CELLULAR
Different
IMMUNOLOGY
43, 202-208 (1979)
Effects of Cortisone on the Humoral to T-Dependent and T-Independent
Immune Response Antigens’
EVANGELIA MANTZOURANISAND YVES BOREL Division
of Immunology, Department of Pediatrics, The Children’s Hospital Harvard Medical School, Boston, Massachusetts, 02115 Received October
Medical
Center,
20, 1978
The effect of the administration of cortisone on the murine humoral immune response to either thymus-dependent (TD) or -independent (TI) antigens was studied in vivo. Whereas the thymus-dependent immune response was markedly suppressed, the thymus-independent immune response was preserved. The opposing effect of steroids on these two types of immune responses appears to be due to the relative independence of thymus-independent antigens Jf a radioresistant cortisone-sensitive accessory cell.
The immunosuppressive effect of corticosteroids on the humoral immune response to T-dependent (TD) antigens is well known (l-3), but there is no information on their influence on the T-independent (TI) antigen immune response in viva. Here we show that cortisone affects both types of immune response differently. The response to TD antigens is, as expected, markedly suppressed by cortisone. In contrast, the immune response to TD antigens remains unchanged or is increased. Reconstitution experiments carried out with cortisone-treated mice, used as either donors or recipients, suggest that the opposing effect of cortisone on both types of immune response might be explained by the action of the steroid on a radioresistant accessory cell. MATERIALS
AND METHODS
Animals. Six- to eight-week-old (C57Bl/6 x DBA/2)F, BDF, mice were obtained from Jackson Laboratory, Bar Harbor, Maine. Cortisone preparation. Cortisone acetate suspension (Merck, Sharp and Dohme, West Point, Pa.) was injected subcutaneously into each mouse as a single dose of 5 or 10 mg. Preparation of antigens and immunization. Sheep red blood cells (SRBC) (Colorado Serum Co. Laboratory, Denver, Colo.) were washed three times and resuspended in 0.15 M saline as a 10% solution and injected intraperitoneally (0.2 ml). Fluorescein isothiocyanate on Celite 10% (Fl) obtained from Calbiochem, San Diego, California, was conjugated with keyhole limpet hemocyanin (KLH; 1 This work was supported by National Institutes of Health Grant AI-AM 1386702and American Cancer Society Grant IM-178. 202 0008-8749/79/030202-07$02.00/O Copyright 6 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
SHORTCOMMUNICATIONS
203
Pacific Biomarine Supply Co., Venice, Calif.) as previously described (10). 0.2 mg, was injected Fluorescein,o keyhole limpet hemocyanin (F&,-KLH), intraperitoneally into each mouse in complete Freund’s adjuvant (CFA). 2,4-Dinitrobenzenesulfonic acid sodium salt (DNP) and 2,4,6-trinitrobenzenesulfonic acid (TNP) were purchased from Eastman Kodak Company, Rochester, New York, and Ficoll400 was obtained from Pharmacia Fine Chemicals, PiscatTNP,,-Lysine-Ficoll, and Fluoresaway, New Jersey. DNPd3- Lysine-Ficoll, cein,,-N-(2-aminoethyl)carbamylmethyl (AECM)-Ficoll were prepared as previously described (6, 8). Five micrograms of each of these antigens was injected intraperitoneally in CFA. Ten to twenty BDF, mice were included in all experimental groups. The subscript numbers indicate the molar ratio of hapten substitution on the carrier. Hemolyticpfaque assay. The Jerne hemolytic plaque assay, using as target either SRBC or SRBC coated with the appropriate hapten (i.e., TNP, Fl) as described