A rapid method for the isolation of functional human T lymphocytes using hydroxyapatite column fractionation

Journal of lmmunologwal Methods. 106 (1988) 169-174 Elsevier

169

JIM 04611

A rapid method for the isolation of functional human T lymphocytes using hydroxyapatite column fractionation S. Tsuru t, M. Taniguchi ], M. Tsugita ], S. Sekiguchi 2 and K. N o m o t o 3 Departments of I Bacteriology, and 'Laboratorv Medicine, National Defense Medical College, Saitama, and ~ Department of Immunology. Medical lnstttute of Bioregulat~on. Kyushu University. Fukuoka. Japan (Received 26 March 1987, revised received 22 June 1987, accepted 7 September 1987)

Passage of peripheral blood lymphocytes through a column of hydroxyapatite resulted in a 7-20-fold depletion of immunoglobulin-bearing cells, a 20-fold depletion of monocytes, and a 1.3-fold enrichment of T cells. The effluent population was virtually devoid of B lymphocyte precursors and monocytes, whereas helper cell and suppressor cell populations remained intact. This method will facilitate the rapid preparation of T-enriched cell populations. Key words: Hydroxyapatite column; Lymph(~yte subpopulation; Fluorescence-activated cell sorter

Introduction

In vitro methods for enriching T ceils from peripheral blood iymphocytes (PBL) depend generally on the presence of known membrane components on B cells. B cells can selectively be killed by treatment with anti-immunoglobulin antibody and complement (Miller et al., 1972) or depleted by passage through an anti-immunoglobulin antibody-coated column (Wigzell et al., 1972), since these ceils bear an immunoglobulin-like structure on their surface. B cells have receptors for a structural component located on the Fc fragment of certain classes of Ig molecule (Fc receptor) and this feature can be utilized for the elimination of B cells. Most such methods require multiple steps and are characterized by poor recoveries. T cells are separated using a polymer or copolymer of ethylene, propylene, vinyl chloride, vinyl acetate, styrene, divinylbenzene, acryionitrile or methyl Correspondence to: S. Tsuru, Department of Bacteriology. National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan.

methacrylate in the form of granules having an acidic (sulfonic, carboxyl, phosphonic or phenol) functional group. However, any traces of residual monomer are cytotoxic to the cells. Therefore, a system separating T cells from the other cells involved in the PBL has been established using hydroxyapatite as the fractionating agent.

Materials and methods

Human peripheral blood lymphocytes Heparinized blood was obtained from apparently healthy individuals. PBL were purified on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient (p=1.076) by centrifugation. After washing in phosphate-buffered saline (PBS) at pH 7.4, the lymphocytes were purified further by passage through columns, to delete B cells a n d / o r monocytes. llydroxyapatite columns and nylon wool columns Sterile hydroxyapatite columns (Pentapack approximately 0.8 g aliquots of washed and dried

0022-1759/88/$03.50 ~:) 1988 Elsevier Science Publishers B.V. (Biomedical Division)

170

hydroxyapatite were packed into the barrel of 1 ml plastic syringes up to the 0.8 ml mark, Asahi Optical Co., Tokyo) were used without any further preparation for the experiments. The columns were rinsed with about 5.0 ml of Dulbecco's PBS containing 5% heat-inactivated fetal calf serum (FCS). A total of 0.5- 1 × 107cells, in a volume of 0.2 ml, were loaded onto the columns and subsequently washed slowly (so as not to generate a fluid hcad) with 5.0 ml of warm (37°C) medium. 5 ml of effluent was collected in 10 ml tubes and the cells pelleted at 290 × g for 10 min in the cold (4°C). Viable cells excluding trypan blue were counted in a hemocytometer. Nylon wool columns (about 0.6 g aliquots of washed and dried nylon wool packed into the barrel of 12 ml plastic syringes) were prepared as described (Julius et al., 1973). Briefly, a total of 1 1 . 5 × 1 0 ~ cells, in a volume of 2.0 ml, were loaded onto the column and subsequently entered the nylon wool with a washing 0.5 1.0 ml of warm medium. Sterilc syringe covcrs were then replaced and the columns left for 45 rain at 37 ° C. Thc colunms were then washed slowly with warm medium and the first 25 ml of effluent were collected in 15 ml tubes, The columns could be re-used after sterilization at 160 ° for 1 h.

