Journal of Immunological Methods, 153 (1992) 73-79
73
© 1992 ElsevierScience Publishers B.V, All rights reserved 0022-1759/92/$05.110
JIM06388
An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays Carin E . G . Havenith, A n n e t t e J. Breedijk, M a r i n a A . M . V e r d a a s d o n k , E d W . A . Kamperdijk and R o b e r t H.J. B e e l e n Department of Cell Biology, Dit,ision of Electronmicroscopy, Vrije Unirersiteit,Amsterdam, Netherlands
(Received 27 January 1992, revised received20 March 1992,accepted 31 March 1992) To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating la-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of la-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays. Key words: Isolation procedure; Nylonwool column; Magneticbead; Sephadex G-10; Dendritic cell; Antigen presentation; T cell
Introduction Various methods for the isolation of antigenprimed T cells suitable for antigen-specific T cell Correspondence to: C.E.G. Havenith, Department of Cell Biology, Division of Electronmieroscopy, Medical Faculty, Vrije Universiteit,Van der Boechorststraat 7, 1081 BT Amsterdam, Netherlands. Tel.: 31-20-5483939; Fax: I}31-206462228. Abbreciations: APC, antigen presenting cells; BSA, bovine serum albumin; DC, dendritic cells; cpm, counts per rain; FCS, fetal calf serum; GT, copolymer glutamine-tyrosine; 3H-TdR, [3H]thymidine; Mab, monoelonalantibody.
proliferation assays have been developed. To deplete primed lymph node or spleen cell suspensions of macrophagcs and of other adherent cells the differenticl attachment to plastic petri dishes has been used (Walker et al., 1983). Also depletion of immunoglobulin-bearing cells from cell suspensions has been described using nylon wool columns (Julius et al., 1973). The method involving the passage of cell suspensions through a column of Sephadex G-10 beads results in the selective retention of antibody-forming cells and of antigen presenting cells (APC) (Ly and Mishell, 1974).
Two of the most common procedures used are combinations of the above mentioned ones, namely a three-step procedure: 1.5 h adherence, a nylon wool column and a Sephadex G-10 column (Walker et al., 1983) and a two-step procedure: a passage through two consecutive columns of Sephadex G-10 (Ly and Mishell, 1974). The use of these two isolation methods gives no satisfactory results in the rat system, sinde the background level responses of antigen-primed T cell-enriched lymph node or spleen cells are invariably high, probably due to contamination with accessory la-positive cells, predominantly B cells and DC. B cells are able to present antigen (Unanue et al., 1984), whereas DC are known to be vet), potent APC capable of given responses at low stimulator to responder ratios (Steinman et al., 1985). To improve the evaluation of the capacity of rat APC to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background responses. Therefore, in the present paper a rapid and reproducible isolation procedure for the isolation of pure T cells is presented for the rat, using a nylon wool column, to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove the last contaminating la-positire cells.
Materials and methods
Animals Inbred male AC1/Mal rats, weighing 180-200 g, were obtained from "Centraal Proefdierenbedrijf" (TNO, Zeist, Netherlands). All animals were kept under routine laboratory conditions and allowed food and water ad libitum.
Immunization procedure AC1/Mal rats, which are high responders for the copolymer glutamine-tyrosine (GT, ICN ImmunoBioiogicals, Lisle, Israel) in an la dependent manner (Amerding et al., 1974), served as source of responder cells for the GT-primed proliferation assay. The rats were anesthetized with ether
(Merck Nederland, Amsterdam, Netherlands) and immunized in both hind footpads with 100 p.g of GT emulsified in 100/zl Freund's complete adjuvant (Difco Laboratories, Detroit, USA).