by others (9-ll), was employed. Direct plaque-forming cells were assayed 5 days after immunization in intact animals or 8 days after immunization in irradiated animals. Indirect plaque-forming cells (13) were revealed by use of rabbit serum to react with mouse IgGi, IgGs,, and IgG, on immunodiffusion. The serum was used at a dilution of l/200. Indirect PFC were determined at the same time as direct PFC and were obtained by subtracting the direct PFC from the total number of PFC. Statistical analysis. Results were analyzed using Student’s t test. For the PFC per spleen or the PFC per lo6 spleen lymphoid cells, the geometric mean of each group and the standard error were calculated. Cell preparation. (a) Peritoneal exudate cells. Three milliliters of thioglycolate medium (Difco Laboratories, Detroit, Mich.) was injected intraperitoneally into each mouse. One week later the mice were sacrificed and the peritoneal exudate (PE) cells were harvested from the peritoneal cavity as described by others (16). The PE cells were 80-90% macrophages by neutral red staining. They were washed twice in Eagle’s minimum essential medium (MEM) fortified with 1% fetal calf serum (FCS) and 1%0heparin, and 3 x lo6 PE cells were infused into either cortisone-treated or untreated animals. (b) Adherent cells were prepared as previously described (20). Briefly, normal mice were sacrificed and their spleens were removed under sterile conditions, mashed in a sterile grinder, and washed in MEM containing 10% fetal calf serum, 0.02 M Hepes buffer, 1% L-glutamine, 1% penicillin, and 1% streptomycin. The spleen cells were adjusted to a concentration of 8 x lo7 cells in 5 ml of MEM. Each 5-ml suspension was plated in a loo-mm sterile plastic petri dish and incubated at 37°C for 2.5 hr. After this incubation, the nonadherent cells were poured off. Each petri dish containing adherent cells was reincubated at 4°C with 2 ml of cold MEM for about 10 min and after the incubation the adherent cells were administered iv to cortisone-treated and untreated mice. (c) Nylon wool-passed cells were prepared as described by Julius (21). RESULTS Effect of cortisone on the immune response of intact animals. Adult (C57 BL/6 x DBAR)F, (BDF,) mice were treated with a single large dose of cortisone 24 hr prior to immunization with TD antigens [SRBC (4) or FI,,-KLH (5)] or TI antigens
204
SHORT COMMUNICATIONS TABLE 1
Different Effect of Cortisone on the Immune Response to T-Dependent (TD) and T-Independent (TI) Antigens in Intact Animals” T-Independent antigens T-Dependent antigens Treatment
SRBC
FLsO-KLH
FLAECMFicoll
DNP4sLysFicoll
TNP,,LysFicoll
778 (138) 595 (121)O 793 (165y
477 (110) 286 (124)’ ND
197 (60) 117 (31)Q ND
123 (20) 124 (49)P ND
Direct PFC/lOB spleen cells (&SE) None Cortisone 5 mg Cortisone 10 mg
593 (134) 9 (12)
8 W
211 (32) 42 (12)” 109 (21)e
255 (122) 175 (81)P 267 (135)g
Indirect PFC/lOB spleen cells (*SE) None Cortisone 5 mg Cortisone 10 mg
168 (39) 29 (29)c 14 (10)b
183 (62) 81 (22)’ 58 (ll)d
46 (19) ND 68 (87)“
a BDF, mice, injected 24 hr previously with 5 or 10 mg cortisone acetate subcutaneously, together with normal untreated mice were immunized intraperitoneally with either TD antigens (SRBC or Fl,,-KLH)orTIantigens(FL,,-AECM-Ficoll, DNP,,-Lys-Ficoll,orTNP,,-Lys-Ficoll). Fivedays later the hemolytic plaque assay was performed to detect direct and the indirect plaque-forming cells (PFC). ND, Not done. b P < 0.001. = P < 0.005. d P < 0.025. eP < 0.05. ‘P < 0.1. II Not statistically significant.
