Possible Metabolic Basis for the Different Immunodeficient States

Proc. Nati Acad. Sci. USA Vol. 79, pp. 3848-3852, June 1982 Medical Sciences

Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase (deoxyadenosine/deoxyguanosine/adenosylhomocysteine/lymphocytes) DENNIS A. CARSON, DONALD B. WASSON, ELLEN LAKOW, AND NAOYUKI KAMATANI Department of Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037 Communicated by Ernest Beutler, March 3, 1982 ABSTRACT An inherited deficiency of adenosine deaminase mechanism. Thus, mouse T lymphoblasts with a mutant form (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes ofthe enzyme insensitive to inhibition by dATP and dGTP are severe combined immunodeficiency disease in humans. A similar also resistant to deoxyadenosine and deoxyguanosine toxicity deficiency in purine nucleoside phosphorylase (Puo phosphoryl- (14, 15). ase; purine-nucleoside:orthophosphate ribosyltransferase, EC The above hypotheses, although offering a cogent explana- 2.4.2.1) engenders a selective cellular immune deficit. To eluci- tion for lymphocyte-specific toxicity in Ado deaminase and Puo date the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting hu- phosphorylase deficiencies, do not explain the immunologic man lymphocytes ofthe Ado deaminase substrates deoxyadenosine differences between the two syndromes. Ado deaminase-defi- and adenosine and the Puo phosphorylase substrate deoxyguano- cient children usually suffer from classical severe combined im- sine. When Ado deaminase was inhibited, micromolar concentra- munodeficiency disease, with absent cellular and humoral im- tions of deoxyadenosine progressively killed nondividing helper mune functions (16). On the contrary, most children with Puo and suppressor-cytotoxic T cells, but not B cells. The toxicity re- phosphorylase deficiency have a selective cellular immune def- quired phosphorylation, with subsequent dATP formation. The icit with normal immunoglobulin levels (2, 17). deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxy- Most in vitro studies concerning the mechanisms of deoxyadenosine, and adenine arabinonucleoside also killed resting T adenosine, adenosine, and deoxyguanosine toxicity have uti- cells. Cell death was unrelated to inhibition of adenosylhomocys- lized rapidly dividinglymphocytes, including both cell lines and teinase (EC 3.3.1. 1) but was preceded by a gradual decline in ATP peripheral blood lymphocytes stimulated with mitogens. How- levels. As much as 1 mM deoxyguanosine did not impair resting ever, the majority of the long-lived T lymphocytes in human lymphocyte viability, despite the synthesis of dGTP. The combi- peripheral blood are actually in a "resting" state (18). In many nation of200 ,M adenosine plus 500 ,uM homocysteine thiolactone but not all systems, these cells can provide helper function killed dividing lymphocytes but had no discernible toxic effect to- without cell division in the induction ofa humoral immune re- ward resting T cells, which accumulated adenosylhomocysteine sponse (19-21). over a 4-hr period but thereafter excreted the nucleoside into the Kredich and Hershfield suggested that increased concentra- culture medium. The different clinical syndromes associated with tions of adenosine or deoxyadenosine in Ado deaminase-defi- genetic deficiencies ofAdo deaminase and Puophosphorylase may cient patients might inhibit biologically important transmeth- be explained by the ability of dATP to kill mature resting T lym- ylation reactions (22, 23). When its normal routes ofcatabolism phocytes by depleting ATP levels. are lacking, adenosine will combine readily with homocysteine in a reversible reaction catalyzed by adenosylhomocysteinase Inherited deficiencies of two enzymes of purine metabolism, (AdoHcyase; EC 3.3.1.1). The thermodynamically favored adenosine deaminase (Ado deaminase; adenosine aminohydro- product, adenosylhomocysteine (AdoHcy), is a potent natural lase, EC 3.5.4.4) and purine nucleoside phosphorylase (Puo inhibitor of transmethylation reactions (22). Additionally, de- phosphorylase; purine-nucleoside:orthophosphate ribosyl- not a substrate for transferase, EC 2.4.2.1), produce immunodeficiency