Preparation of T cell-enriched cell populations Isolation of T cell-enriched cell populations of the draining popliteal lymph nodes or the spleens of GT-primed rats 2 weeks after immunization was performed following three different methods. After the lymph nodes or spleens had been minced and pressed gently through a monodur gauze (100 /~m; Stokvis&Smits, IJmuiden, Netherlands), the total cell suspension (designated as total cells) was split into three equal portions to compare the three different isolation procedures. The first isolation method, performed according to Ly and Misheli (1974), uses a 1.5 h adherence step and two times a Sephadex G-10 column. The cells were cultured in culture medium for 1.5 h at 37°C in tissue culture flasks (Greiner, Aiphen a.d. Rijn, Netherlands). Culture medium comprised complete RPMI 1640 medium (Flow Laboratories, lrvine Ayrshire, Scotland), 10% heat inactivated fetal calf serum (FCS), 20 mM L-glutamine, penicillin 50 U/ml, and streptomycin 50/~g/ml (all from Gibco Ltd, Pairsley, Scotland). The collected non-adherent cells were resuspended in 2 ml culture medium and placed on top of a Sephadex G-10 column. Briefly, a Sephadex G-10 column was made in a barrel of a 10 ml syringe (Sherwood Medical, Crawley, UK) plugged with glass wool and filled with 6 ml of autoclaved Sephadex G-10 (Pharmacia, Uppsala, Sweden), swollen for four times in saline. The column was rinsed with 25 mi of warm culture medium before the cells were placed on the column, by mixing the top 2 ml of Sephadex with the cell suspension. The cells were allowed to penetrate the Sephadex bed for 10 rain at 37°C, the top 2 ml were mixed again and penetration was allowed for another 5 min at 37°C. Then the column was washed through with 20-25 ml of warm culture medium. The effluent cells were placed on top of a second Sephadex G-10 column and the same procedure was performed as above. Subsequently again the effluent cells (designated as adherence/sephadex/sephadex) were collected.
The second isolation method was performed following a three differed step, namely 1.5 h adherence, a nylon wool column and a Sephadex G-10 column, according to Walker et al. (1983). The cells were cultured in culture medium for 1.5 h at 37°C in tissue culture flasks (Greiner). The collected non-adherent cells were loaded onto a nylon wool column (maximal 2 x l0 s cells per column) and washed into the nylon wool with 0.5-1 ml of warm (37°C) culture medium according to the method of Julius et al. (1973). Nylon wool columns comprised of about 1.0 g aliquots of nylon wool (Leuko-Pak Leukocyte Filters, Fenwal Laboratories, Illinois, U.S.A) packed into the barrels of 10 ml plastic syringes (Sherwood Medical) up to the 8 mi mark. These columns were autoclaved, rinsed with 20 ml warm culture medium and placed in an incubator at 37°C at least 1 h before use. After loading the cells onto the column, the column was replaced in the incubator and left for 45 min. The column was then washed slowly with warm culture medium and the first 20 ml of effluent was collected. The effluent cells were resuspended in 2 ml culture medium and placed on top of a Sephadex G-10 column (see above). The effluent cells (designated as adherence/nyion/sephadex) were collected. The third and newly developed isolation method comprised a nylon wool column and a removal of la-positive cells making use of the monocional antibody (Mab) OX-6 (a gift from Dr. Barclay, Oxford, UK) and of magnetic beads coated with sheep anti-mouse lgG (Dynal, Oslo, Norway). The cell suspension was loaded onto a nylon wool column (see above). After collecting the effluent cells of the nylon wool column, the cells were washed twice in RPMI 1640 medium with 0.5% bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, USA) and incubated for 15 rain on ice with 20 p.I ascites of OX-6 in 1 ml medium with 0.5% BSA. The cells were washed twice and incubated for 15 min on ice with magnetic beads coated with sheep anti-mouse IgG (Dynal). The number of magnetic beads used was 30-40% of the total amount of effluent cells. The la-positive cells attached to the magnetic beads were removed using a magnet (Dynal). The non-attaching cell population (designated as nylon/anti-la beads) was collected.
The T cell-enriched cell populations acquired with the three above described methods w e r e washed twice, the purity was phenotypically evaluated in cytocentrifuge preparations and functionally they were compared as responder cells in the GT-primed T cell proliferation assay, using dendritic cells as APC.
