Brief Definitive Report

I N H I B I T I O N OF M A T U R A T I O N OF H U M A N P R E C U R S O R L Y M P H O C Y T E S BY C O F O R M Y C I N , A N I N H I B I T O R OF T H E E N Z Y M E A D E N O S I N E BY J E A N - J A C Q U E S

BALLET,

RICHARD

INSEL, EZIO M E R L E R ,

DEAMINASE* AND F R E D

S. R O S E N

(From the Division of Immunology, Department of Medicine, Children's Hospital and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115)

High concentrations of adenosine are known to be toxic to fibroblasts and lymphocytes under conditions of in vitro culture (1, 2). Normally, accumulation of adenosine nucleotides in all mammalian cells is prevented by the presence of adenosine deaminase, an aminohydrolase which converts adenosine to inosine (3). A genetically determined deficiency of adenosine deaminase has been associated with the autosomal recessive form of severe combined immunodeficiency, a syndrome in which precursor lymphocytes fail to mature into T cells and B cells (4-7). Erythrocytes of affected infants convert exogenous adenosine to AMP and ATP at an abnormally increased rate as a consequence of the enzyme defect, and fail to form inosine from the exogenous adenosine (8). These metabolic disturbances can be mimicked in normal erythrocytes by coformycin (8), a potent competitive inhibitor of adenosine deaminase (9, 10). In this study, the effects of coformycin were examined on the in vitro function of normal lymphocytes.

Materials and Methods Isolation of Lymphocytes. Lymphocytes were obtained from tonsils, thymuses, and peripheral blood. Tonsils from children (4- to 10-yr old) undergoing elective tonsillectomy and thymus biopsies (obtained with informed parental consent from children undergoing thoracic surgery) were teased into single cell suspensions. The cells were filtered through sterile glass wool and washed before fractionation in RPMI-1640 medium containing antibiotics as previously described (11). Blood from normal adult donors was collected in heparin (100 U/ml), and sedimented with dextran (1 ml of 6% Macrodex 70000/10 ml blood). The leukocytes, in medium TC-199 containing 10% heat-inactivated serum (from an A B + donor), were passed over a column of glass beads which was prewarmed at 37°C and nonadhering cellswere collected and washed three times in TC-199. Fractionation of Lymphocytes. Lymphocytes were suspended at a concentration of no more than 10~ cells/ml in 17% bovine serum albumin. The cell suspension was added to the top of the gradient, centrifuged, and cells from each layer harvested (11). Purified B cells were prepared from layers seven and eight which were rosetted with sheep red cells (E), and nonrosetted cells were retained at the gradient interface in Ficoll-Triosil (density 1.080). After fractionation procedures, cells were washed three times in medium TC-199 before use. Rosettes with E. Washed E were adjusted to 1% vol/vol concentration in magnssium-free phosphate-buffered saline. Suspensions of lymphocytes (2 × 10~ cells/ml) containing 10% absorbed fetal calf serum were added to an equal volume of a 1% suspension of E. The suspensions were incubated at 37°C (20 min), centrifuged at 200 g (5 min), and stored at 4°C (at least 1-2 h). Rosettes * This investigation was supported by grant AI-05877 from the National Institute of Health, U. S. Public Health Service. THE J O U R N A L OF E X P E R I M E N T A L M E D I C I N E " VOLUME

