Isolation o f T Lymphocyte Subsets from Peripheral Blood Using Monoclonal A ntilym phocyte Antibodies*

Transcription

1 ANNALS O F CLINICAL A N D LABORATORY SC IE N C E, Vol. 15, No. 1 Copyright 1985, Institute for Clinical Science, Inc. Isolation o f T Lymphocyte Subsets from Peripheral Blood Using Monoclonal A ntilym phocyte Antibodies* RAMAN PATEL, M.D., AGNES SO RIA N O,t LOLA L O E B,t M AM DOOH GHONEU M, P h.d., and DANIEL NAJERA, B.S. The Division of Nephrology, Department of Medicine, King-Drew Medical Center and the Charles R. Drew Postgraduate Medical School, Los Angeles, CA ABSTRACT M onoclonal an tisera against h e lp e r (serum OKT4) and su p p resso r (serum OKT8) T cells were em ployed in a com plem ent-dependent lymphocytotoxicity reaction to isolate helper and suppressor T cells from p eripheral blood m ononuclear cells. W hen prepared by this m ethod, helper and suppressor T cells had a purity of 67.8 percent and 78.8 percent, respectively. Approximately 56 percent of viable suppressor and 33 percent of viable helper T cells were lost during the procedure. Introduction The central role of T lymphocytes in th e reg u latio n of norm al im m une respose has been established. The ability of T cells to perform a given immunoregulatory function is related to th e p resence of specific surface m em brane d e term in an ts. T hus, in the m ouse Ly-1 d e te rm in a n t is associated w ith the h elper-inducer and Ly-23 w ith suppressor-cytotoxic T cells.3 In m an, also, developm ent of m onoclonal antibodies against well-defined T cell surface determ inants has led to the identification of h e lp e r and su p p resso r T lym phocyte subsets analogous to those described in * Supported by MBRS Grant No. 2S , from the National Institutes of H ealth, Bethesda, MD. t Pre-med. student participants. 66 the m ouse.7,8,9 In both species, alteration of T lym phocyte subsets has b een associated w ith a variety of pathological states, and their characterization in disease has been widely studied.1 The present authors have been interested in the study of T lymphocyte subsets in autoim m unity and have em ployed a relatively sim ple m ethod for th e p rep a ra tio n of helper and suppressor T enriched cells. O ur m ethod is described in this paper. M aterials and M ethods I s o l a t io n o f P e r i p h e r a l B l o o d M o n o n u c l e a r C e l l s Twenty to 40 ml of blood w ere drawn by venipuncture into test tubes containing preservative-free heparin (10 xg per ml)*. T he blood was d ilu te d w ith an * Gibco, Santa Clara, CA /85/ $00.90 Institute for Clinical Science, Inc.

2 ISOLATION OF T LYMPHOCYTE SUBSETS 67 equal volum e of H ank s b uffered salt solution (HBSS) and 10 ml volumes were lay ered upon th re e ml ficoll-hypaque solution.2 The tubes were centrifuged at 1500 RPM for 30 m inutes in a Sorvall GLC-2 m odel centrifuge, and the in te r face co n tain in g th e m ononuclear cells was a sp ira te d and w ashed tw ice w ith phosphate buffered saline, ph 7.4 (PBS). The cells w ere resuspended in RPM I m edium containing five p e rc e n t fetal calf serum (FCS) and adjusted to a concentration of 50 million cells p er ml. P r e p a r a t i o n o f T - e n r i c h e d C e l l s T-enriched cells w ere prepared using the nylon wool adherence techniq u e.5 Briefly, 0.6 g of nylon wool was packed into the barrel of a 10 ml syringe to the six ml mark, rinsed with 50 ml medium (p re h e a te d to 37 C), and in cu b ated at 37 C for one hour. O ne to two ml of p e rip h e ra l blood m ononuclear cells (PBMN) (concentration 50 million cells p e r ml) w ere placed onto th e wool, allow ed to p erco late, and th e syringe incubated at 37 C for an additional hour. T -enriched cells w ere eluted by dropwise (one drop p er sec) addition of p re heated culture m edium. The cells were w ashed tw ice w ith PBS and ad ju sted to a concentration of two m illion cells per ml. P r e p a r a t i o n o f T C e l l S u b s e t s Two one ml volum es of T -enrich ed cells (concentration two million cells per ml) w ere placed in each of two five ml test tu b es n u m b e re d 1 and 2. O ne hundred ml each of appropriately diluted m onoclonal sera OKT4 (specific for helper T subset) and OKT8 (specific for suppressor T subset)* were added to test tubes 1 and 2, respectively, and in cu * O rth o D iagnostics, R aritan, NJ. b a te d at 37 C for 10 m in u te s.610 Two hundred ml of rabbit serum as a source of com plem ent were then added to each tu b e and th e tubes w ere in cu b ated at 37 C for an additional 45 minutes. The cells were centrifuged at 100 RPM for 10 m in u tes, w ashed once w ith PBS, and re su sp e n d e d in one ml of cu ltu re m edium. Test tube 1 contained viable suppressor and dead helper T cells; test tu b e 2 consisted of viable h e lp e r and dead suppressor T cells. In order to separate viable cells from the dead, 0.5 ml of cell suspension (containing one m illion cells) was lay ered onto 0.4 ml ficoll-hypaque solution in Fisher centrifuge tubes (no ) and the tubes were centrifuged at looog for two m inutes in a Fisher centrifuge m odel 59. D u rin g ficoll-hypaque gradient centrifugation, viable cells collect in the interface while dead cells are driven into a pellet at the bottom of the test tube. The interface is aspirated, pooled, and washed once with PBS. The cells are th en adjusted to the desired concentration. Results Y i e l d o f T - e n r i c h e d C e l l s O ur results in six experim ents using the nylon wool adherence technique are shown in table I. The mean yield of T- enriched cells was 40.7 ± 5.8 percent with a range of 25.1 to 60.2 percent. The B cells decreased by a m ean of 61.5 p ercent, and T-enriched cells had a m ean B cell concentration of 11.2 ± 0.8 percent (range 8.2 to 13.1 percent), as deterim ined by the presence of surface im m u noglobulin positive cells.4 Y i e l d o f H e l p e r a n d S u p p r e s s o r T C e l l s Approximate yields of helper and suppressor T cells w ere determ in ed as fol-