A nlisera Monoclonal antibodies specific for Leu 2a, Leu 3a, Leu 4, Leu 12 and Leu M3 were purchased from the Becton Dickinson Monoclonal Center (Mountain View, CA). These antibodies were fluorescein-conjugated. The specificity of these monoclonal antibodies has been well documented (Ledbetter et al., 1981). Contamination of surface immunoglobulin (lg) bearing cells was detected by' staining with phycoerythrin (PE) conjugated antiH L A - D R (Becton Dickinson). Fluorescence staining of cells Single-cell suspensions (from which the erythrocytes had been lysed by 0.85% a m m o n i u m chloride) were stained in 96-well flexible microtiter plates (Dynatech Laboratories, Dynatech, Alexandria, VA). For staining, 106 target cells were either added to the FITC-conjugated antibodies, or both FITC- and PE-conjugated antibodies. After 30 min of incubation on ice, cells

were washed three times and analyzed. The medium (RPMI 1640, Grand Island Biological Corp., Grand Island, NY) was supplemented with 3% heat-inactivated newborn calf serum. 10 mM Hepes, 0.1% sodium azide and used throughout the procedure.

Analvsis of cell preparations Analysis of fluorescence-binding cells was carried out with a fluorescence-activated cell sorter (FACS Ii. Becton Dickinson Electronics Laboratory, Mountain View, ('A) (Bonner et al., 1972). Excitation of both dyes at 488 nm by the FACS argon ion laser produced maximum fluorescence emission at 525 nm for FIT(" and 575 nm for PE. Cells were processed at 2000-3000 cells/s and the intensity of fluorescence was recorded for each individual cell on the pulse height analyzer. The level of background fluorescence was determined by analyzing T cell-enriched preparations treated with FITC-conjugated anti-human lg, without the intermediate sera from normal controls as negative control. Thus, the percentage of labelled cells was determined by counting the number of cells giving fluorescence signals above the background and dividing by the total number of viable cells examined. Light scattering signals distinguished viable from non-viable lymphocytes. The calculation by the FACS analyzer was based on an analysis of 5 -10 × 104 individual viable cells. Antibody assay Antibody-forming cultures were carried out using a microadaptation of the method described by Callard (1979). Control cultures contained 4 x 105 PBL/welI. In limiting dilution experiments, 1.5 x 105 T cells/well were recombined with various numbers of irradiated "1 cells (cells were subjected to 1500 rads of irradiation using a Toshiba ~"J('o source at a dose rate of 300 rads/min). T and B cell populations were separated by cell sorting using the rnonoclonal anti-Leu 5b antibody. A minimum of eight cultures was performed for each group. Purified influenza virus ( A / P R S ) was used as the antigen. The cultures were incubated in a humidified atmosphere of 5% CO~ in air at 3 7 0 ( ` for 6 days. The cells were then washed twice and resuspended in 0.2 ml of RPMI 1640 containing 20 mM Hepes and 5% FCS and in-

171 cubated for a further 18 h at 3 7 ° C in humidified air. The culture supernatant was then removed and stored at - 2 0 ° C before assay for specific anti-influenza antibody. Specific anti-influenza antibody in the culture supernatant was determined by solid-phase enzyme immunoassay (EIA) performed in flat-bottom microtiter plates as described by Callard and Smith (1981). The analysis was performed on limiting dilutions of T helper (Th) cells. Th cells were diluted over a range to yield a significant percentage of cultures unable to produce specific antibody. Results were expressed as the percentage of negative cultures relative to the number of added T cells. A culture was scored as positive for production of antibody if the culture supernatant bound more than the mean of the replicate control culture wells plus twice the standard deviation. Control wells contained 1.5 x 105 B cells plus antigen. Control cultures normally gave less than 0.5 n g / m l to 2 n g / m l of specific antibody. Positive cultures ranged from approximately 5-200 n g / m l . Th cell frequencies were calculated from semi-log plots of the percentage of negative wells versus the number of added T cells. Cvtotoxic assay Cytotoxic T lymphocytes (CTL) were generated in one-way mixed lymphocyte cultures using previously described methods (Zarling and Bach, 1976), with the following modifications: 25 ml of PBL, which had been passed through a hydroxyapatite column, at 1 x 106 cells/ml were mixed with an equal volume of X-irradiated (2000 rad) lymphocytes (2 x 10 6 cells/ml) from an aliogeneic donor in a 75 cmz plastic tissue culture flask. After 3 days of incubation at a 45° angle and at 3 7 ° C in humidified air containing 5% CO 2, the cells were gently resuspended and the flask was incubated in a vertical position for an additional 3 days. On day 6, cultures were harvested for assay. Viability, as demonstrated by trypan blue dye exclusion, was at least 95%. Allogeneic lymphocytes, as target cells, at 1-2 x 106 cells/ml were incubated in 25 cm 2 flasks at 37 ° C in humidified air containing 5% CO 2. On day 3, 50% of culture medium was replaced with fresh medium containing sufficient phytohemagglutinin (PHA) to bring the medium in target cell suspensions to a final