143, 1976

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were c o u n t e d in a N e u b a u e r h e m o c y t o m e t e r . At l e a s t 200 l y m p h o c y t e s were counted, a n d r e s u l t s a r e e x p r e s s e d as t h e n e a r e s t p e r c e n L Cellular Culture. L y m p h o c y t e s were s u s p e n d e d i m m e d i a t e l y a f t e r f r a c t i o n a t i o n in m e d i u m RPMI-1640 s u p p l e m e n t e d w i t h 15% h e a t - i n a c t i v a t e d h u m a n A B + s e r u m a t 1 or 2 x 10" l y m p h o cytes/ml. 1 ml c u l t u r e s in 16 x 125 m m plastic c u l t u r e t u b e s were m a i n t a i n e d a t 37°C in 5% CO~ in air. Mitogens and Antigens. B a c t o - p h y t o h e m a g g l u t i n i n P (PHA) (Difco L a b o r a t o r i e s , Detroit, Mich.) a n d pokeweed m i t o g e n (PWM) ( G r a n d I s l a n d Biological Co., G r a n d Island, N. Y.) were u s e d at a final dilution of 1/100 of t h e stock solution. C o n c a n a v a l i n A (Con A) ( P h a r m a c i a Fine C h e m i c a l s , U p p s a l a , Sweden) w a s u s e d at 5 ftg/ml of culture. T e t a n u s toxoid (T.T.) ( M a s s a c h u s e t t s Biological Laboratory, Boston, Mass.) w a s u s e d at a c o n c e n t r a t i o n of 10 p g / m l culture. L y m p h o c y t e m i t o g e n i c factor (LMF) w a s produced a n d u s e d as p r e v i o u s l y described (12). Measurement of['~H]Thymidine Incorporation. C e l l u l a r proliferation w a s m e a s u r e d by counti n g TCA-precipitable ['~Hlthymidine on filter p a p e r d i s k s w h i c h were a s s a y e d for r a d i o a c t i v i t y in duplicate (11). Irnmunoglobulin G Synthesis. B-cell or p r e c u r s o r l y m p h o c y t e p o p u l a t i o n s were c u l t u r e d at 2 x 10" cells/ml in RPMI-1640 c o n t a i n i n g 15% h u m a n h e a t - i n a c t i v a t e d s e r u m from a A B + donor. On t h e 6th d a y of c u l t u r e , cells were w a s h e d a n d r e s u s p e n d e d in m e d i u m TC-199 (deficient in valine, leucine, a n d isoleucine) to w h i c h 2 tLCi/ml 1'4C|leucine, a n d 1 t~Ci/ml 1'4C]valine a n d p~C]isoleucine were added. 2 d a y s l a t e r s u p e r n a t e s were h a r v e s t e d a n d n e w l y formed IgG w a s m e a s u r e d (13). R e s u l t s a r e e x p r e s s e d as t h e difference in c o u n t s per m i n u t e of t h e goat a n t i r a b b i t s e r u m a n d t h e n o r m a l r a b b i t s e r u m precipitates. Coformycin. Coformycin (9, 10) w a s a gift from Dr. H. U m e z a w a ( I n s t i t u t e of Microbial C h e m i s t r y , Tokyo, J a p a n ) . D i l u t i o n s were m a d e w i t h s a l i n e a n d 0.1 ml of d i l u t e d solution w a s added to 1-ml c u l t u r e s . L y m p h o c y t e s in c u l t u r e were e i t h e r exposed for 1/2 h to coformycin or d u r i n g t h e e n t i r e course of t h e e x p e r i m e n t .

Results Effect of Coformycin on T- and B-Cell Functions. When peripheral blood lymphocytes were rosetted with E, the number of E rosettes formed in the presence of coformycin (37%) did not differ significantly from the number of E rosettes formed in the absence of coformycin (44%). Similarly, coformycin had no effect on the rosetting of thymus cells. Coformycin in concentrations higher than 1 × 10-:' M inhibited the rate of [3H]thymidine incorporation into unfractionated blood lymphocytes, stimulated with PHA but had no effect when used in ion concentrations lower than 1 x 10-~' M (Table I). A concentration of 1 x 10-s M was chosen for all subsequent experiments. Coformycin did not inhibit responses to Con A, PWM, or T.T. TABLE I

Responses of T Cells and B Cells Coformycin T-cellmitogens PHA* Con A* PWMT T.T.* B-cellmitogen LMF§

0 2,075 106,720 34,365 88,352 119,214

± ± ± ± ±

10 :' M 492 5,176 1,002 4,700 1,097

29,386 ± 526

69,670 31,236 79,812 113,221

± ± ± ±

4,792 526 3,911 5,095

10 " M

107,373 25,834 77,983 126,254

± ± ± ±

6,140 212 3,397 4,524

10 7 M

10 ~ M

99,834 ± 3,678 32,746 ± 3,166 -

92,816 ± 8,166 26,846 ± 6,658 -

30,272 ± 120

Results expressed in net counts per minute and per 10~ cells plus one standard deviation. {'~H]thymidine incorporation measured on the 3rd (for P H A , Con A, and P W M ) and the 7th days of culture (forT.T. and L M F plus T.T.). Controls = (average ± I SD) = 2,075 ± 492. * Unfractionated lymphocytes. T-cell enriched (albumin layers 4-5). § B ceils (albumin layers 6-8 depleted of E-reactive cellsL