Preparation of dendritic cells Cell suspensions of the spleen were enriched for dendritic cells (DC) according to the method of Knight et al. (1986). Briefly, the spleen was minced and digested in culture medium containing collagenase/DNA~ (150 U/ml collagenase type Ill, 10 U/ml DNAse type I, both from Sigma Chemical Co.) for 90 rain at 37°C. The collected cells were cultured overnight and enriched for DC using a discontinuous gradient (14.5% w / v Nycodenz, Nycomed, Oslo, Norway). After this gradient, centrifuged at 600 × g at 20°C for 20 rain, cells were collected from the interphase. These cells, viability greater than 95% and purified to about 70% DC, were used as antigen presenting cells in the GT-primed T cell proliferation assay. DC were defined according to Havenith et ai. (1992), namely strongly la-positive cells having acid phosphatase activity in a central spot near the nucleus and extending cell processes.
GT-primed T cell proliferation assay Responder cells (1, 2 or 4 × 105 T cell-enriched lymph node or spleen cells/well) were plated into flat-bottomed 96-well culture plates (Greiner). GT was added at a final concentration of 100 p.g/ml. Splenic DC, irradiated with 3000 rad (~°Co), were added in different concentrations to the responder cells in triplicate, into a final volume of 200 p.I. The culture medium was supplemented with 50 p.M fl-mercaptoethanol (Merck Nederland). The cultures were incubated for 3 days at 37°C in a humidified atmosphere of 5% CO 2 in air. After adding 1 p.Ci of [3H] thymidine per well (3H-TdR, Amersham, UK), the amount of 3H-TdR uptake was determined 18 h later using an automatic cell harvester to collect the cultured cells and analyzed in a liquid scintillation counter. Data, mean values of triplicate wells, are expressed in counts per min (cpm).
Staining procedures Detection of antigen on cytocentrifuge preparations of spleen dendritic cells and the different T cell-enriched populations was done by an indirect immunoperoxidasc method and a double staining method of la antigens and acid phosphatase activity as described in detail by Havenith et al. (1992). Used were the Mabs OX-6 ( a l a ) and OX-19 combined with OX-52 (pan T cell markers; a gift from Dr. Barclay, Oxford, UK), and His 14 (pan B cell marker; a gift from Dr. Nieuwenhuis, Groningen, Netherlands).
Statistical analysis Differences between the percentages of OX19/52-positive cells and the background responses of the different isolated T cell populations were tested with the Mann-Whitney U t e ~
Results
loo ANS
s0
~' "i
NB
e0 40
20 0 0X-19/52
HIs 14
OX-6
monoclonal a ~ . ~ Y
Fig. I. Expression of OX-19/52, Hi~ ~t~t. ~ (~.6 ~.~1'diffet~n~ T cell-enrichedlymph node cell popla~'~t~;. ~C: ~otal0ymph node) cell suspension. ASS: cells isolated with an adherence step and two times Sephadex G-10 column. ANS: cells isolated with an adherence step, a nylon wool column and a Sephadex G-10 column. NB: cells isolated with a nylon wool column and an indirect method with magnetic beads to remove la-positivecells. The data represent mean percentages + SD of positive cells for the monoclonal antibodies OX19/'52, His 14 and OX-6(n = 3).
