Blut, Band 32, Seite 353-360 (1976) Abteilung Klinische Immunologic und Transfusionsmedizin der Universit~it Giessen and Department of Microbiology, Mayo Clinic and Mayo Medical School, Rochester, Minnesota, USA.
Inhibitory Activity of Medium-dialyzed Transfer Factor on Lymphocyte Blastogenesis Christian Mueller-Eckhardt and Roy E. Ritts jr. Summary Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFma). Several batches of TUna were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Histoplasmin). No transfer of immunologic reactivity by TFma was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells. Zusammenfassung Menschlicher Transferfaktor wurde yon normalen Spendern mit ausgepr~gten verz6gerten Hautreaktionen gegen gew6hnliche mikrobielle Antigene dutch Dialyse yon Lymphozytenextrakt gegen Gewebekulturmedium hergestellt ( T F a ) . Verschiedene Pr~tparationen yon TFma wurden auf ihre F~ihigkeit untersucht, die DNASynthese yon unsensibilisierten Lymphozyten in vitro in Anwesenheit der korrespondierenden Antigene (PPD, SKSD, Candida, Histoplasmin) spezifisch zu steigern. In keinem Fall konnte ein Transfer immunologischer Reaktivit~t durch TFma nachgewiesen werden. Jedoch lieB sich h~iufig ein signifikanter, unspezifischer Hemmeffekt des TFma in Lymphozytenkulturen feststellen, die mit Antigenen, PHA oder allogeneischen Zellen stimuliert wurden. Key words: Transfer factor, lymphocyte culture.
It has been demonstrated by Lawrence and associates that dialyzable transfer factor (TF), a putative initiator of cellular immunity, is able of converting uncommitted or naive lymphocytes to an antigen-responsive state subsequent to exposure to antigen [9, 10]. As a pre-existing moiety of circulating leukocytes, TF is liberated in 15 to 30 min following contact with antigen or subsequent to cell disruption or dialysis of cell lysate and is thought to have an immunologically specific effect. Structurally, it represents a small dialyzable molecule (molecular weight 20 • 20 mm induration) to one or several of the antigens under study. The antigens were (a) Tuberculin ( P P D ) (Parke Davis and Co., Detroit, Michigan), 0.1 ml (containing 5 units of PPD) ; (b) Monilia albicans, allergenic extract (Hollister-Stier Laboratory, Spokane, Washington), 0.1 ml of a 1 : 100 dilution in sterile, pyrogen-free saline; (c) Stredofokinase-StredOtodornase(SK-SD) (Varidase | (Lederle Laboratory Division, Pearl River, New York) diluted in sterile saline to contain 50 units of SK and 12.5 units of SD in 0.1 ml; (d) HistodOlasmin (Parke Davis and Co.), 0.1 ml in sterile saline. In addition to delayed cutaneous reactivity, the responses of all but one TF donor were assessed in the in vitro lymphocyte transformation test. In one donor (D. G., TF No. VII), only the lymphocyte transformation test was performed. Four batches of TF were prepared from two donors (L. B. and K. L.) with documented in vivo potency of previous waterdialyzed TF preparations. After each of three administrations, L. B.'s TF had conferred both Candida and SK-SD reactivity and K. L.'s TF histoplasmin reactivity on recipients unresponsive to these antigens. Donor L. B. had consistently yielded active TF when blood was drawn 2 4 4 8 h following skin testing, but on two occassions when she was not skintested immediately prior to blood collection, no conversion of skin reactivity was caused in the recipient by TF. Preparalion ofdialyzable TF. 400 ml of venous blood were collected into disposable plastic syringes and anticoagulated with preservative-free heparin (Panheparin| Abbot Lab., North Chicago, Illinois; final concentration 100 units/ml) or into Fenwall plastic bags containing 2250 units of heparin in 30 ml of stabilizer. For preparation of TF No. I to No. IV, the heparinized blood was mixed with 1/6 of its volume with a 6% dextran/saline solution (Gentran 75 | Travenol) and