THE SEPARATION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE-DEFICIENT ERYTHROCYTES FROM THE BLOOD OF HETEROZYGOTES FOR GLUCOSE-6-PHOSPHATE-DEHYDROGENASE DEFICIENCY

189

THE SEPARATION OF GLUCOSE-6-

PHOSPHATE-DEHYDROGENASE-DEFICIENT ERYTHROCYTES FROM THE BLOOD OF HETEROZYGOTES FOR GLUCOSE-6-

PHOSPHATE-DEHYDROGENASE DEFICIENCY* ERNEST BEUTLER M.D. Chicago CHAIRMAN, DEPARTMENT

OF

MEDICINE,

CITY OF HOPE MEDICAL

CENTER;

ASSOCIATE CLINICAL PROFESSOR OF MEDICINE, UNIVERSITY OF SOUTHERN CALIFORNIA

MARYELLEN C. BALUDA M.S. Pittsburg RESEARCH

TECHNICIAN,

DEPARTMENT OF MEDICINE, CITY OF HOPE MEDICAL CENTER

IN 1960 Ohno et al. demonstrated that the two X-chromosomes of females of various mammalian species were different (Ohno and Hauschka 1960, Ohno and Makino 1961). One was hyperpyknotic and formed the chromatin body, while the other resembled the autosomes. This observation led Lyon (1961), who was working with sexlinked coat-colour genes, to suggest that the hyperpyknotic X-chromosome was genetically inactive, that inactivation took place in early embryonic life, and that the same X-chromosome remained inactivated in the progeny of each cell of the embryo once inactivation had taken place. We had independently formulated the same hypothesis, on the basis of Ohno’s observations, to explain certain troublesome features regarding the genetics of a sex-linked mutation of man-glucose-6-phosphate-dehydrogenase (G.-6-P.D.) deficiency (Beutler 1962). We reasoned that if one of the X-chromosomes of the female heterozygote was inactivated, then females with intermediate enzyme activity in the peripheral blood should have two red-cell .populations. These would consist of one population of enzyme-deficient red cells, and one population of normal red cells. The disappearance of glutathione from these cells when challenged with acetylphenylhydrazine, and the reduction of methaemoglobin in them, was found to The curves obtained were occur in two components. identical with those obtained with artificial mixvirtuallv tures of normal and enzyme-deficient red cells (Beutler et al. 1962). Attempts to demonstrate by histochemical means that two cell populations were present were technically unsatisfactory (Beutler 1962, Beutler and Fairbanks 1962, Beutler et al. 1962). We have now succeeded in separating enzyme-deficient red cells from the blood of Negro women who are heterozygous for G.-6-P.D. deficiency. This was accomplished by studying the red cells of a subject who was heterozygous for this abnormality and for sickle-cell haemoglobin. Since the sickle-cell trait does not measurably shorten the lifespan of erythrocytes, these cells may be considered in every way comparable to cells from other heterozygotes for G.-6-P.D. deficiency, but the presence of sickle haemoglobin made it possible to separate the cells with a high

proportion

of

methasmoglobin

from those with

(enzyme-deficient cells)

low proportion of methaemoglobin (normal cells). This could be accomplished because methsemoglobin-containing sickle cells will not undergo sickling when the oxygen tension is decreased (Itano 1950, a

Beutler 1961). Methods

Subjects Two Negro

women

with the sickle-cell trait and with inter-

* This paper was presented, in part, at the IXth European Congress of Hæmatology, in August, 1963. Beutler, Dern, and Baluda

(1963).