3 68 PATEL, SORIANO, LOEB, GHONEUM, AND NAJERA E x p e r im e n t TABLE I Results of Nylon Wool Separation of Lymphocytes in Six Patients P re - n y lo n Wool PBM C* S J g r+ t P o st- n y lo n T - e n r ic h e d Wool S I g ND ND ND ND M e a n ls e m ± ± ± * P e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s, i n m i l l i o n s. { Surface I g b e a r i n g c e l l s, i n p e r c e n t. ND = n o t d o n e. lows. Follow ing ficoll-hypaque sep aration of viable and dead cells, the num ber of cells in the interface and in the cell pellet were determ ined in each of the two test tubes. O ur results of a representative experim ent are shown in table II. It can be seen that following treatm ent of T -en rich ed cells w ith serum OKT4 plus com plem ent (test tube #1) 200,000 cells (assumed to be suppressor T cells) w ere present in the interface (viability greater than 98 percent). The cell pellet consisted of 640,000 cells, of w hich 250,000 w ere viable cells. If one assumes that viable cells in the pellet represent suppressor T and dead cells the helper T cells, then approximately 450,000 viable suppressor T cells (200,000 cells in interface plus 250,000 viable cells in pellet) per one m illion T -enriched cells w ere obtained, a yield of 45 percent. However, of th ese a significant n u m b er (250,000 cells or 55.6 percent) were lost in the pellet along with dead helper T cells. Similarly, the yield of helper T cells (after treatm ent of T-enriched cells with serum OKT8 plus com plem ent) was calculated to be 630,000 cells per one million T-enriched cells (63 percent yield), of w hich approxim ately 210,000 cells (33.3 percent) w ere lost in the pellet. The ratio of helper to suppressor T cells (1.4) is norm al for our laboratory.6 P u r it y o f H e l p e r a n d S u p p r e s s o r T C e l l s The purity of the helper and suppressor T -en rich ed p rep a ra tio n s was assessed by testing each with monoclonal sera OKT3, OKT4, and OKT8 in a complem ent dependent lymphocytotoxicity assay.610 O ur results are shown in table III. Serum OKT3 (specific for all T cells) reacted with 88.2 percent of helper and 97.6 percent of suppressor T cells; serum OKT4 (specific for helper T cells) reacted with 67.8 percent of helper and 12.3 percen t of su p p resso r T cells; and serum OKT8 (specific for su p p resso r T cells) was cytotoxic for 78.8 p e rc e n t of su p p resso r and 13.5 p e rc e n t of h e lp e r T cells. As controls, 3.7 percent and 5.2 p e rc e n t of h e lp e r T and su p p resso r T cells respectively w ere killed by rabbit com plem ent alone. TABLE I I Separation of Viable and Dead T Cells on Ficoll-Hypaque Gradient Following Treatment of T Lymphocytes with 0KT Monoclonal Antisera and Rabbit Serum T e s t T ube N o. P r o c e d u r e R e s u l t s 1 m i l l i o n T c e l l s 1. V i a b l e T s i n c e l l s i n (1 ) + s e ru m OKT4 i n t e r f a c e = 2 0 0, r a b b i t s e ru m 2. C e l l s i n p e l l e t = 6 4 0, V i a b l e c e l l s i n p e l l e t = 2 5 0, D ead c e l l s i n p e l l e t «3 9 0, m i l l i o n T c e l l s 5. V i a b l e T h c e l l s i n (2 ) + s e ru m OKT8 i n t e r f a c e = 4 2 0, r a b b i t s e ru m 6. C e l l s i n p e l l e t = 4 3 0, V i a b l e c e l l s i n p e l l e t = 2 1 0, D e a d c e l l s i n p e l l e t = 2 2 0, = Y i e l d o f T s ( 4 5 0,0 0 0 p e r 1 m i l l i o n T - e n.r ic h e d c e l l s ) 3 = V i a b l e T s l o s t i n p e l l e t = Y i e l d o f T h ( 6 3 0,0 0 0 p e r 1 m i l l i o n T - e n r i c h e d c e l l s ) 7 = V i a b l e Th l o s t i n p e l l e t T s = S u p p r e s s o r T c e l l s Th = H e l p e r T c e l l s