allowed to sediment at room temperature. After 45-60 min the cells of the supernatant leukocyte-rich plasma were separated on a sterile Ficol-Hypaque gradient by centrifugation for 30 rain at 400 • g. An almost pure fraction of lymphocytic cells, practically free of granulocytes, with approximately 80% small lymphocytes was collected from the interphase. For the preparation of TF No. V to VIII, the blood was diluted with an equal volume of sterile, pyrogen-free saline and immediately separated on a Ficoll-Hypaque gradient as described above. By this modification the yield of lymphocytic ceils was considerably increased. The ceils were then washed twice in sterile saline, adjusted to a final concentration of 1 • 10s lymphocytes/ml and frozen-thawn 10 times in dry ice-acetone. After addition of nonpyrogenic crystalline DNAse (Worthington Biochemical Corporation, Freehold, New Jersey; final concentration 1 mg/ml) and sterile MgSO4 (final concentration 5 mM) the cell extract was incubated for 30 min at 37~ C in a waterbath. Thereafter the lysates were dialyzed against either 100 volumes of sterile, pyrogen-free water, the dialysis fluid freeze-dried
Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis
355
and the remaining material reconstituted with normal saline to the original volume of the cell lysate (TF No. I-IV) or dialyzed against sterile culture medium (RPMI 1640) for 18 h at 4 ~C (ratio of dialysate to dialysis volume 1 : 5 or 1 : 10). All TF samples were passed through 0.45 b~m membrane filters (Millipore Corp., Bedford, Massachusetts) and stored in 5 ml aliquots at ~ 2 5 ~ C until used. Great care was taken during the entire procedure to maintain sterile conditions. Preparation of lymphocyte cultures. Lymphocytes for culture were separated from venous heparinized blood diluted with equal amounts of sterile culture medium (RPMI 1640), centrifuged on a Ficoll-Hypaque gradient, washed twice in RPMI 1640 and adjusted to a lymphocyte concentration of 0.5 • 106 cells/ml in RPMI 1640 with 15% heat-inactivated fetal calf serum (Reheis Chemical Co., Phoenix, Arizona), Penicillin (100 units/ml) and Streptomycin (100 b~g/ml; Difco, Detroit, Michigan). The cultures were set up in triplicates in sterile microtiter wells (Microtest II Tissue Culture Plates, Falcon, Division of BioQuest, Cockeysville, Maryland). In each well 0.2 ml of the cell suspension (1 • 105 lymphocytes), 10 bd of antigen or mitogen and 20 to 100 bd of TF were placed. Appropriate controls were included. In one series of experiments the lymphocyte concentration was increased to 1 • 106/ml of culture medium with 30% of fetal calf serum, 200 units of penicillin and 200 btg/ml of streptomycin. 0.1 ml of the cell suspension were mixed with 0.1 ml of mediumdialyzed TF, resulting in identical cell concentration (5 • 105/ml) as above. The cultures were incubated at 37~ C with a flow rate of 4 1/min of air and 0.4 1/min of CO~ for four to seven days. Twelve to 18 h after labeling with 1 btCi of aH-thymidine the cultures were harvested by a multiple well aspirator on filter paper (Grade 934 AH Glass fiber filter, Reeve Angel, Clifton, New Jersey) and counted in a Beckman Liquid Scintillation Counter.
Preparation of anligens and mitogensfor in vitro use (a) Tuberculin (PPD), low in preservative (Parke, Davis and Co., lot no. 974775, courtesy of Dr. Devlin), 10 ~xg/well in 10 bd of RPMI 1640; (b) Streptokinase-Streptodornase (Lederle), dialyzed versus sterile saline to remove the preservative; 1 unit of SK and 0.25 unit of SD in 10 btl per well; (c) "Candidin" (Monilia albicans, Hollister-Stier), dialyzed versus sterile saline, 10 bd/well of a 1 : 5 dilution in RPMI 1640; (d) Histoplasmin (Parke, Davis and Co.), dialyzed versus sterile saline, 10 ixt/well, undiluted; (e) Bacto-Phytobemagglutinin M (Difco) (PHA), dilution 1 : 25 and 1 : 100 in RPMI 1640; 10 hal/well.