[DNP,,-Lys-Ficoll(6), TNP,,-Lys-Ficoll(7), and Fl,,-AECM-Ficoll (S)]. The hemolytic plaque assay was used to detect direct (12) as well as indirect antibodyforming cells (13). The number of antibody-forming cells was expressed per million spleen lymphoid cells, since a single dose of 5 mg of cortisone resulted in a marked shrinkage of spleen size (14, 15) to about one-third of normal size. [spleen weight of cortisone (5 mg) treated mice: 36 f 4 mg]. Table 1 shows that administration of 5 or 10 mg of cortisone induced a profound decrease in the number of direct as well as indirect plaque-forming cells in response to TD antigens only. In contrast, the immune response to TI antigens was almost unaffected. This effect of cortisone on the TD immune response was time dependent. Thus, 1 week after 5 mg of cortisone was administered, although the spleen weight of the cortisone-treated animals was still about half the normal, the immune response to SRBC or to Fl,,-KLH was not significantly different from that of controls. [Immune response to Fl,,-KLH: untreated mice, 217 (+37) PFC/106 spleen cells; cortisone (5 mg)-treated mice, 147(k38) PFC/lOs spleen cells. Immune response to SRBC: untreated mice, 1254 (&158) PFC/106 spleen cells cortisone (5 mg)-treated mice, 904 (+216) PFC/106 spleen cells]: Two weeks after administration of cortisone, however, both the spleen weight and the immune response to TD antigens were normal. When a low dose of DNP,,-Lys-Ficoll (1 pg) was given to
205
SHORT COMMUNICATIONS
cortisone-treated mice, an enhancement of the immune response was found [untreated mice, 257 (~92) PFC/106 spleen cells; cortisone (5 mg)-treated mice, 1247 (+637) PFC/106 spleen cells], as previously reported with a higher dose of cortisone (10 mg) in vitro (16). This enhanced immune response by cortisone was not observed with doses either higher or lower than 1 pg of DNP,,-Lys-Ficoll. Similarly, no increase was noticed in response to various doses of TD antigens. Adoptive transfer of spleen cell suspensions from cortisone-treated donors to irradiated animals. Suspensions of spleen cells from donor animals treated with 5 mg of cortisone 24 hr previously were transferred to lethally irradiated (850 R) syngeneic recipient hosts. Each recipient received 40 x lo6 spleen cells iv and was immunized immediately after the cell transfer with either SRBC, Fl*,-KLH, or Fl,,-AECM-Ficoll. This procedure completely restored to normal levels the immune response of the cortisone-treated group to both TD antigens, SRBC and F&,,-KLH, whereas the response to Fl,,-AECM-Ficoll was enhanced (Table 2). When the above adoptive transfer experiment was carried out 1 week after cortisone treatment, the immune response of the cortisone-treated group to TD antigens was enhanced while that to both TI antigens (Fl,,-AECM-Ficoll and DNP,,-Lys-Ficoll) was unchanged (Table 2B). These experiments suggest that a radioresistant cell is directly affected by cortisone in vivo, as was shown in vitro by others (16, 17), since this cell appears to be an obvious candidate provided by the lethally irradiated host (18, 19). TABLE 2 Reconstitution Experiment in Lethally Irradiated Hosts with Spleen Cell Suspensions from Donors Treated with Cortisonea Direct PFCkpleen (*SE) T-Independent antigens T-Dependent antigens Treatment A None Cortisone 5 mg B None Cortisone 5 mg
SRBC 20,408 (8,100) 23,408 (6,400)d
F&KLH
FL,-AECMFicoll
DNP,,Ficoll
276,108 (50,400) 192,300 (27,200)“
8,760 (3,100) 31,500 (2,500)b
ND
33,400 (12,200) 77,800 (15,800) 106,100 (17,OoO)C 156,109 (29,400)’
62,100 (28,200) 57,300 (7,loO)d
6,640 (1,100) 6,380 (2,000)d
D Spleen cells, 40 x 109, from either normal BDF, donors or donors which had received subcutaneously 5 mg of cortisone acetate (A) 24 hr previously or(B) 8 days previously were transferred to irradiated (850 R) syngeneic mice. Immediately after cell repopulation, the recipients were challenged with either one of the following antigens: SRBC or Fl,-KLH, as TD antigens, or Fl,,AECM-Ficoll or DNP,-Lys-FicolI as TI antigens. Eight days later the direct and indirect plaque-forming cells were tested by hemolytic plaque assay. The indirect-plaque forming cells (PFC spleen cells 2 SE) for experiment A were as follows: For SRBC-untreated mice: 35,900 (8,380); cortisone (5 mg)-treated mice: 46,208 (14,400). For Fl,,-KLH-untreated mice: 235,400 (75,800); cortisone (5 mg)-treated mice: 266,900 (54,300). For Fl,,-Ficoll-untreated mice: 70 (1,500); cortisone (5 mg)-treated: 7,039 (2,010). The difference between untreated and treated groups for each antigen was not statistically significant. *P < 0.001. CP < 0.005. d Not statistically significant.