4 ISOLATION OF T LYMPHOCYTE SUBSETS 69 S e ru m TA BLE I I I Specificity of Helper and Suppressor T Cells Evaluated by Lymphocytoxicity Assay Using Monoclonal OKT Anti-T Lymphocyte Antisera and Rabbit Complement P e r c e n t C y t o t o x i c i t y A g a i n s t H e l p e r T C e l l s S u p p r e s s o r T C e l l s R a b b i t c o m p le m e n t OKT OKT OKT OKT 3 = M o n o c lo n a l a n t i s e r u m a g a i n s t T c e l l s. OKT 4 = M o n o c lo n a l a n t i s e r u m a g a i n s t h e l p e r T c e l l s. OKT 8 = M o n o c lo n a l a n t i s e r u m a g a i n s t s u p p r e s s o r T c e l l s. Comments A sim ple tech n iq u e has b een d e scribed for th e isolation of helper and suppressor T-enriched cells from peripheral blood m ononuclear cells. O ur results indicate a relatively high degree of purity of th e T cell subsets p re p a re d by this m ethod. A m ajor disadvantage of the tech n iq u e is th e loss of viable h e lp e r (33.3 percent) and suppressor (55.6 percent) T cells in the pellet along with the dead cells. Following separation of viable and dead lym phocytes over ficollhypaque gradient, th e cell interface (containing viable cells) was practically devoid of any contam inating dead lym phocytes (viability over 98 p ercent). Rather, the viable cells tended to move faster into the separation m edium and thereby contam inate the pellet and give lower yields of helper and suppressor T cells. C onceivably, loss of viable cells m ight be m inim ized if cell separation w ere to be p e rfo rm ed at a som ew hat lower centrifugal force, for example, 300 to 400g for 15 to 20 m inutes rather than at 1000g for two m inutes as has been done by us. C learly, the crucial step appears to be the separation of viable and dead cells on the ficoll-hypaque gradient. W hether or not alternate m ethods of cell preparation such as the panning tech nique11 would give better yields is unknown. Com parative studies using monoclonal antisera from different sources, have not been perform ed by the present authors. However, similar values for the percentages of T helper and T suppressor cells in normal hum an peripheral blood have been obtained w ith m onoclonal antibodies Leu-3 (specific for helper T cells (and Leu-2 (specific for suppressor T cells), respectively.* T h erefo re, it should be feasible to use monoclonal anti-t helper and anti-t suppressor antibodies from different sources for the preparation of T cell subsets provided that such antibodies are reactiv e in th e com plem entdependent lymphocytotoxicity assay. References 1. B a c h, M-A. and B a c h, J-F. : The use of monoclonal anti-t cell antibodies to study T cell imbalances in human diseases. Clin. Exp. Immunol. 45:449, B o y u m, A.: Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. Suppl. 21:97, C a n t o r, H. and W e i s s m a n, I.: Developm ent and function of thymocytes and T lymphocytes. Prog. Allergy 20:2011, G r e y, H. M., R a b e l i n o, E., and P ir o f s k y, B.: Immunoglobulins on the surface of lymphocytes IV. Distribution in hypogam m aglobulinem ia, cellular im m une deficiency, and chronic lym phatic leukemia. J. Clin. Invest. 50:2368, H e n r y, C.: Nylon wool adherence. Selected methods in Cellular Immunology. Mishell, B. B. and Shiigi, S. M., eds. San Francisco, W. H. Freeman, 1980, pp P a t e l, R. and W i l l i a m s C.: T cell and T cell su b set d eterm in ation in norm al peripheral blood: comparison of the indirect immunofluorescence and com plem ent-dependent lym phocytotoxicity techniques. Experientia. In press. 7. R e i n h e r z, E. L., K u n g, P. C., G o l d s t e i n, G., and S c h l o s s m a n, S. F. : A monoclonal antibody with selective reactivity with functionally mature human thymocytes and all peripheral human T cells. J. Immunol. 123:1312, * B ecton-dickinson Co., Mountain View, CA, source book sections 4.16 and 4.2.

5 70 PATEL, SORIANO, LOEB, GHONEUM, AND NAJERA 8. R e i n h e r z, E. L., K u n g, P. C., G o l d s t e i n, G., and S c h l o s s m a n, S. F.: Further characterization of the human inducer T cell subset defined by m onoclonal antibody. J. Immunol. 123-, , R e i n h e r z, E. L., K u n g, P. C., G o l d s t e i n, G., and S c h l o s s m a n, S. F.: A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum term ed TH 2. J. Im m unol. 124:1301, V a n W a u w e, J. and G o s s e n s, J. : M onoclonal antihuman T lymphocyte antibodies: Enumeration and characterization o f T cell su b sets. Immunology 42:157, W y s o c k i, L. J. and S a t o, V. L.: Panning for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. 75:2844, 1978.