Mixed lymphocyte culture (MLC). Stimulating cells were inactivated with mitomycin C (Nutritional Biochemical Corp., Cleveland, Ohio) at a final concentration of 250 b~g/mlfor 30 rain at 37~ C. Responding cells and stimulating ceils of four unrelated persons were separated in an identical manner as described for the lymphocyte culture. 0.1 ml of responding and 0.1 ml of stimulating cell suspensions in a final concentration of 1 • 106 cells/ml were incubated with or without 40 bd of four different batches of TF (No. V to VIII) for five days and then processed as outlined above. Results
The effect of T F on ]ymphoblastogenesis. A total of 52 lymphocyte suspensions of 31 individuals were tested for lymphoblastogenesis in response to the four antigens u n d e r study. Kinetic studies showed that the maximum reactions with P P D and Candida occurred after 6 to 7 days of culture. Thus, a culture time of six days was adopted. P H A cultures were terminated after 3 days. I n preliminary experiments, lymphocytes of antigen-positive and antigen-negative donors were cultured with three different batches of water-dialyzed and mediumdialyzed T F in the presence or absence of antigens. No significant differences between cultures supplemented with water-dialyzed T F as compared to medium-dialyzed T F were observed. Therefore, all further experiments were carried out exclusively with medium-dialyzed TF.
43 44 45 46
43 44 45 46
43 44 45 46
VI
VII
VIII
2.27 13,83 0.70 1.27
1.14 10.36 2.76 0.77
0.80 6.46 2.94 1.41
1.36 7.56 3.32 2.29
1.35 12.09 0.95 " 2.48
1.06 6.65 1.77 0.81
0.87 4.92 1.19 1.09
1.77 4.70 3.19 2.42
Inhibitory Activity of Medium-dialyzed Transfer Factor on Lymphocyte Blastogenesis Christian Mueller-Eckhardt and Roy E. Ritts jr. Summary Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFma). Several batches of TUna were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Histoplasmin). No transfer of immunologic reactivity by TFma was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells. Zusammenfassung Menschlicher Transferfaktor wurde yon normalen Spendern mit ausgepr~gten verz6gerten Hautreaktionen gegen gew6hnliche mikrobielle Antigene dutch Dialyse yon Lymphozytenextrakt gegen Gewebekulturmedium hergestellt ( T F a ) . Verschiedene Pr~tparationen yon TFma wurden auf ihre F~ihigkeit untersucht, die DNASynthese yon unsensibilisierten Lymphozyten in vitro in Anwesenheit der korrespondierenden Antigene (PPD, SKSD, Candida, Histoplasmin) spezifisch zu steigern. In keinem Fall konnte ein Transfer immunologischer Reaktivit~t durch TFma nachgewiesen werden. Jedoch lieB sich h~iufig ein signifikanter, unspezifischer Hemmeffekt des TFma in Lymphozytenkulturen feststellen, die mit Antigenen, PHA oder allogeneischen Zellen stimuliert wurden. Key words: Transfer factor, lymphocyte culture.