PHA concentration of 1.2 ffg/ml. On day 5, fresh medium was added. On day 6, 5 x 106 cells from each flask were removed and were gently centrifuged. The supernatants were aspirated off and 250 ttCi of 51Cr (New England Nuclear, Boston, MA) added to the cell pellets. The cells were gently resuspended and incubated in a 37 ° C water bath for 1 h with intermittent gentle agitation. The suspension was washed once in medium, incubated in 7.0 ml medium for 90 min in a 3 7 ° C water bath, layered over a Ficoll-Hypaque density gradient, and centrifuged for 20 rain at 1000 x g. Cells from the interface were harvested, washed twice in medium, and resuspended at 1 x 105 cells/ml in medium. Viability, as measured by trypan blue dye exclusion, was > 98%. Spontaneous release of 5~Cr from these targets was usually less than 15% in an 8 h assay.

Results

Removal of B cells B cell depletion from PBL was accomplished by passage through either a hydroxyapatite column or a nylon wool column. Aliquots of cells before and after loading onto the columns were directly stained with either Leu 12 as a marker for B cells, Leu M3 as a marker for m o n o c y t e / m a c r o p h a g e antigen, or Leu 4 as a marker for T cells (Table I). PBL contained 72-75% T cells, 10-15% B cells and 8-15% m o n o c y t e / m a c r o p h a g e series before loading onto the column. The nylon wool column effluent cells showed a 2-5-fold depletion of B cells, a 5-9-fold depletion of monocytes a n d / o r macrophages and a 1.2-fold enrichment of T cells. In 15 experiments, the effluent populations contained between 84-90% T cells, 2-5% B cells and 4.0- 4.8% m o n o c y t e s / m a c rophages. The recovery of T cells was between 59 and 76%. In 15 experiments, the hydroxyapatite column effluent cells showed a 7-20-fold depletion of B cells, a 16 24-fold depletion of monocytes/macrophages with a complementary 1.3-fold enrichment of T cells. The recoveries of T cells in these experiments were 87-90%, which were superior to those by nylon wool passage. The viability of the column effluent cells was always > 90% (as assessed by trypan blue dye exclusion), irre-

172 TABLE I RECOVERY OF T CELLS AF'I"ER HYDROXYAPATITE FILTRATION Exp.

no. of cells filtered

% Stained cells before filtration "

Column

% Stained cells after filtration "

B

Mo/M,~

T

1

1 × 107

10

11

2

1

X 10 7

12

3

0.5

× 10 7

4

0.5

5

% Recover3' of viable cells

B

Mo/Mq,

T

Total

"1"

72

Nylon h HA"

5.0 1.0

4.0 0.6

90 97

34 75

68 87

8

73

Nylon HA

4.0 0.8

4.4 0.4

84 95

45 82

70 89

10

15

75

Nylon HA

2.0 0.8

4.5 0.8

87 t~6

45 74

76 90

X 10 7

12

14

75

Nylon HA

2.0 0.6

4.6 0.6

85 97

60 80

64 89

1 x 108

15

10

75

Nylon HA

5.0 2.0

4.8 0.6

90 95

37 84

59 90

a Direct stain with either FITC-conlugated anti-Leu 12, anti-Leu M3 or anti-Leu 4 antibodies. Data presented represent the mean of the three experiments. t, Cells filtered through nylon wool columns at 37 °C as described the materials and methods section. ~ Cells from this experiment were used as a source of cooperation. See Table II and Fig. 1.

