. The capacity to express increased DTH could be adoptively transferred to naive recipients by systemic injection pooled spleen and peritoneal exudate cells. Moreover, the expression of augmented responses was inhibited by transfer of cells from normally immunized donors. Although augmentation of DTH was accompanied by increased lymphocyte proliferative responses to OVA in vitro, there was no similar effect on T cell responses to polyclonal mitogens. The data support the view that augmentation of DTH by CsA is attributable to suppressor cell dysfunction, but that this is unlikely to be a non-specific suppressor cell impairment.     

Lymphocyte recruitment in vaccinia virus-induced cutaneous delayed-type hypersensitivity.

Our goal was to study the small lymphocytes that were recruited to cutaneous DTH lesions in order to determine if there existed a subset of small lymphocytes which preferentially migrated into cutaneous inflammatory sites rather than into lymphoid tissues. Lymphocytes were radiolabelled and injected i.v. into sensitized recipient rats on which DTH lesions were induced by the i.d. injection of vaccinia virus. Small lymphocytes, from unstimulated and antigen-stimulated LNs, migrated in large numbers to LNs but only to a modest extent to the DTH lesions. Lymphoblasts, from antigen-stimulated nodes, migrated to DTH lesions well, but poorly to normal LNs. The ratio of the radioactivity in the lesions to that in LNs was used as an index of the preferential migration of the cells. This ratio for small lymphocytes was 0.8:1, while that of lymphoblasts was 34:1. Lymphocytes from the blood and spleen were better at entering lesions than the small LNCs, and their ratio was higher. Peritoneal exudate lymphocytes induced by the i.p. injection of virus migrated very well to the DTH lesions and poorly into LNs, and produced a ratio of 130:1. The peritoneal lymphocytes that migrated into the lesions were primarily small lymphocytes. Negative selection for either surface Ig-, W3/25-, MRC OX-8- or MRC OX-6- cells had little effect on the accumulation of the cells within the DTH lesions. In conclusion, it is suggested that there exists a subset of small lymphocytes with receptors on their surfaces which cause them to migrate selectively to inflammatory sites rather than to LNs. An inflammatory exudate is a rich source of this subset of small lymphocytes, while only a small proportion of small LNCs being to this subset.