206
SHORT COMMUNICATIONS TABLE 3 Administration of Accessory Cells to Cortisone-Treated Mice” Direct PFC/lOGspleen cells (*SE)
T-Dependent antigens Treatment None Cortisone 5 mg Cortisone and 3 x adherent cells Cortisone and 3 x PE cells Cortisone and 3 x cells from cortisone-treated
SRBC
Fl,,-KLH
T-Independent antigen DNP,,-LysFicoll
957 (13O)b 95 (37)
302 (46)8 62 ( 19)h
600 (65)’ 434 (104)”
360 ( 130)d
ND
ND
467 ( 132)e
164 (38)’
426 (164)’
106 1Og 106 mice
101 (33)f
(1Adult BDF, mice were treated with 5 mg of cortisone subcutaneously. Then, 24 hr later, they were administered intravenously 3 x lo6 peritoneal exudate (PE) cells and, immediately afterwards, were immunized ip with either SRBC or Fl,,-KLH (TD antigens) or DNP,,-Lys-Ficoll (TI antigen). In addition, a group of cortisone-treated mice received adherent spleen cells and another group received 3 x lo6 peritoneal exudate cells derived from cortisone-treated mice prior to immunization with SRBC. Controls were treated with 5 mg of cortisone 24 hr previously and normal untreated mice. The direct hemolytic plaque-forming assay was performed 5 days after immunization. Values represent 19 S PFC/lO” of spleen cells (*SE). *-lP values are as follows: c - d, CO.01; b - d,
Different
IMMUNOLOGY
43, 202-208 (1979)
Effects of Cortisone on the Humoral to T-Dependent and T-Independent
Immune Response Antigens’
EVANGELIA MANTZOURANISAND YVES BOREL Division
of Immunology, Department of Pediatrics, The Children’s Hospital Harvard Medical School, Boston, Massachusetts, 02115 Received October
Medical
Center,
20, 1978
The effect of the administration of cortisone on the murine humoral immune response to either thymus-dependent (TD) or -independent (TI) antigens was studied in vivo. Whereas the thymus-dependent immune response was markedly suppressed, the thymus-independent immune response was preserved. The opposing effect of steroids on these two types of immune responses appears to be due to the relative independence of thymus-independent antigens Jf a radioresistant cortisone-sensitive accessory cell.