disease in oxyadenosine itself, although AdoHcyase, human beings (1-3). Previous experiments have attempted to inactivates the enzyme and inhibits the hydrolysis of endoge- determine the reasons for lymphocyte-specific toxicity in both nously formed AdoHcy (23). Because some transmethylation Ado deaminase and Puo phosphorylase deficiency (4-12). In probably occurs throughout the cell cycle, inhibition ofAdoHcy affected patients, T lymphocytes may selectively phosphorylate hydrolysis could impair the survival of both resting and prolif- either deoxyadenosine or deoxyguanosine, which are excreted erating lymphocytes. by other tissues unable to phosphorylate the nucleosides (4). The goals of the present investigations were (i) to compare The T lymphocytes progressively sequester dATP and dGTP, in normal, resting human T and B lymphocytes the toxicity and because in this cell type deoxynucleoside-phosphorylating ac- metabolism of deoxyadenosine, adenosine, deoxyguanosine, tivity exceeds dATP- and dGTP-degrading activity (4, 9-12). and related analogs, (ii) to ascertain the relative roles of nu- dATP and dGTP are established potent inhibitors of mam- cleoside phosphorylation and AdoHcy accumulation in mediat- malian ribonucleoside-diphosphate reductase (EC 1.17.4.1) ing any observed toxic effects, and (iii) thereby to identify the (13). The toxicity ofdeoxyadenosine and deoxyguanosine toward metabolic basis for the contrasting phenotypes associated with dividing, immature T lymphocytes may depend upon this genetic deficiencies ofAdo deaminase and Puo phosphorylase. The publication costs ofthis article were defrayed in part by page charge Abbreviations: Ado deaminase, adenosine deaminase; Puo phospho- payment. This article must therefore be hereby marked "advertise- rylase, purine nucleoside phosphorylase; AdoHcy, adenosylhomocys- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. teine; AdoHcyase, adenosylhomocysteinase. 3848 Downloaded by guest on September 29, 2021 Medical Sciences: Carson et al. Proc. Natl. Acad. Sci. USA 79 (1982) 3849 MATERIALS AND METHODS RESULTS Lymphocyte Culture. Peripheral blood mononuclear cells Deoxyadenosine Toxicity. Resting human lymphocytes in- were isolated from heparinized blood of normal volunteers by cubated at 370C in RPMI 1640 medium supplemented with 20% Ficoll/Hypaque sedimentation as described previously (24). autologous plasma remained 90% viable for at least 4 days. After being washed three times in RPMI 1640 medium (Flow When the tight-binding Ado deaminase inhibitor deoxycofor- Laboratories, McLean, VA) the cells were resuspended at a mycin was added to the same cultures at 0.1 uM, viability was density of 106 per ml in RPMI 1640 medium supplemented with still 80% after 4 days. Under these conditions, the addition of 20% autologous plasma, penicillin at 100 units/ml, strepto- increasing concentrations of deoxyadenosine to the medium mycin at 100 ,ug/ml, and 2 mM glutamine. One-milliliter ali- produced a dose- and time-dependent loss ofcell viability, with- recovered cells. One micromo- quots were dispersed in 12 x 75 mm plastic tubes (Falcon 2058) out altering the total number of 40%, and 10 deoxy- or After various time periods, lar deoxyadenosine killed an average of kLM with without added nucleosides. adenosine killed 60% of the cells at 4 days (Fig. 1). were removed and the percentage of cells nonadherent cells Nondividing T lymphocytes bearing the Leu-2a and Leu-3a excluding 0.1% trypan blue was determined in a hemocytometer. antigens were sensitive to deoxyadenosine toxicity. Thus, a 4- Peripheral blood mononuclear cells were depleted of T lym- day incubation ofunfractionated peripheral blood mononuclear phocytes before culturing by rosetting with neuraminidase- cells with concentrations of deoxyadenosine that killed 60% of treated sheep erythrocytes, followed by centrifugation through the cells did not alter the percentage of viable lymphocytes Ficoll/Hypaque (25). Approximately 70% of the cells at the in- binding fluoresceinated monoclonal anti-Leu-3a (55-60%) or terphase stained with fluoresceinated F(ab')2 polyvalent goat anti-Leu-2a (22-32%). The T-cell-depleted, B-lymphocyte-en- anti-human Ig (26). riched cell population was markedly more resistant to deoxy- The effects of nucleosides on phytohemagglutinin-induced adenosine toxicity than unfractionated lymphocytes were (Fig. lymphocyte proliferation were determined as described (24). 