Cell composition of the different isolated T cell populations The cell counts of the different populations of T cells of the lymph nodes of GT-primed animals (total cells, adherence/sephadex/sephadex, adherence/nylon/sephadex, nylon/anti-la beads) are shown in Fig. 1. These data show that the optimal T cell enrichment (percentage of OX19/52-positive cells) is achieved with method nyion/anti-la beads (98%), followed by method adherence/nylon/sephadex (85%), whereas with method adherence/sephadex/sephadex (62%) hardly any purification is achieved compared to total cells (54%). Method nylon/anti-la beads differed significantly from method adherence/ nylon/sephadex and a d h e r e n c e / s e p h a d e x / sephadex ( p
73
© 1992 ElsevierScience Publishers B.V, All rights reserved 0022-1759/92/$05.110
JIM06388
An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays Carin E . G . Havenith, A n n e t t e J. Breedijk, M a r i n a A . M . V e r d a a s d o n k , E d W . A . Kamperdijk and R o b e r t H.J. B e e l e n Department of Cell Biology, Dit,ision of Electronmicroscopy, Vrije Unirersiteit,Amsterdam, Netherlands
(Received 27 January 1992, revised received20 March 1992,accepted 31 March 1992) To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating la-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of la-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays. Key words: Isolation procedure; Nylonwool column; Magneticbead; Sephadex G-10; Dendritic cell; Antigen presentation; T cell
Introduction Various methods for the isolation of antigenprimed T cells suitable for antigen-specific T cell Correspondence to: C.E.G. Havenith, Department of Cell Biology, Division of Electronmieroscopy, Medical Faculty, Vrije Universiteit,Van der Boechorststraat 7, 1081 BT Amsterdam, Netherlands. Tel.: 31-20-5483939; Fax: I}31-206462228. Abbreciations: APC, antigen presenting cells; BSA, bovine serum albumin; DC, dendritic cells; cpm, counts per rain; FCS, fetal calf serum; GT, copolymer glutamine-tyrosine; 3H-TdR, [3H]thymidine; Mab, monoelonalantibody.
proliferation assays have been developed. To deplete primed lymph node or spleen cell suspensions of macrophagcs and of other adherent cells the differenticl attachment to plastic petri dishes has been used (Walker et al., 1983). Also depletion of immunoglobulin-bearing cells from cell suspensions has been described using nylon wool columns (Julius et al., 1973). The method involving the passage of cell suspensions through a column of Sephadex G-10 beads results in the selective retention of antibody-forming cells and of antigen presenting cells (APC) (Ly and Mishell, 1974).
Two of the most common procedures used are combinations of the above mentioned ones, namely a three-step procedure: 1.5 h adherence, a nylon wool column and a Sephadex G-10 column (Walker et al., 1983) and a two-step procedure: a passage through two consecutive columns of Sephadex G-10 (Ly and Mishell, 1974). The use of these two isolation methods gives no satisfactory results in the rat system, sinde the background level responses of antigen-primed T cell-enriched lymph node or spleen cells are invariably high, probably due to contamination with accessory la-positive cells, predominantly B cells and DC. B cells are able to present antigen (Unanue et al., 1984), whereas DC are known to be vet), potent APC capable of given responses at low stimulator to responder ratios (Steinman et al., 1985). To improve the evaluation of the capacity of rat APC to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background responses. Therefore, in the present paper a rapid and reproducible isolation procedure for the isolation of pure T cells is presented for the rat, using a nylon wool column, to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove the last contaminating la-positire cells.
Materials and methods
Animals Inbred male AC1/Mal rats, weighing 180-200 g, were obtained from "Centraal Proefdierenbedrijf" (TNO, Zeist, Netherlands). All animals were kept under routine laboratory conditions and allowed food and water ad libitum.
Immunization procedure AC1/Mal rats, which are high responders for the copolymer glutamine-tyrosine (GT, ICN ImmunoBioiogicals, Lisle, Israel) in an la dependent manner (Amerding et al., 1974), served as source of responder cells for the GT-primed proliferation assay. The rats were anesthetized with ether
(Merck Nederland, Amsterdam, Netherlands) and immunized in both hind footpads with 100 p.g of GT emulsified in 100/zl Freund's complete adjuvant (Difco Laboratories, Detroit, USA).
