Int. Archs Allergy appl. Immun. 60: 178-185 (1979)
In vivo Induction of Carrier-Specific Cyclophosphamide-Sensitive Suppressor Cells for Cell-Mediated Immunity in Mice A. M. Attcillah, A. Ahmed and K. W. Sell Immunology Department, Naval Medical Research Institute, Bethesda, Md.
Introduction Regulatory factors play an important role in a wide variety of complex biological systems, both in terms of inducing, augment ing, limiting, or suppressing a given response. Studies of the immune system in
mice have provided considerable evidence for the existence of such regulatory cells and factors [2, 5, 8, 12, 13], Thus, suppressor cells have been shown to be implicated in nearly all recognized mechanisms of immunoregulation. These include the mainte nance of immunological tolerance [15], an
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Abstract. Experiments were carried out to study the sequential events that follow the immunization of mice. To accomplish this goal, a reliable in vitro lymphoid-cell, spon taneous-proliferation assay was established. Mice were immunized with 2,4-dinitrophenylbovine serum albumin (DNP-BSA) or DNP-OVA albumin (OVA) in saline and subse quently received a secondary immunization at various intervals thereafter. 24 h after challenge, popliteal lymph node cells (PLN) from these mice were cultured in vitro for 24 h with 1 ¿«Ci of 3H-thymidine (3H-TdR). Kinetic studies revealed that carrier-specific, peak spontaneous proliferation occurred on day 6 following immunization, whereas no proliferation occurred on day 10. The proliferating cells were T cells. Prior treatment of mice with cyclophosphamide (CY) 3 days before immunization prolonged the ability of PLN CY from immunized mice to proliferate on day 10 after immunization. Addition of 5 X 103 thymocytes from day-10 DNP-BSA or DNP-OVA immunized mice to 1 X 105 PLN CY cells from day-10 immunized mice with homologous antigen caused a marked inhibition of the uptake of 3H-TdR, in contrast to thymocytes from day-6 immunized mice or from day-10 immunized mice that had been treated with 100 mg/kg CY 3 days before immunization. Our results suggest that the natural sequence of events following immunization with either DNP-BSA or DNP-OVA without adjuvant involves proliferation of T-lymphoid cells, which are subsequently turned off by a separate subset of thymusderived suppressor T cells. These suppressor cells were found to be carrier-specific and CYsensitive.
hi vivo Induction of CY-Sensitive Suppressor Cells
Materials and Methods Mice C57BL/10 Sn male mice, 4-8 weeks old, were purchased from The Jackson Laboratories, Bar Harbor, ME., and used throughout these studies.
Antigens
BSA and OVA were coupled with DNP by the method of Eisen et al. [4]. The number of DNP groups per mole of BSA or OVA was calculated as described [4], It was found that DNP-BSA con sisted of 16 DNP groups per mole of BSA and 5 DNP groups per mole of OVA. Immunization Baseline studies were carried out in efforts to determine the optimum primary and secondary challenge doses. Groups of mice were immunized with 100 fig of DNP-BSA or saline subcuta neously (s.c.) in the right footpad in a volume of 0.1 ml. At varying intervals thereafter, they were challenged with 40 fig of DNP-BSA in the left footpad. Controls received saline. Carrier specific ity was determined by challenge with 40 itg of DNP-OVA. A similar protocol was used when pri mary immunization was established with DNPOVA. Each group consisted of no less than 6 mice. Measurement of DTH in vivo in vivo DTH was assayed by the measurement of footpad thickness after secondary challenge in the left footpad by the use of dial-gauge calipers. Three measurements were made at 24 h and the arithmetic mean and percent standard error (SE) calculated. The percent increase in footpad thick ness was calculated as follows: % increase in footpad thickness = units of measurement after skin testing units of measurement before skin testing ^ units of measurement before skin testing C ell P reparation
Mice were killed by cervical dislocation and the regional popliteal lymph nodes or thymus ex cised using aseptic techniques. Mononuclear cell preparations were obtained by the gentle teasing of the lymph nodes and passing them over a 60mesh sterile stainless steel screen in Hanks’ balanc ed salt solution kept at 4 °C. Cell suspensions were washed three times and resuspended in cul ture medium consisting of RMPI 1640 (Grand Island Biological Co., Grand Island, N.Y.) contain ing L-glutamine, 100 U/ml of penicillin, 100 fig / ml of streptomycin, and supplemented with 5% heat-inactivated (56 °C, 30 min) fetal bovine
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/26/2018 1:56:59 AM
tigenic competition [1], the genetic control of immune responses [3], the phenomenon of chronic allotype suppression, and the regulation of the antibody response to anti gens [7]. It has previously been reported that animals that demonstrate humoral immunity to sheep red blood cells are usually refractory to the induction of delayed-type hypersensitivity (DTH) to the same antigen [9J. Several explanations have been postulated for this lack of cell-mediat ed immunity (CMI). These include the abil ity of antibody or antigen-antibody complex es to inhibit the activated T cells [10] or, after primary immunization, the develop ment of antigen-specific T cells which will subsequently suppress the induction of CMI [16]. While it is clearly established that T cells can suppress immune responses, the target and mechanism of this T cell-mediated sup pression are still unclear. Tada et al. [14] and Herzenberg et al. [7] have provided evidence which indicates the helper T cells are the target of T cell-mediated suppres sion. It was the object of this study to develop an easy, reliable in vitro suppressor T-cell assay using simple hapten-protein conjugat es which correlate in vitro suppressor cell activity with in vivo DTH activity. Evi dence will be presented which corroborates the findings of Tada et al. [14] and Herzen berg et al. [7] that the suppression is T cellmediated.