The immunosuppressive effect of corticosteroids on the humoral immune response to T-dependent (TD) antigens is well known (l-3), but there is no information on their influence on the T-independent (TI) antigen immune response in viva. Here we show that cortisone affects both types of immune response differently. The response to TD antigens is, as expected, markedly suppressed by cortisone. In contrast, the immune response to TD antigens remains unchanged or is increased. Reconstitution experiments carried out with cortisone-treated mice, used as either donors or recipients, suggest that the opposing effect of cortisone on both types of immune response might be explained by the action of the steroid on a radioresistant accessory cell. MATERIALS
AND METHODS
Animals. Six- to eight-week-old (C57Bl/6 x DBA/2)F, BDF, mice were obtained from Jackson Laboratory, Bar Harbor, Maine. Cortisone preparation. Cortisone acetate suspension (Merck, Sharp and Dohme, West Point, Pa.) was injected subcutaneously into each mouse as a single dose of 5 or 10 mg. Preparation of antigens and immunization. Sheep red blood cells (SRBC) (Colorado Serum Co. Laboratory, Denver, Colo.) were washed three times and resuspended in 0.15 M saline as a 10% solution and injected intraperitoneally (0.2 ml). Fluorescein isothiocyanate on Celite 10% (Fl) obtained from Calbiochem, San Diego, California, was conjugated with keyhole limpet hemocyanin (KLH; 1 This work was supported by National Institutes of Health Grant AI-AM 1386702and American Cancer Society Grant IM-178. 202 0008-8749/79/030202-07$02.00/O Copyright 6 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
SHORTCOMMUNICATIONS
203
Pacific Biomarine Supply Co., Venice, Calif.) as previously described (10). 0.2 mg, was injected Fluorescein,o keyhole limpet hemocyanin (F&,-KLH), intraperitoneally into each mouse in complete Freund’s adjuvant (CFA). 2,4-Dinitrobenzenesulfonic acid sodium salt (DNP) and 2,4,6-trinitrobenzenesulfonic acid (TNP) were purchased from Eastman Kodak Company, Rochester, New York, and Ficoll400 was obtained from Pharmacia Fine Chemicals, PiscatTNP,,-Lysine-Ficoll, and Fluoresaway, New Jersey. DNPd3- Lysine-Ficoll, cein,,-N-(2-aminoethyl)carbamylmethyl (AECM)-Ficoll were prepared as previously described (6, 8). Five micrograms of each of these antigens was injected intraperitoneally in CFA. Ten to twenty BDF, mice were included in all experimental groups. The subscript numbers indicate the molar ratio of hapten substitution on the carrier. Hemolyticpfaque assay. The Jerne hemolytic plaque assay, using as target either SRBC or SRBC coated with the appropriate hapten (i.e., TNP, Fl) as described by others (9-ll), was employed. Direct plaque-forming cells were assayed 5 days after immunization in intact animals or 8 days after immunization in irradiated animals. Indirect plaque-forming cells (13) were revealed by use of rabbit serum to react with mouse IgGi, IgGs,, and IgG, on immunodiffusion. The serum was used at a dilution of l/200. Indirect PFC were determined at the same time as direct PFC and were obtained by subtracting the direct PFC from the total number of PFC. Statistical analysis. Results were analyzed using Student’s t test. For the PFC per spleen or the PFC per lo6 spleen lymphoid cells, the geometric mean of each group and the standard error were calculated. Cell preparation. (a) Peritoneal exudate cells. Three milliliters of thioglycolate medium (Difco Laboratories, Detroit, Mich.) was injected intraperitoneally into each mouse. One week later the mice were sacrificed and the peritoneal exudate (PE) cells were harvested from the peritoneal cavity as described by others (16). The PE cells were 80-90% macrophages by neutral red staining. They were washed twice in Eagle’s minimum essential medium (MEM) fortified with 1% fetal calf serum (FCS) and 1%0heparin, and 3 x lo6 PE cells were infused into either cortisone-treated or untreated animals. (b) Adherent cells were prepared as previously described (20). Briefly, normal