1). T Cell Phenotyping. At the initiation of culture, and at in- The toxicity of deoxyadenosine toward resting human pe- tervals thereafter, the peripheral blood lymphocytes were ripheral blood lymphocytes was preceded by a slow but pro- stained with directly fluoresceinated monoclonal antibodies gressive accumulation ofdATP over at least a 2-day period (Fig. anti-Leu-2a (Becton Dickinson, Sunnyvale, CA), which detects 2). Accompanying the increase was a fall in intracellular ATP suppressor and cytotoxic T cells, and anti-Leu-3a, which detects levels, which anteceded cell death by 1-2 days. Human eryth- helperT cells (26, 27). The cells were counterstainedwith ethid- rocytes incubated with deoxyadenosine also accumulated dATP ium bromide at 0.1 ,ug/ml. After washing, the percent viable over a 2-day period. However, ATP levels did not change (not (ethidium bromide-negative) lymphocytes staining with the shown). monoclonal antibodies was determined by fluorescence micros- Effect of Deoxyguanosine. Deoxyguanosine concentrations copy. up to 1 mM were nontoxic toward resting peripheral blood lym- Nucleotide Concentrations. Washed cells were extracted phocytes (Table 1) and did not detectably impair lymphocyte with 0.4 M perchloric acid at 4°C' and then neutralized and fro- responsiveness to phytohemagglutinin after removal ofthe nu- zen as described (28, 29). Concentrations of ATP, dATP, and cleoside by washing. Resuspension ofthe lymphocytes daily in dGTP were determined by high-performance liquid chroma- fresh medium supplemented with 1 mM deoxyguanosine still (11). The formation of adenine nucleoside triphos- left the cells 90% viable after 4 days of incubation. In lympho- tography cyte cultures containing 100 ,uM or 1 mM deoxyguanosine, re- phate analogs was deduced from the appearance of new ultra- spectively, dGTP levels rose to 200 pmol per 107 cells, and 800 violet-absorbing peaks in the triphosphate region, following pmol per 107cells after 24 hr, without any change in ATP levels. incubation of lymphocytes for 4-24 hr with the various com- pounds, as described (29). AdoHcy Formation. Peripheral blood mononuclear cells at a density of 2 x 106 per ml were incubated in complete medium containing 20% autologous plasma supplemented with nucleo- 80- sides. Between 1 and 72 hr thereafter, the cells were harvested, and AdoHcyase activity was measured in the nucleoside syn- thetic direction as described by Kredich and Hershfield (22, 23). *, 60- Enzyme activity is expressed as nmol ofAdoHcy formed per min = per mg of protein. The protein content was estimated by the Lowry method (30). cL 40- To measure AdoHcy formation by intact cells, lymphocyte suspensions were incubated in complete medium supple- 20- mented with 100-200 ,uM adenosine (in some cases radioac- tively labeled), 100-500 AM homocysteine thiolactone, and 0.1 ,M deoxycoformycin. Subsequently, the cells were pelleted, 0- I_ J washed, and extracted as described (28, 29). Additionally, cis- 0 10 20 30 40 160 diols in the culture medium, including AdoHcy, were isolated Deoxyadenosine Concentration (MAM) on a boronate affinity column (31) and then fractionated on a /LBondapak C18 column (Waters Associates) eluted with 3% FIG. 1. Deoxyadenosine toxicity toward nondividing peripheral acetonitrile in water (vol/vol) at a flow rate of 1 ml/min. blood lymphocytes. Either unfractionated peripheral blood lympho- AdoHcy emerged at 15.5 min. Concentrations were determined cytes (e) or lymphocytes enriched in B cells by prior removal of the sheep erythrocyte-rosetting fraction (o) were incubated in RPMI 1640 by comparison of absorbance at 254 nm with AdoHcy standards medium containing 20% autologous plasma, 0.1 ,uM deoxycoformycin, that were processed identically, and by measuring radioactivity and various concentrations of deoxyadenosine. After 4 days of incu- of the column effluent. bation, the percent viable nonadherent cells was determined. Downloaded by guest on September 29, 2021 3850 Medical Sciences: Carson et aL Proc. Natl. Acad. Sci. USA 79 (1982)