It has been demonstrated by Lawrence and associates that dialyzable transfer factor (TF), a putative initiator of cellular immunity, is able of converting uncommitted or naive lymphocytes to an antigen-responsive state subsequent to exposure to antigen [9, 10]. As a pre-existing moiety of circulating leukocytes, TF is liberated in 15 to 30 min following contact with antigen or subsequent to cell disruption or dialysis of cell lysate and is thought to have an immunologically specific effect. Structurally, it represents a small dialyzable molecule (molecular weight 20 • 20 mm induration) to one or several of the antigens under study. The antigens were (a) Tuberculin ( P P D ) (Parke Davis and Co., Detroit, Michigan), 0.1 ml (containing 5 units of PPD) ; (b) Monilia albicans, allergenic extract (Hollister-Stier Laboratory, Spokane, Washington), 0.1 ml of a 1 : 100 dilution in sterile, pyrogen-free saline; (c) Stredofokinase-StredOtodornase(SK-SD) (Varidase | (Lederle Laboratory Division, Pearl River, New York) diluted in sterile saline to contain 50 units of SK and 12.5 units of SD in 0.1 ml; (d) HistodOlasmin (Parke Davis and Co.), 0.1 ml in sterile saline. In addition to delayed cutaneous reactivity, the responses of all but one TF donor were assessed in the in vitro lymphocyte transformation test. In one donor (D. G., TF No. VII), only the lymphocyte transformation test was performed. Four batches of TF were prepared from two donors (L. B. and K. L.) with documented in vivo potency of previous waterdialyzed TF preparations. After each of three administrations, L. B.'s TF had conferred both Candida and SK-SD reactivity and K. L.'s TF histoplasmin reactivity on recipients unresponsive to these antigens. Donor L. B. had consistently yielded active TF when blood was drawn 2 4 4 8 h following skin testing, but on two occassions when she was not skintested immediately prior to blood collection, no conversion of skin reactivity was caused in the recipient by TF. Preparalion ofdialyzable TF. 400 ml of venous blood were collected into disposable plastic syringes and anticoagulated with preservative-free heparin (Panheparin| Abbot Lab., North Chicago, Illinois; final concentration 100 units/ml) or into Fenwall plastic bags containing 2250 units of heparin in 30 ml of stabilizer. For preparation of TF No. I to No. IV, the heparinized blood was mixed with 1/6 of its volume with a 6% dextran/saline solution (Gentran 75 | Travenol) and allowed to sediment at room temperature. After 45-60 min the cells of the supernatant leukocyte-rich plasma were separated on a sterile Ficol-Hypaque gradient by centrifugation for 30 rain at 400 • g. An almost pure fraction of lymphocytic cells, practically free of granulocytes, with approximately 80% small lymphocytes was collected from the interphase. For the preparation of TF No. V to VIII, the blood was diluted with an equal volume of sterile, pyrogen-free saline and immediately separated on a Ficoll-Hypaque gradient as described above. By this modification the yield of lymphocytic ceils was considerably increased. The ceils were then washed twice in sterile saline, adjusted to a final concentration of 1 • 10s lymphocytes/ml and frozen-thawn 10 times in dry ice-acetone. After addition of nonpyrogenic crystalline DNAse (Worthington Biochemical Corporation, Freehold, New Jersey; final concentration 1 mg/ml) and sterile MgSO4 (final concentration 5 mM) the cell extract was incubated for 30 min at 37~ C in a waterbath. Thereafter the lysates were dialyzed against either 100 volumes of sterile, pyrogen-free water, the dialysis fluid freeze-dried
Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis
355