mice were sacrificed and their spleens were removed under sterile conditions, mashed in a sterile grinder, and washed in MEM containing 10% fetal calf serum, 0.02 M Hepes buffer, 1% L-glutamine, 1% penicillin, and 1% streptomycin. The spleen cells were adjusted to a concentration of 8 x lo7 cells in 5 ml of MEM. Each 5-ml suspension was plated in a loo-mm sterile plastic petri dish and incubated at 37°C for 2.5 hr. After this incubation, the nonadherent cells were poured off. Each petri dish containing adherent cells was reincubated at 4°C with 2 ml of cold MEM for about 10 min and after the incubation the adherent cells were administered iv to cortisone-treated and untreated mice. (c) Nylon wool-passed cells were prepared as described by Julius (21). RESULTS Effect of cortisone on the immune response of intact animals. Adult (C57 BL/6 x DBAR)F, (BDF,) mice were treated with a single large dose of cortisone 24 hr prior to immunization with TD antigens [SRBC (4) or FI,,-KLH (5)] or TI antigens
204
SHORT COMMUNICATIONS TABLE 1
Different Effect of Cortisone on the Immune Response to T-Dependent (TD) and T-Independent (TI) Antigens in Intact Animals” T-Independent antigens T-Dependent antigens Treatment
SRBC
FLsO-KLH
FLAECMFicoll
DNP4sLysFicoll
TNP,,LysFicoll
778 (138) 595 (121)O 793 (165y
477 (110) 286 (124)’ ND
197 (60) 117 (31)Q ND
123 (20) 124 (49)P ND
Direct PFC/lOB spleen cells (&SE) None Cortisone 5 mg Cortisone 10 mg
593 (134) 9 (12)
8 W
211 (32) 42 (12)” 109 (21)e
255 (122) 175 (81)P 267 (135)g
Indirect PFC/lOB spleen cells (*SE) None Cortisone 5 mg Cortisone 10 mg
168 (39) 29 (29)c 14 (10)b
183 (62) 81 (22)’ 58 (ll)d
46 (19) ND 68 (87)“
a BDF, mice, injected 24 hr previously with 5 or 10 mg cortisone acetate subcutaneously, together with normal untreated mice were immunized intraperitoneally with either TD antigens (SRBC or Fl,,-KLH)orTIantigens(FL,,-AECM-Ficoll, DNP,,-Lys-Ficoll,orTNP,,-Lys-Ficoll). Fivedays later the hemolytic plaque assay was performed to detect direct and the indirect plaque-forming cells (PFC). ND, Not done. b P < 0.001. = P < 0.005. d P < 0.025. eP < 0.05. ‘P < 0.1. II Not statistically significant.
[DNP,,-Lys-Ficoll(6), TNP,,-Lys-Ficoll(7), and Fl,,-AECM-Ficoll (S)]. The hemolytic plaque assay was used to detect direct (12) as well as indirect antibodyforming cells (13). The number of antibody-forming cells was expressed per million spleen lymphoid cells, since a single dose of 5 mg of cortisone resulted in a marked shrinkage of spleen size (14, 15) to about one-third of normal size. [spleen weight of cortisone (5 mg) treated mice: 36 f 4 mg]. Table 1 shows that administration of 5 or 10 mg of cortisone induced a profound decrease in the number of direct as well as indirect plaque-forming cells in response to TD antigens only. In contrast, the immune response to TI antigens was almost unaffected. This effect of cortisone on the TD immune response was time dependent. Thus, 1 week after 5 mg of cortisone was administered, although the spleen weight of the cortisone-treated animals was still about half the normal, the immune response to SRBC or to Fl,,-KLH was not significantly different from that of controls. [Immune response to Fl,,-KLH: untreated mice, 217 (+37) PFC/106 spleen cells; cortisone (5 mg)-treated mice, 147(k38) PFC/lOs spleen cells. Immune response to SRBC: untreated mice, 1254 (&158) PFC/106 spleen cells cortisone (5 mg)-treated mice, 904 (+216) PFC/106 spleen cells]: Two weeks after administration of cortisone, however, both the spleen weight and the immune response to TD antigens were normal. When a low dose of DNP,,-Lys-Ficoll (1 pg) was given to
205
SHORT COMMUNICATIONS