s p e c t i v e o f the v i a b i l i t y in the s t a r t i n g p o p u l a t i o n . If d e f i b r i n a t e d b l o o d was u s e d i n s t e a d o f h e p a r i n i z e d b l o o d the h y d r o x y a p a t i t e c o l u m n y i e l d e d a 1 0 - 1 5 - f o l d d e p l e t i o n o f l g - b e a r i n g cells. In o r d e r to d e t e c t s u r f a c e a n t i g e n s o n cells b e f o r e a n d a f t e r p a s s a g e t h r o u g h the c o l u m n s , three m o n o c l o n a l a n t i b o d i e s , t e r m e d Leu 2a, Leu 3a a n d Leu 11 w e r e used in this e x p e r i m e n t ( T a b l e 11). Cells s h o w n in T a b l e I, exp. 1 w e r e used for this s t a i n i n g e x p e r i m e n t . F u n c t i o n a l l y . Leu 2a defines the h u m a n s u p p r e s s o r / c y t o t o x i c subset, Leu 3a d e f i n e s the h u m a n h e l p e r / i n d u c e r subset, w h i l e

Leu 11 d e f i n e s the h u m a n n a t u r a l killer ( N K ) cell s u b s e t in P B L . T h e r e f o r e , Leu 2a is c o n s i d e r e d to be e q u i v a l e n t to O K T 8 a n d Leu 3a is c q u i v a l e n I to O K T 4. T h e s e a n t i b o d i e s w e r e f l u o r e s c e i n - c o n j u g a t e d . E f f l u e n t cells o b t a i n e d f o l l o w i n g hyd r o x y a p a t i t e c o l u m n p a s s a g e c o n t a i n e d a b o u t the s a m e p r o p o r t i o n s o f T cell s u b s e t s as w e r e p r e s e n t b e f o r e the p r o c e d u r e . H o w e v e r , p a s s a g e t h r o u g h n y l o n w o o l a p p e a r s to affect the p r o p o r t i o n of t h e s e cells, in that there is a c o n s i d e r a b l e d e c r e a s e in the n u m b e r of Leu 11 ' cells. It was p o s s i b l e to e l u t e B cells f r o m the colu m n s by c o m p r e s s i o n but there was h e a v y c o n t a m i n a t i o n w i t h d e a d cells.

TABLE II DISTRIBUTION OF T CELL SUBSETS AFTER HYDROXYAPATITE FILTRATION Antibody

Leu2a Leu3a Leu 11

% Stained cells a Before filtration

After filtration Hydroxyapatite

Nylon

28_+ 8 45+10 15± 7

27+3 50+5 15+5

40+5 55+5 5+_2

" Direct stain with either FITC-conjugated anti-Leu 2a. antiLeu 3a, anti-Leu 11 antibodies. Mean ± 1 SD.

Recovery of helper cell function The hydroxyapatite column effluent population c o n t a i n e d all the c o o p e r a t i n g a c t i v i t y f o u n d in w h o l e l y m p h o c y t e s . T o d e t e r m i n e the f r e q u e n c y of h e l p e r T ( T h ) cells specific for A / P R 8 i n f l u e n z a virus, 1.5 × 10 5 o f i r r a d i a t e d l y m p h o c y t e s was add e d to 1.5 × 10 5 e f f l u e n t cells in e a c h well a n d c h a l l e n g e d w i t h A / P R 8 . C o n t r o l s o f 1.5 × 10 5 B l y m p h o c y t e s plus a n t i g e n ( w i t h o u t a n y a d d e d i r r a d i a t e d l y m p h o c y t e s ) w e r e set up to e s t a b l i s h b a c k g r o u n d levels of s t i m u l a t i o n . C o m p a r i s o n o f

173 TABLE III COMPARISON OF F R E Q U E N C I E S OF A U T O L O G O U S IIELPER T CELLS IN A / P R 8 ANTIBODY RESPONSES

1.5-fold increase in T cells observed in the hydroxyapatite effluent population compared to whole lymphocyte population.

Cells

Frequency × 10 5 of Th cells

Recover)' of cytotoxic precursor cell activity

Whole Effluent ~

1.46 + 0.4 0.80 + 0.3

CTL activity was completely recovered in the hydroxyapatite effluent population. Following 6 days incubation with allogeneic lymphocytes, the cytotoxic activities of either unfiltered cells or hydroxyapatite effluent ceils were measured against 5tCr-labelled PHA-generated lymphoblastoid targets. The values for percent cytotoxicity at each attacker:target cell ratio were plotted for each of the cell populations and the slopes of the lines compared (Fig. 1). The results obtained with this system parallelled those obtained with the cooperative system. The hydroxyapatite effluent population had nearly twice as much precursor cell activity (25% cytotoxicity/106 sensitized cells) as the whole lymphocyte population (15% cytotoxicity/106 sensitized ceils), a result which parallels the 1.5-fold enrichment of T cells in the effluent population.