100- 1201 50 S80- ,1 A 40cD

I-

eB x 0 12 301

_= C 2 T. 4 = CL C 40- 8 20 0I cl- 0 20 4 2 10

0 04 Days Hours FIG. 2. Levels of ATP and dATP in deoxyadenosine-treated lym- FIG. 3. Levels of AdoHcy in the cells and medium of adenosine phocytes. Nondividing peripheral blood lymphocytes were incubated plus L-homocysteine thiolactone-treated lymphocytes. Peripheral blood as 0.1 described in Fig. 1 in medium supplemented with pM deoxy- lymphocytes at a density of 2 x 106 per ml were incubated at 37°C in coformycin (9), 0.1 deoxycoformycin plus 20 ,uM deoxyadenosine ,tM medium supplemented with 0.1 ,uM deoxycoformycin, 100 PM aden- (o), 0.1 ,uM deoxycoformycin plus 50 pM deoxyadenosine (i), and 0.1 osine, and 200 pM homocysteine thiolactone. At various time periods, MMtM deoxycoformycin plus 50 ,uM deoxyadenosine plus 100 PM deoxy- AdoHcy concentrations in the cells (e) and medium (A) and the percent cytidine (o). At the times indicated, the percent initial concentration initial cytosolic AdoHcyase activity () were determined. of ATP (-) and the ratio of dATP to ATP (--) were determined in each cell pellet by high-performance liquid chromatography. The initial ATP concentration was 700 pmol per 106 cells. decreased the rate of accumulation of dATP, most likely via competitive inhibition of deoxyadenosine phosphorylation by Effects of Adenosine plus Homocysteine. In deoxycofor- deoxycytidine kinase (EC 2.7.1.74). mycin-supplemented medium, as much as 200 ,uM adenosine Toxicity of Adenine Nucleoside Analogs. The adenine nu- and 500 ,M homocysteine thiolactone had no effect upon the cleoside analogs 2-chlorodeoxyadenosine (29), 2-fluorodeoxy- viability ofresting peripheral blood lymphocytes during a 4-day adenosine (29), adenine arabinonucleoside, formycin A, 3'- incubation period (Table 1) or upon their subsequent response deoxyadenosine, and tubercidin each killed at least 50% ofrest- to phytohemagglutinin after washing and resuspension in fresh ing peripheral blood lymphocytes at concentrations '510 ,M medium. Nevertheless, the T cells did accumulate substantial during a 4-day incubation period. The toxicity of formycin A, AdoHcy intracellularly during the first 4 hr ofculture (Fig. 3). adenine arabinonucleoside, and 3'-deoxyadenosine required However, internal AdoHcy concentrations fell markedly with 0.1 ,uM deoxycoformycin supplementation of the cultures to longer incubation in the absence ofdetectable toxicity, and de- inhibit deamination. Notably, 100 ,uM concentrations of the spite the continued presence of adenosine and homocysteine Ado deaminase-resistant adenine nucleoside analogs 6-meth- thiolactone in the medium. Accompanying the decline in cel- ylmercaptopurine ribonucleoside (Sigma) and 6-methylmercap- lular AdoHcy was (i) the appearance of AdoHcy in the culture topurine deoxyribonucleoside (29) did not impair the viability medium and (ii) a gradual decrease in cytosolic AdoHcyase ac- of resting lymphocytes, although the same drugs at 1-5 AM tivity (Fig. 3). inhibited phytohemagglutinin-induced mitogenesis and the Deoxycytidine Prevention of Deoxyadenosine Toxicity. The growth of T lymphoblastoid cell lines (not shown). addition of deoxycytidine to the culture medium in increasing Comparative Effects of Nucleosides Toward Resting and concentrations from 20 to 100 ,uM prevented the toxicity of Proliferating Lymphocytes. A comparison of the ratio of nu- deoxyadenosine toward nondividing human peripheral blood cleoside concentrations killing 50% of resting cells and those lymphocytes (Fig. 4). The deoxycytidine did not block deoxy- inhibiting phytohemagglutinin-induced lymphocyte mitogen- adenosine transport across the cell membrane, because the par- esis by 50% emphasized the marked differences in the toxicity tial inactivation of AdoHcyase induced in deoxyadenosine- ofthe various agents. As shown in Table 1, for deoxyadenosine treated cultures was not altered by deoxycytidine supplemen- and 2-chlorodeoxyadenosine, the ratio was 1. On the contrary, tation (not shown). Rather, as shown in Fig. 2, the deoxycytidine deoxyguanosine, and the combination ofadenosine plus homo-