and the remaining material reconstituted with normal saline to the original volume of the cell lysate (TF No. I-IV) or dialyzed against sterile culture medium (RPMI 1640) for 18 h at 4 ~C (ratio of dialysate to dialysis volume 1 : 5 or 1 : 10). All TF samples were passed through 0.45 b~m membrane filters (Millipore Corp., Bedford, Massachusetts) and stored in 5 ml aliquots at ~ 2 5 ~ C until used. Great care was taken during the entire procedure to maintain sterile conditions. Preparation of lymphocyte cultures. Lymphocytes for culture were separated from venous heparinized blood diluted with equal amounts of sterile culture medium (RPMI 1640), centrifuged on a Ficoll-Hypaque gradient, washed twice in RPMI 1640 and adjusted to a lymphocyte concentration of 0.5 • 106 cells/ml in RPMI 1640 with 15% heat-inactivated fetal calf serum (Reheis Chemical Co., Phoenix, Arizona), Penicillin (100 units/ml) and Streptomycin (100 b~g/ml; Difco, Detroit, Michigan). The cultures were set up in triplicates in sterile microtiter wells (Microtest II Tissue Culture Plates, Falcon, Division of BioQuest, Cockeysville, Maryland). In each well 0.2 ml of the cell suspension (1 • 105 lymphocytes), 10 bd of antigen or mitogen and 20 to 100 bd of TF were placed. Appropriate controls were included. In one series of experiments the lymphocyte concentration was increased to 1 • 106/ml of culture medium with 30% of fetal calf serum, 200 units of penicillin and 200 btg/ml of streptomycin. 0.1 ml of the cell suspension were mixed with 0.1 ml of mediumdialyzed TF, resulting in identical cell concentration (5 • 105/ml) as above. The cultures were incubated at 37~ C with a flow rate of 4 1/min of air and 0.4 1/min of CO~ for four to seven days. Twelve to 18 h after labeling with 1 btCi of aH-thymidine the cultures were harvested by a multiple well aspirator on filter paper (Grade 934 AH Glass fiber filter, Reeve Angel, Clifton, New Jersey) and counted in a Beckman Liquid Scintillation Counter.
Preparation of anligens and mitogensfor in vitro use (a) Tuberculin (PPD), low in preservative (Parke, Davis and Co., lot no. 974775, courtesy of Dr. Devlin), 10 ~xg/well in 10 bd of RPMI 1640; (b) Streptokinase-Streptodornase (Lederle), dialyzed versus sterile saline to remove the preservative; 1 unit of SK and 0.25 unit of SD in 10 btl per well; (c) "Candidin" (Monilia albicans, Hollister-Stier), dialyzed versus sterile saline, 10 bd/well of a 1 : 5 dilution in RPMI 1640; (d) Histoplasmin (Parke, Davis and Co.), dialyzed versus sterile saline, 10 ixt/well, undiluted; (e) Bacto-Phytobemagglutinin M (Difco) (PHA), dilution 1 : 25 and 1 : 100 in RPMI 1640; 10 hal/well.
Mixed lymphocyte culture (MLC). Stimulating cells were inactivated with mitomycin C (Nutritional Biochemical Corp., Cleveland, Ohio) at a final concentration of 250 b~g/mlfor 30 rain at 37~ C. Responding cells and stimulating ceils of four unrelated persons were separated in an identical manner as described for the lymphocyte culture. 0.1 ml of responding and 0.1 ml of stimulating cell suspensions in a final concentration of 1 • 106 cells/ml were incubated with or without 40 bd of four different batches of TF (No. V to VIII) for five days and then processed as outlined above. Results
The effect of T F on ]ymphoblastogenesis. A total of 52 lymphocyte suspensions of 31 individuals were tested for lymphoblastogenesis in response to the four antigens u n d e r study. Kinetic studies showed that the maximum reactions with P P D and Candida occurred after 6 to 7 days of culture. Thus, a culture time of six days was adopted. P H A cultures were terminated after 3 days. I n preliminary experiments, lymphocytes of antigen-positive and antigen-negative donors were cultured with three different batches of water-dialyzed and mediumdialyzed T F in the presence or absence of antigens. No significant differences between cultures supplemented with water-dialyzed T F as compared to medium-dialyzed T F were observed. Therefore, all further experiments were carried out exclusively with medium-dialyzed TF.
43 44 45 46
43 44 45 46
43 44 45 46
VI
VII
VIII
2.27 13,83 0.70 1.27
1.14 10.36 2.76 0.77
0.80 6.46 2.94 1.41
1.36 7.56 3.32 2.29
1.35 12.09 0.95 " 2.48
1.06 6.65 1.77 0.81
0.87 4.92 1.19 1.09
1.77 4.70 3.19 2.42