Effluent cells depleted of lg-bearing cells by passage through hydroxyapatite column.

the cooperating activity of whole lymphocytes with the activity of the hydroxyapatite column effluent showed that the effluent cells were nearly twice as active (Table III). This is consistent with the nearly

60

50

40

Discussion .'..2_

30

o~

2O

0.5

2 E,'T r a t i o

Fig. 1. Cytotoxicity of hydroxyapatite column filtered lymphocytes and unfiltered lymphocytes sensitized in vitro. Hydroxyapatite column filtered (e) or unfiltered ( O ) lymphocytes were sensitized in vitro for 6 days with 1000 rad irradiated lymphocytes. Various members of sensitized cells were then incubated with 5 x 106 51Cr-labelled lymphoblasts for 8 h. The effector-to-target ( E / T ) ratio was 0.5 : 1, 1 : 1 and 2 : 1. Specific lysis of all lymphocytes was < 5.3% and the spontaneous 51Cr release was always < 14.7%. The population of cytotoxic cells in the hydroxyapatite effluent population used in this experiment is shown in Table I, exp. 1. The points in this figure represent the mean specific 51Cr release+ standard deviation from four experiments.

Nylon wool columns have been used as a rapid and reproducible approach for the depletion of Ig-bearing cells from peripheral blood cell suspensions. However, some contaminating Ig-bearing cells remain and the time required for the fractionation limits the use of this method. An attempt to remove the contaminating Ig-bearing cells from the effluent population by passage through hydroxyapatite resulted in further depletion. The contaminating Ig-bearing ceils removed by such passage through hydroxyapatite had fewer membrane-associated Ig determinants than did the vast majority of the lg-bearing cells in the peripheral blood cell suspensions and may represent a subpopulation of B cells which are less efficient in either precursor or memory cell function. The immunofluorescent staining results agreed with findings of the functional assay for B precursor or B memory cell activity. 80-90% of the T cells in the PBL were recovered in the column effluents. The T cell loss was not selective for T cell subpopulations in-

174 v o l v e d in c y t o t o x i c p r e c u r s o r o r c o o p e r a t o r activities. since the a c t i v i t y o f the r e c o v e r e d p o p u l a t i o n e q u a l l e d that o f w h o l e p e r i p h e r a l b l o o d l e u k o cytes, w h e n e x p r e s s e d as a f u n c t i o n of the n u m b e r o f T cells in the e x p e r i m e n t s . A l t h o u g h the basis for the s e l e c t i v e r e t e n t i o n of I g - b e a r i n g cells a n d m a c r o p h a g e s o n these hyd r o x y a p a t i t e c o l u m n s r e m a i n s to be d e t e r m i n e d , the p r o c e d u r e we h a v e d e s c r i b e d p r o v i d e s a r a p i d a n d e f f e c t i v e m e t h o d for the i s o l a t i o n of f u n c tional T cells f r o m h u m a n p e r i p h e r a l b l o o d .

References Bonner, W.A.. Hullet. H.R., Sweet, R.G. and Herzenberg, I..A. (1972) Fluorescence activated cell sorting. Rev. Sci. Instrum. 43. 404. Callard, R.E. (1979) Specific in vitro antibody response to influenza virus by human hlcg)d lymphocytes. Nature 282. 734.

Callard, R.E. and Smith, C.M. (1981) llistocompatibility requirements for T cell help in specific in vitro antibody responses to influenza virus by human blood lymphocytes. Eur. J. lmmunol. 11,206. Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. lmmunol. 3, 645. Ledbetter, JA., Evans, R.L., Lipinski, M., Lunningham-Randies, C., Goc~, R.A. and Herzenberg, I,.A. (1981) Evolutiona~ conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulation in mouse and man. J. Exp. Med. 153, 310. Miller, J.F.A.P., Sprent, J., Basten, A. and Warner, N.L. (1972) Selective cytotoxicity of anti-kappa serum for B lymphocytes. Nature New Biol. 237. 18. Wigzell, H., Sundquist, K.D. and Yoshida, T.O. (1972) Separation of cells according to surface antigens by the use of antibody-coated columns. Fractionation of cells carrying immunoglobulins and blo