Table 1. Effects of nucleosides and hydroxyurea on resting and proliferating human lymphocytes Approximate ID50, ,uM Resting Proliferating I)60 resting/ Drug cells cells ID50 proliferating Deoxyadenosine (+ deoxycoformycin) 5-10 5-10 1 Deoxyguanosine >1,000 30 >33 Adenosine (+ homocysteine thiolactone) >200 40 >5 2-Chlorodeoxyadenosine 0.01-0.02 0.01-0.02 1 Cytosine arabinonucleoside >6 0.03 >200 Hydroxyurea >10,000 300 >33 The ID0 is the concentration of nucleoside killing 50% of the input cells, as measured by 0.1% trypan blue exclusion after 4-day incubation in RPMI 1640 medium supplemented with 20% autologous plasma. The ID50 forproliferating cells is the concentration inhibitingby 50% the incorporation of tritiated leucine into acid-precipitable material in lymphocytes stimulated 72 hr earlier with phytohemagglutinin (24). Downloaded by guest on September 29, 2021 Medical Sciences: Carson et al. Proc. Natl. Acad. Sci. USA 79 (1982) 3851 The resistance of nondividing lymphocytes to the noxious 80 effects of adenosine plus homocysteine deserves comment,

a) considering that the same combination is lethal to rapidly di- 8 viding human lymphoblastoid cell lines (22, 23). Our results 60 a) indicate that the ability to excrete newly synthesized AdoHcy c C.) averted a sustained increase in lymphocyte AdoHcy concen- a) trations. Hoffman et al. have also reported that adenosine- 40 and homocysteine-treated rat liver cells release AdoHcy (34). Indeed, AdoHcy excretion may represent a physiological re- 20 sponse that protects differentiated cells from internal AdoHcy accumulation. 0 20 40 60 80 100 In the absence ofan inhibitor of Puo phosphorylase, one must Deoxycytidine Concentration (MM) be circumspect in interpreting the metabolic effects of deoxyguanosine on human lymphocytes. Nonetheless, concentrations FIG. 4. Deoxycytidine prevention of deoxyadenosine toxicity. Resting lymphocyte cultures containing 50 ,M deoxyadenosine and of deoxyguanosine that produced intracellular levels of dGTP 0.1 j.&M deoxycoformycin received increasing concentrations of deoxy- equivalent to or higher than concentrations of dATP that were cytidine. The percent viable cells was determined after 4 days of lethal to resting lymphocytes did not decrease cell viability. incubation. These results suggest a fundamental distinction in the modes of toxicity of dATP and dGTP. The exact mechanisms by which deoxyadenosine nucleotides cysteine, inhibited lymphocyte proliferation but did not kill kill mature resting T cells remain uncertain. Accompanying resting cells. dATP formation in lymphocytes was a fall in ATP levels that anteceded cell death by 1-2 days. In pharmacologic concentra- DISCUSSION tions, dATP may act as an ATP antagonist that impedes adenine These experiments suggest that the different immunologic phe- nucleotide synthesis or increases ATP breakdown. We have notypes associated with genetic deficiencies of Ado deaminase recently found that dATP, but not dGTP, markedly increases and Puo phosphorylase may be caused by the destruction of the activity ofa cytoplasmic ribonucleotidase purified from hu- mature, nondividing T lymphocytes by deoxyadenosine but not man lymphocytes. The eventual death ofmature T lymphocytes deoxyguanosine. As little as 1 ,M deoxyadenosine killed non- incubated with deoxyadenosine probably result from the cu- dividing human peripheral blood T lymphocytes incubated in mulative malregulation of multiple adenine nucleotide-depen- medium supplemented with autologous plasma and the tight- dent metabolic conversions. binding Ado deaminase inhibitor deoxycoformycin. Resting On a functional level, Gelfand and co-workers previously lymphocyte populations enriched in B cells were severalfold showed that deoxyguanosine inhibited T lymphoblast growth, more resistant to deoxyadenosine toxicity. Under similar con- phytohemagglutinin-induced cell proliferation, and T suppres- ditions, neither 1 mM deoxyguanosine nor 200 AM adenosine sor cell activity at concentrations that did not affect T helper cell plus homocysteine thiolactone impaired lymphocyte survival. activity and the differentiation of B cells to an