cortisone-treated mice, an enhancement of the immune response was found [untreated mice, 257 (~92) PFC/106 spleen cells; cortisone (5 mg)-treated mice, 1247 (+637) PFC/106 spleen cells], as previously reported with a higher dose of cortisone (10 mg) in vitro (16). This enhanced immune response by cortisone was not observed with doses either higher or lower than 1 pg of DNP,,-Lys-Ficoll. Similarly, no increase was noticed in response to various doses of TD antigens. Adoptive transfer of spleen cell suspensions from cortisone-treated donors to irradiated animals. Suspensions of spleen cells from donor animals treated with 5 mg of cortisone 24 hr previously were transferred to lethally irradiated (850 R) syngeneic recipient hosts. Each recipient received 40 x lo6 spleen cells iv and was immunized immediately after the cell transfer with either SRBC, Fl*,-KLH, or Fl,,-AECM-Ficoll. This procedure completely restored to normal levels the immune response of the cortisone-treated group to both TD antigens, SRBC and F&,,-KLH, whereas the response to Fl,,-AECM-Ficoll was enhanced (Table 2). When the above adoptive transfer experiment was carried out 1 week after cortisone treatment, the immune response of the cortisone-treated group to TD antigens was enhanced while that to both TI antigens (Fl,,-AECM-Ficoll and DNP,,-Lys-Ficoll) was unchanged (Table 2B). These experiments suggest that a radioresistant cell is directly affected by cortisone in vivo, as was shown in vitro by others (16, 17), since this cell appears to be an obvious candidate provided by the lethally irradiated host (18, 19). TABLE 2 Reconstitution Experiment in Lethally Irradiated Hosts with Spleen Cell Suspensions from Donors Treated with Cortisonea Direct PFCkpleen (*SE) T-Independent antigens T-Dependent antigens Treatment A None Cortisone 5 mg B None Cortisone 5 mg
SRBC 20,408 (8,100) 23,408 (6,400)d
F&KLH
FL,-AECMFicoll
DNP,,Ficoll
276,108 (50,400) 192,300 (27,200)“
8,760 (3,100) 31,500 (2,500)b
ND
33,400 (12,200) 77,800 (15,800) 106,100 (17,OoO)C 156,109 (29,400)’
62,100 (28,200) 57,300 (7,loO)d
6,640 (1,100) 6,380 (2,000)d
D Spleen cells, 40 x 109, from either normal BDF, donors or donors which had received subcutaneously 5 mg of cortisone acetate (A) 24 hr previously or(B) 8 days previously were transferred to irradiated (850 R) syngeneic mice. Immediately after cell repopulation, the recipients were challenged with either one of the following antigens: SRBC or Fl,-KLH, as TD antigens, or Fl,,AECM-Ficoll or DNP,-Lys-FicolI as TI antigens. Eight days later the direct and indirect plaque-forming cells were tested by hemolytic plaque assay. The indirect-plaque forming cells (PFC spleen cells 2 SE) for experiment A were as follows: For SRBC-untreated mice: 35,900 (8,380); cortisone (5 mg)-treated mice: 46,208 (14,400). For Fl,,-KLH-untreated mice: 235,400 (75,800); cortisone (5 mg)-treated mice: 266,900 (54,300). For Fl,,-Ficoll-untreated mice: 70 (1,500); cortisone (5 mg)-treated: 7,039 (2,010). The difference between untreated and treated groups for each antigen was not statistically significant. *P < 0.001. CP < 0.005. d Not statistically significant.
206
SHORT COMMUNICATIONS TABLE 3 Administration of Accessory Cells to Cortisone-Treated Mice” Direct PFC/lOGspleen cells (*SE)
T-Dependent antigens Treatment None Cortisone 5 mg Cortisone and 3 x adherent cells Cortisone and 3 x PE cells Cortisone and 3 x cells from cortisone-treated
SRBC
Fl,,-KLH
T-Independent antigen DNP,,-LysFicoll
957 (13O)b 95 (37)
302 (46)8 62 ( 19)h
600 (65)’ 434 (104)”
360 ( 130)d
ND
ND
467 ( 132)e
164 (38)’
426 (164)’
106 1Og 106 mice
101 (33)f
(1Adult BDF, mice were treated with 5 mg of cortisone subcutaneously. Then, 24 hr later, they were administered intravenously 3 x lo6 peritoneal exudate (PE) cells and, immediately afterwards, were immunized ip with either SRBC or Fl,,-KLH (TD antigens) or DNP,,-Lys-Ficoll (TI antigen). In addition, a group of cortisone-treated mice received adherent spleen cells and another group received 3 x lo6 peritoneal exudate cells derived from cortisone-treated mice prior to immunization with SRBC. Controls were treated with 5 mg of cortisone 24 hr previously and normal untreated mice. The direct hemolytic plaque-forming assay was performed 5 days after immunization. Values represent 19 S PFC/lO” of spleen cells (*SE). *-lP values are as follows: c - d, CO.01; b - d,