Preparation of T cell-enriched cell populations Isolation of T cell-enriched cell populations of the draining popliteal lymph nodes or the spleens of GT-primed rats 2 weeks after immunization was performed following three different methods. After the lymph nodes or spleens had been minced and pressed gently through a monodur gauze (100 /~m; Stokvis&Smits, IJmuiden, Netherlands), the total cell suspension (designated as total cells) was split into three equal portions to compare the three different isolation procedures. The first isolation method, performed according to Ly and Misheli (1974), uses a 1.5 h adherence step and two times a Sephadex G-10 column. The cells were cultured in culture medium for 1.5 h at 37°C in tissue culture flasks (Greiner, Aiphen a.d. Rijn, Netherlands). Culture medium comprised complete RPMI 1640 medium (Flow Laboratories, lrvine Ayrshire, Scotland), 10% heat inactivated fetal calf serum (FCS), 20 mM L-glutamine, penicillin 50 U/ml, and streptomycin 50/~g/ml (all from Gibco Ltd, Pairsley, Scotland). The collected non-adherent cells were resuspended in 2 ml culture medium and placed on top of a Sephadex G-10 column. Briefly, a Sephadex G-10 column was made in a barrel of a 10 ml syringe (Sherwood Medical, Crawley, UK) plugged with glass wool and filled with 6 ml of autoclaved Sephadex G-10 (Pharmacia, Uppsala, Sweden), swollen for four times in saline. The column was rinsed with 25 mi of warm culture medium before the cells were placed on the column, by mixing the top 2 ml of Sephadex with the cell suspension. The cells were allowed to penetrate the Sephadex bed for 10 rain at 37°C, the top 2 ml were mixed again and penetration was allowed for another 5 min at 37°C. Then the column was washed through with 20-25 ml of warm culture medium. The effluent cells were placed on top of a second Sephadex G-10 column and the same procedure was performed as above. Subsequently again the effluent cells (designated as adherence/sephadex/sephadex) were collected.
The second isolation method was performed following a three differed step, namely 1.5 h adherence, a nylon wool column and a Sephadex G-10 column, according to Walker et al. (1983). The cells were cultured in culture medium for 1.5 h at 37°C in tissue culture flasks (Greiner). The collected non-adherent cells were loaded onto a nylon wool column (maximal 2 x l0 s cells per column) and washed into the nylon wool with 0.5-1 ml of warm (37°C) culture medium according to the method of Julius et al. (1973). Nylon wool columns comprised of about 1.0 g aliquots of nylon wool (Leuko-Pak Leukocyte Filters, Fenwal Laboratories, Illinois, U.S.A) packed into the barrels of 10 ml plastic syringes (Sherwood Medical) up to the 8 mi mark. These columns were autoclaved, rinsed with 20 ml warm culture medium and placed in an incubator at 37°C at least 1 h before use. After loading the cells onto the column, the column was replaced in the incubator and left for 45 min. The column was then washed slowly with warm culture medium and the first 20 ml of effluent was collected. The effluent cells were resuspended in 2 ml culture medium and placed on top of a Sephadex G-10 column (see above). The effluent cells (designated as adherence/nyion/sephadex) were collected. The third and newly developed isolation method comprised a nylon wool column and a removal of la-positive cells making use of the monocional antibody (Mab) OX-6 (a gift from Dr. Barclay, Oxford, UK) and of magnetic beads coated with sheep anti-mouse lgG (Dynal, Oslo, Norway). The cell suspension was loaded onto a nylon wool column (see above). After collecting the effluent cells of the nylon wool column, the cells were washed twice in RPMI 1640 medium with 0.5% bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, USA) and incubated for 15 rain on ice with 20 p.I ascites of OX-6 in 1 ml medium with 0.5% BSA. The cells were washed twice and incubated for 15 min on ice with magnetic beads coated with sheep anti-mouse IgG (Dynal). The number of magnetic beads used was 30-40% of the total amount of effluent cells. The la-positive cells attached to the magnetic beads were removed using a magnet (Dynal). The non-attaching cell population (designated as nylon/anti-la beads) was collected.
The T cell-enriched cell populations acquired with the three above described methods w e r e washed twice, the purity was phenotypically evaluated in cytocentrifuge preparations and functionally they were compared as responder cells in the GT-primed T cell proliferation assay, using dendritic cells as APC.