179
Attallah/Ahmed/Scll
serum. The cell counts were determined using a Coulter counter and the cell concentration adjust ed to 1 X 106/ml, or as indicated. Spontaneous Cell Proliferation Assay The lymph node cell suspension (1 X 106/ml) in 0.2 ml was dispersed in each well of a micro titer plate (Linbro Corp., New Haven, Conn.) in trip licate. The cultures were then incubated at 37 °C in a humidified atmosphere of 5%> C 0 2 in air for varying periods of time. Each culture received 1 «Ci of 3H-TdR (methyl-3H-thymidinc, spec. act. 1.9Ci/mmol, Schwartz-Mann, Orangeburg, N.Y.) in 20 [A of media at varying periods of time (as indicated in the Results section) prior to harvest. Cells were harvested using the Multiple Automat ed Sample Harvester, as previously described [6], The mean uptake of 3H-TdR and the SE were cal culated [6], Stimulation indices (SI) were calculat ed by dividing the mean counts per minute (cpm) of experimental cultures by the mean cpm of the control cultures. When assaying for suppressor cells, the percent inhibition was calculated by the formula: (Mean cpm of target cells with effector or suppressor cell) 1--------------------- —------------- ------------- x 100. Mean cpm of target cells Target cells denote the lymph node cells (LNC) without the addition of thymocytes, and effector or suppressor cells denote the thymocytes which are added to the LNC. Antisera Anti-6>-scrum was obtained from Litton Bionctics, Rockville, MD. The cells were treated with anti-0-serum in the presence of a preselected lot of rabbit complement (C').
Results Correlation of DTH with in vitro Proliferation of Lymphoid Cells Baseline experiments were carried out to correlate the existence of DTH in vivo (by measurement of the footpad thickness) and the spontaneous proliferation of lymphoid cells in vitro (as assayed by the uptake of
3H-TdR). Groups of C57BL/10 mice were immunized with 100 ,ug of DNP-BSA or DNP-OVA in the right footpad. At varying days postimmunization, the mice were chal lenged with 40 /
In vivo Induction of Carrier-Specific Cyclophosphamide-Sensitive Suppressor Cells for Cell-Mediated Immunity in Mice A. M. Attcillah, A. Ahmed and K. W. Sell Immunology Department, Naval Medical Research Institute, Bethesda, Md.
Introduction Regulatory factors play an important role in a wide variety of complex biological systems, both in terms of inducing, augment ing, limiting, or suppressing a given response. Studies of the immune system in
mice have provided considerable evidence for the existence of such regulatory cells and factors [2, 5, 8, 12, 13], Thus, suppressor cells have been shown to be implicated in nearly all recognized mechanisms of immunoregulation. These include the mainte nance of immunological tolerance [15], an
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/26/2018 1:56:59 AM
Abstract. Experiments were carried out to study the sequential events that follow the immunization of mice. To accomplish this goal, a reliable in vitro lymphoid-cell, spon taneous-proliferation assay was established. Mice were immunized with 2,4-dinitrophenylbovine serum albumin (DNP-BSA) or DNP-OVA albumin (OVA) in saline and subse quently received a secondary immunization at various intervals thereafter. 24 h after challenge, popliteal lymph node cells (PLN) from these mice were cultured in vitro for 24 h with 1 ¿«Ci of 3H-thymidine (3H-TdR). Kinetic studies revealed that carrier-specific, peak spontaneous proliferation occurred on day 6 following immunization, whereas no proliferation occurred on day 10. The proliferating cells were T cells. Prior treatment of mice with cyclophosphamide (CY) 3 days before immunization prolonged the ability of PLN CY from immunized mice to proliferate on day 10 after immunization. Addition of 5 X 103 thymocytes from day-10 DNP-BSA or DNP-OVA immunized mice to 1 X 105 PLN CY cells from day-10 immunized mice with homologous antigen caused a marked inhibition of the uptake of 3H-TdR, in contrast to thymocytes from day-6 immunized mice or from day-10 immunized mice that had been treated with 100 mg/kg CY 3 days before immunization. Our results suggest that the natural sequence of events following immunization with either DNP-BSA or DNP-OVA without adjuvant involves proliferation of T-lymphoid cells, which are subsequently turned off by a separate subset of thymusderived suppressor T cells. These suppressor cells were found to be carrier-specific and CYsensitive.
hi vivo Induction of CY-Sensitive Suppressor Cells
Materials and Methods Mice C57BL/10 Sn male mice, 4-8 weeks old, were purchased from The Jackson Laboratories, Bar Harbor, ME., and used throughout these studies.