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When purified B cells were cultured with T-cell LMF, the presence of coformycin did not affect the proliferative response of B cells (Table I). IgG synthesis by LMF-stimulated B cells was similar in the presence of coformycin (1,324 cpm) or in its absence (1,160 cpm). Effects of Coformycin on Precursor Cells. Cells with precursor characteristics were present in albumin gradient layers 1-3 of tonsil lymphocytes (7). Inability of these precursor cells to react with E, high spontaneous [3H]thymidine incorporation, and inability to react with PHA were some of their characteristics. All lymphoid cells in layer one of albumin gradients exhibited these characteristics, while cells from layers two and three were contaminated with T cells. Precursor cells differentiated during 10 days of culture into T cells (14) (Fig. 1). The presence of coformycin inhibited the appearance of E-reactive cells (Fig. 1). This inhibition did not correlate with the number of cells in culture nor with the number of dead cells. In a typical example, on the 7th day, in the control culture, there were 5 × 105 cells, 55% of which excluded trypan blue and 34% of which were E positive. In the coformycin-treated culture, there were 5 × 105 cells, 44% of which excluded trypan blue and only 1% of which were E positive. The spontaneous incorporation of [~H]thymidine in precursor cells was decreased by coformycin (Table II). Coformycin also inhibited PHA-induced prolif7o 6o

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DAYS OF CULTURE FIG. 1. Effect of coformycin on E reactivity of human precursor cells in culture. Results expressed in percents of E-rosetting lymphocytes at various time intervals of culture. (O) in the absence of, or (©) in the presence of coformycin (10 -~ M). (Average results obtained from four different tonsil lymphocytes from albumin gradient layers one and two. Bars indicate two standard deviations.)

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TABLE II Effect of Coformycin on Spontaneous [3H]Thymidine Incorporation or Lectin Induced Proliferative Response Exp. 1"

In the absence of coformycin In the presence of coformycin

Exp. 25

Exp. 3§

Control

PHA

Control

PHA

Control

PHA

6,236 +- 457

5,185 -+ 260

2,430 -+ 164

37,943 _+ 3,121

3,181 -+ 60

48,930 _+ 3,718

1,636 -+ 59

1,189 _+ 52

440 -+ 47

55,496 ± 4,663

1,434 _+ 26

374 -+ 141

Results expressed in net counts per m i n u t e and * PHA added on day 4, cultures h a r v e s t e d on day 5 PHA added on day 7, cultures h a r v e s t e d on day § P H A added on day 0, cultures h a r v e s t e d on day

per 106 cells -+ one standard deviation. 7. 10. 7.

erative responses of the precursor cells (Table II) when PHA was added to the culture on day 4 or day 7. However, in experiments where PHA was added at the beginning of culture, coformycin did not alter the proliferative response of the cells harvested after 7 or 10 days of culture. Precursor cells synthesized IgG at day 8 in culture in the presence of 1 x 10-6 M of coformycin, an amount that inhibited inception of E rosetting and PHA proliferation (28,024 cpm vs. 19,680 cpm in control culture). Discussion and S u m m a r y A limiting concentration of coformycin, 1 x 10-~ M, a potent inhibitor of adenosine deaminase, has no apparent effect on T- or B-cell function in vitro, although larger amounts of this drug inhibit proliferative responses to mitogens. On the other hand, 1 x 10-6 M of this inhibitor has a profound effect on precursor lymphocytes. Lymphoid cells, which have neither T- nor B-cell characteristics have been identified in blood, tonsils, bone marrow, and thymus and have been observed to mature under conditions of in vitro culture into typical T or B lymphocytes (14, 15). On the one hand, maturation of these precursor lymphocytes into T cells, as defined by E rosetting, and proliferation in the presence of PHA, is very sensitive to the presence of coformycin. On the other hand, maturation of precursor lymphocytes into immunoglobulin-secreting cells is not impaired by coformycin. The metabolic effects of coformycin which are ultimately responsible for the inhibition of T-cell maturation are not known at present. Infants affected with severe combined immunodeficiency have lymphoid cells with precursor characteristics but no ~r very few mature T cells and B cells (7). The experiments reported here have mimicked in vitro the alterations observed in infants affected with this disease. From these findings it may be surmised that the B-cell defect in adenosine deaminase deficiency may be secondary to the failure of T-cell maturation. Indeed, the B-cell number is often normal in adenosine deaminase-deficient infants (5). A child with a genetically determined deficiency of nucleoside phosphorylase, the next enzyme in the salvage pathway which converts inosine to hypoxanthine, has a profound T-cell deficiency but normal numbers of B cells (16). Further studies of purine metabolism in lymphocytes are clearly needed to define the remarkable immunosuppressive

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effects of t h e s e metabolic d i s t u r b a n c e s induced by chemical inhibitors or genetically d e t e r m i n e d e n z y m e deficiencies in the p u r i n e m e t a b o l i c p a t h w a y s . I n t h e p r e s e n t e x p e r i m e n t s , it w a s o b s e r v e d t h a t t h e presence of P H A in c u l t u r e s of p r e c u r s o r cells o v e r c a m e the effects of coformycin. T h i s w a s p r o b a b l y a t t r i b u t a b l e to the r e c r u i t m e n t effect of s m a l l n u m b e r s of cells (2-6%) which escaped the effect of coformycin a n d m a t u r e d into T cells. Received for publication 3 February 1976.