antibody-secret- All three nucleosides inhibited phytohemagglutinin-induced ing stage (17, 21). These authors concluded that Puo phos- lymphocyte mitogenesis and killed established human T lym- phorylase deficiency leads to a selective impairment of prolif- phoblastoid cell lines (4, 8-12, 22-24). eration-dependent T cell functions (35). In contrast, Uberti et The Ado deaminase-inhibited peripheral blood lymphocytes al. demonstrated that deoxyadenosine inhibited leucine uptake incubated with deoxyadenosine slowly but progressively ac- during the first day after lymphocyte stimulation with phyto- cumulated dATP over at least 2 days. Hence, even at the mi- hemagglutinin (36), whereas Hayward showed that the nucleo- cromolar deoxyadenosine concentrations measured in the side prevented the synthesis ofsoluble helper factors by T cells plasma of Ado deaminase-deficient children (32), the net rate (37). of dATP formation by mature T lymphocytes exceeds its com- Recently, Kefford and Fox have reported that resting lym- bined rates of degradation and utilization. The enzyme pri- phocytes treated with nontoxic concentrations ofthe adenosine marily phosphorylating deoxyadenosine in peripheral blood T deaminase inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine were cells at lethal nucleoside concentrations was probably deoxy- also killed by micromolar deoxyadenosine concentrations (38). cytidine kinase. In support of this conclusion, exogenous de- Hence the toxic effects of deoxyadenosine toward resting lym- oxycytidine inhibited dATP formation and prevented deoxy- phocytes cannot be attributed to a unique property of adenosine toxicity at concentrations that did not alter deoxy- deoxycoformycin. adenosine-induced inactivation of intracellular AdoHcyase. In sum total, the most likely explanation for the different im- In addition to deoxyadenosine, the adenine nucleoside an- munologic phenotypes associated with Ado deaminase and Puo alogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, ade- phosphorylase deficiency relates to the contrasting effects of nine arabinonucleoside, formycin A, tubercidin, and 3'-deoxy- dATP and dGTP, rather than to direct interference with adenosine killed nondividing human peripheral blood lympho- AdoHcy catabolism by adenosine or deoxyadenosine. Deoxy- cytes. In contrast, the adenine nucleoside analogs 6-methyl- guanosine is toxic primarily towards dividing cells, especially mercaptopurine ribonucleoside and 6-methylmercaptopurine the immature thymocytes that preferentially sequester dGTP. deoxyribonucleoside did not impair resting lymphocyte survival In contrast, the dATP formed from deoxyadenosine depletes at concentrations severalfold higher than those that killed di- ATP levels in both dividing thymocytes and resting mature T viding cells. The 6-methylmercaptopurine ribonucleoside, un- lymphocytes. Puo phosphorylase-deficient patients may have like the former nucleosides, accumulates in cells primarily, al- enough T lymphocytes that enter and complete the maturation though not exclusively, as the 5'-monophosphate derivative process, despite ribonucleoside-diphosphate reductase inhibi- (33). All together, the results show that phosphorylation, and tion by dGTP, to permit proper T cell help of B lymphocyte suggest that triphosphate formation, is required for the killing proliferation and antibody production. In children with Ado de- ofresting T lymphocytes by deoxyadenosine and related drugs. aminase deficiency, the accumulated dATP kills both prolifer- Downloaded by guest on September 29, 2021 3852 Medical Sciences: Carson et atPProc. Natl. Acad. Sci. USA 79 (1982) ating thymocytes and mature resting T cells, with ablation of 17. Martin, D. W., Jr., & Gelfand, E. W. (1981) Annu. Rev. all immunologic functions under T cell control. Biochem. 5O% 845-875. 18. Carson, D. (1981) in Textbook ofHematology, eds. Williams, W. We thank Ms. A. Milne for typing the manuscript. This work was J., Beutler, E., Eisler, A. J. & Rundles, R. W. (McGraw-Hill, supported by National Institutes of Health Grants GM 23200, AM New York), 3rd- Ed., in press. 07144, and RR 00833. N. 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