Antigens
BSA and OVA were coupled with DNP by the method of Eisen et al. [4]. The number of DNP groups per mole of BSA or OVA was calculated as described [4], It was found that DNP-BSA con sisted of 16 DNP groups per mole of BSA and 5 DNP groups per mole of OVA. Immunization Baseline studies were carried out in efforts to determine the optimum primary and secondary challenge doses. Groups of mice were immunized with 100 fig of DNP-BSA or saline subcuta neously (s.c.) in the right footpad in a volume of 0.1 ml. At varying intervals thereafter, they were challenged with 40 fig of DNP-BSA in the left footpad. Controls received saline. Carrier specific ity was determined by challenge with 40 itg of DNP-OVA. A similar protocol was used when pri mary immunization was established with DNPOVA. Each group consisted of no less than 6 mice. Measurement of DTH in vivo in vivo DTH was assayed by the measurement of footpad thickness after secondary challenge in the left footpad by the use of dial-gauge calipers. Three measurements were made at 24 h and the arithmetic mean and percent standard error (SE) calculated. The percent increase in footpad thick ness was calculated as follows: % increase in footpad thickness = units of measurement after skin testing units of measurement before skin testing ^ units of measurement before skin testing C ell P reparation
Mice were killed by cervical dislocation and the regional popliteal lymph nodes or thymus ex cised using aseptic techniques. Mononuclear cell preparations were obtained by the gentle teasing of the lymph nodes and passing them over a 60mesh sterile stainless steel screen in Hanks’ balanc ed salt solution kept at 4 °C. Cell suspensions were washed three times and resuspended in cul ture medium consisting of RMPI 1640 (Grand Island Biological Co., Grand Island, N.Y.) contain ing L-glutamine, 100 U/ml of penicillin, 100 fig / ml of streptomycin, and supplemented with 5% heat-inactivated (56 °C, 30 min) fetal bovine
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/26/2018 1:56:59 AM
tigenic competition [1], the genetic control of immune responses [3], the phenomenon of chronic allotype suppression, and the regulation of the antibody response to anti gens [7]. It has previously been reported that animals that demonstrate humoral immunity to sheep red blood cells are usually refractory to the induction of delayed-type hypersensitivity (DTH) to the same antigen [9J. Several explanations have been postulated for this lack of cell-mediat ed immunity (CMI). These include the abil ity of antibody or antigen-antibody complex es to inhibit the activated T cells [10] or, after primary immunization, the develop ment of antigen-specific T cells which will subsequently suppress the induction of CMI [16]. While it is clearly established that T cells can suppress immune responses, the target and mechanism of this T cell-mediated sup pression are still unclear. Tada et al. [14] and Herzenberg et al. [7] have provided evidence which indicates the helper T cells are the target of T cell-mediated suppres sion. It was the object of this study to develop an easy, reliable in vitro suppressor T-cell assay using simple hapten-protein conjugat es which correlate in vitro suppressor cell activity with in vivo DTH activity. Evi dence will be presented which corroborates the findings of Tada et al. [14] and Herzen berg et al. [7] that the suppression is T cellmediated.
179
Attallah/Ahmed/Scll
serum. The cell counts were determined using a Coulter counter and the cell concentration adjust ed to 1 X 106/ml, or as indicated. Spontaneous Cell Proliferation Assay The lymph node cell suspension (1 X 106/ml) in 0.2 ml was dispersed in each well of a micro titer plate (Linbro Corp., New Haven, Conn.) in trip licate. The cultures were then incubated at 37 °C in a humidified atmosphere of 5%> C 0 2 in air for varying periods of time. Each culture received 1 «Ci of 3H-TdR (methyl-3H-thymidinc, spec. act. 1.9Ci/mmol, Schwartz-Mann, Orangeburg, N.Y.) in 20 [A of media at varying periods of time (as indicated in the Results section) prior to harvest. Cells were harvested using the Multiple Automat ed Sample Harvester, as previously described [6], The mean uptake of 3H-TdR and the SE were cal culated [6], Stimulation indices (SI) were calculat ed by dividing the mean counts per minute (cpm) of experimental cultures by the mean cpm of the control cultures. When assaying for suppressor cells, the percent inhibition was calculated by the formula: (Mean cpm of target cells with effector or suppressor cell) 1--------------------- —------------- ------------- x 100. Mean cpm of target cells Target cells denote the lymph node cells (LNC) without the addition of thymocytes, and effector or suppressor cells denote the thymocytes which are added to the LNC. Antisera Anti-6>-scrum was obtained from Litton Bionctics, Rockville, MD. The cells were treated with anti-0-serum in the presence of a preselected lot of rabbit complement (C').
Results Correlation of DTH with in vitro Proliferation of Lymphoid Cells Baseline experiments were carried out to correlate the existence of DTH in vivo (by measurement of the footpad thickness) and the spontaneous proliferation of lymphoid cells in vitro (as assayed by the uptake of
3H-TdR). Groups of C57BL/10 mice were immunized with 100 ,ug of DNP-BSA or DNP-OVA in the right footpad. At varying days postimmunization, the mice were chal lenged with 40 /