Preparation of dendritic cells Cell suspensions of the spleen were enriched for dendritic cells (DC) according to the method of Knight et al. (1986). Briefly, the spleen was minced and digested in culture medium containing collagenase/DNA~ (150 U/ml collagenase type Ill, 10 U/ml DNAse type I, both from Sigma Chemical Co.) for 90 rain at 37°C. The collected cells were cultured overnight and enriched for DC using a discontinuous gradient (14.5% w / v Nycodenz, Nycomed, Oslo, Norway). After this gradient, centrifuged at 600 × g at 20°C for 20 rain, cells were collected from the interphase. These cells, viability greater than 95% and purified to about 70% DC, were used as antigen presenting cells in the GT-primed T cell proliferation assay. DC were defined according to Havenith et ai. (1992), namely strongly la-positive cells having acid phosphatase activity in a central spot near the nucleus and extending cell processes.
GT-primed T cell proliferation assay Responder cells (1, 2 or 4 × 105 T cell-enriched lymph node or spleen cells/well) were plated into flat-bottomed 96-well culture plates (Greiner). GT was added at a final concentration of 100 p.g/ml. Splenic DC, irradiated with 3000 rad (~°Co), were added in different concentrations to the responder cells in triplicate, into a final volume of 200 p.I. The culture medium was supplemented with 50 p.M fl-mercaptoethanol (Merck Nederland). The cultures were incubated for 3 days at 37°C in a humidified atmosphere of 5% CO 2 in air. After adding 1 p.Ci of [3H] thymidine per well (3H-TdR, Amersham, UK), the amount of 3H-TdR uptake was determined 18 h later using an automatic cell harvester to collect the cultured cells and analyzed in a liquid scintillation counter. Data, mean values of triplicate wells, are expressed in counts per min (cpm).
Staining procedures Detection of antigen on cytocentrifuge preparations of spleen dendritic cells and the different T cell-enriched populations was done by an indirect immunoperoxidasc method and a double staining method of la antigens and acid phosphatase activity as described in detail by Havenith et al. (1992). Used were the Mabs OX-6 ( a l a ) and OX-19 combined with OX-52 (pan T cell markers; a gift from Dr. Barclay, Oxford, UK), and His 14 (pan B cell marker; a gift from Dr. Nieuwenhuis, Groningen, Netherlands).
Statistical analysis Differences between the percentages of OX19/52-positive cells and the background responses of the different isolated T cell populations were tested with the Mann-Whitney U t e ~
Results
loo ANS
s0
~' "i
NB
e0 40
20 0 0X-19/52
HIs 14
OX-6
monoclonal a ~ . ~ Y
Fig. I. Expression of OX-19/52, Hi~ ~t~t. ~ (~.6 ~.~1'diffet~n~ T cell-enrichedlymph node cell popla~'~t~;. ~C: ~otal0ymph node) cell suspension. ASS: cells isolated with an adherence step and two times Sephadex G-10 column. ANS: cells isolated with an adherence step, a nylon wool column and a Sephadex G-10 column. NB: cells isolated with a nylon wool column and an indirect method with magnetic beads to remove la-positivecells. The data represent mean percentages + SD of positive cells for the monoclonal antibodies OX19/'52, His 14 and OX-6(n = 3).
Cell composition of the different isolated T cell populations The cell counts of the different populations of T cells of the lymph nodes of GT-primed animals (total cells, adherence/sephadex/sephadex, adherence/nylon/sephadex, nylon/anti-la beads) are shown in Fig. 1. These data show that the optimal T cell enrichment (percentage of OX19/52-positive cells) is achieved with method nyion/anti-la beads (98%), followed by method adherence/nylon/sephadex (85%), whereas with method adherence/sephadex/sephadex (62%) hardly any purification is achieved compared to total cells (54%). Method nylon/anti-la beads differed significantly from method adherence/ nylon/sephadex and a d h e r e n c e / s e p h a d e x / sephadex ( p