References 1. Green, H., and T. S. Chan. 1973. Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase. Science (Wash. D. C.). 182:836. 2. Hirschhorn, R., J. Grossman, and G. Weissman. 1970. Effect of cyclic 3'-5'-adenosine monophosphate and theophylline on lymphocyte transformation. Proc. Soc. Exp. Biol. Med. 133:1361. 3. Parks, R. E., Jr., G. W. Crabtree, C. M. Kong, R. P. Agarwal, K. C. Agarwal, and E. M. Scholar. 1975. Incorporation of analog purine nucleosides into the formed elements of human blood: erythrocytes, platelets and lymphocytes. Ann. N. Y. Acad. Sci. 255:412. 4. Giblett, E. R., J. Anderson, F. Cohen, B. Pollara, and H. J. Meuwissen. 1972. Adenosine deaminase deficiency in two patients with severely impaired cellular immunity. Lancet. 2:1067. 5. Meuwissen, H. J., B. Pollara, and R. J. Pickering. 1975. Combined immunodeficiency associated with adenosine deaminase deficiency. J. Pediatr. 86:169. 6. Parkman, R., E. W. Gelfand, F. S. Rosen, A. Sanderson, and R. Hirschhorn. 1975. Severe combined immunodeficiency and adenosine deaminase deficiency. N. Engl. J. Med. 292:714. 7. Gatien, J. G., E. E. Schneeberger, R. Parkman, and E. Merler. 1975. Isolation on discontinuous gradients of bovine albumin of a subpopulation of human lymphocytes exhibiting precursor characteristics. Eur. J. Immunol. 5:306. 8. Agarwal, R. P., G. W. Crabtree, R. E. Parks, Jr., J. A. Nelson, R. Keightley, R. Parkman, F. S. Rosen, R. C. Stern, and S. H. Polmar. 1976. Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency. J. Clin. Invest. 57: in press. 9. Ohno, M., N. Yagisawa, S. Shibahara, S. Kondo, K. Maeda, and H. Umezawa. 1974. Synthesis of coformycin. J. Am. Chem. Soc. 96:4326. 10. Nakamura, H., G. Koyama, Y. Iltaka, M. Ohno, N. Yagisawa, S. Kondo, K. Maeda, and H. Umezawa. 1974. Structure of coformycin; an unusual nucleoside of microbial origin. J. Am. Chem. Soc. 96:4327. 11. Geha, R. S., F. S. Rosen, and E. Merler. 1973. Identification and characterization of subpopulations of lymphocytes in human peripheral blood after fractionation on discontinuous gradients of albumin: the cellular defect in X-linked agammaglobulinemia. J. Clin. Invest. 52:1726. 12. Geha, R. S., and E. Merler. 1974. Human lymphocyte mitogenic factor: synthesis by sensitized thymus-derived lymphocytes, dependence of expression on the presence of antigen. Cell. Immunol. 10:86, 13. Geha, R. S., E. E. Schneeberger, F. S. Rosen, and E. Merler. 1973. Interaction of human thymus-derived and non-thymus-derived lymphocytes in vitro. Induction of proliferation and antibody synthesis in B lymphocytes by a soluble factor released from antigen stimulated T lymphocytes. J. Exp. Med. 138:1230.

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14. Gatien, J. G., E. E. Schneeberger, and E. Merler. 1975. Analyses of human thymocyte subpopulations using discontinuous gradients of albumin: precursor lymphocytes in human thymus. Eur. J. I m m u n o l . 5:313. 15. Chess, L., H. Levine, R. P. MacDermott, and S. F. Schlossman. 1975. Immunologic functions of isolated human lymphocyte subpopulations. VI. Further characterization of the surface Ig negative, E rosette negative (Null Cell) subset. J. I m m u n o l . 115"1483. 16. Giblett, E., A. J. Amman, D. Wara, R. Sandman, and L. K. Diamond. 1975. Nucleoside phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet 1:1010.