mediate red-cell G.-6-P.D. levels, therefore presumably heterozygous for G.-6-P.D. deficiency (subjects no. 1 and 2), one subject with the sickle trait but with a normal red-cell G.-6-P.D. level (subject no. 3), and two normal subjects (subjects no. 4 and 5) were studied. 8 ml. of blood was drawn into 2 ml. acidcitrate-dextrose (N.I.H. formula B) and stored at 4°C before use. All studies were carried out within 24 hours of taking the blood samples. Oxidation of Haemoglobin by Nitrite and its Reduction The blood sample was centrifuged, the plasma was removed, and the cells were treated with 1 % sodium-nitrite solution to convert their haemoglobin to methsemoglobin. The buffy coat was removed and the red cells were washed five times in 7-10 volumes of 0-9% NaCl solution. They were then incubated in a This system containing glucose and Nile-blue sulphate. system has been described in detail elsewhere (Beutler and Baluda 1963, Beutler et al. 1963). It permits normal erythrocytes to reduce methaemoglobin rapidly, but allows G.-6-P.D.deficient cells to do so only very slowly. There is no interaction between normal and enzyme-deficient red cells; each cell reduces methxmoglobin at a rate which is closely related to its G.-6-P.D. activity (Beutler and Baluda 1963). Millipore-filtration Studies These were carried out bv a modification of the method of Jandl et al. (1961). Plasma from a compatible donor was used as a suspending medium for the sickling of red cells and their subsequent filtration. It was prepared from blood drawn into ethylenediamine tetra-acetic acid and freed of suspended material by high-speed centrifugation and millipore filtration (Jandl et al. 1961). Plasma stored in the frozen state before use was found preferable to fresh plasma because the nonspecific methaemoglobin-reducing action (Beutler and Baluda 1963) of such plasma was less. After use, the suspending plasma was recovered, reprocessed, and reused. A 45 mm. millipore-filter disc, with a mean pore size of 5 fL, was employed in these studies. The filtration assembly was prepared by thoroughly moistening the sintered glass portion with 0-9% NaCl solutions, and placing the filter disc into position while moistened with saline solution. The top of the inflow assembly was sealed with a rubber disc, and the space above the filter was flushed out thoroughly with the water-saturated 10% C02;90°onitrogen gas mixture employed. Hypodermic needles were pushed through the rubber disc for this purpose, as suggested to us by Dr. J. H. Jandl. The filter disc was moistened with plasma just before filtration of the cell mixture. Filtration was carried out under a pressure of 82

Hg. Preliminary studies with this system indicated that virtually no deoxygenated cells from a sickle-trait (S.-A.) subject passed through the millipore filter. If a mixture of equal numbers of s.-A. cells and normal cells was passed through the filter, each at a concentration of 0-15%, slightly over half of the normal cells passed the filter up to a suspension volume of 15 ml. At higher volumes, the passage of a large proportion of normal cells was impeded in the presence of sickle cells, presumably because the cells clogged the filter pores. Microdeterminations of G.-6-P.D. and Methaemoglobin G.-6-P.D. activity was measured by a micro-adaptation of the method of Marks (1958).t The concentration of glucose-6phosphate was reduced to 0-05 M. This does not affect the The volume of all other enzyme activity (Beutler 1960). reactants was reduced to 1/10 of the usual quantity except that mm.

either 5 or 10. litres of hasmolysate was used. Assays were carried out in the Zeiss PMQ n spectophotometer, using a microcuvette with a critical volume of approximately 0-24 ml. and a path length of 1-000 cm. The results were expressed as optical density (o.D.) units per gramme haemoglobin, as referred to the usual 2-5 ml. assay system. In effect, this was achieved by dividing enzyme activity by 10, to compensate for the reduced distribution volume of the hoemolysate, In carrying out methsemoglobin determinations the same microcuvettes were employed. A slit-width of 0.02-0.05 mm. +Normal values

(mean-1 s.n.)=15.9 +- 2.4. Negro hemizygote (mean 4;1 s.D.)=27±l’5.

G.-6-P.D. deficient

190 the monochrometer of the PMQ 11 spectrophotometer was found to be satisfactory. Methsmoglobin and haemoglobin determinations were carried out by a micromodification of the method of Evelyn and Malloy (1938). 0-3 ml. of M/60 phoson

phate buffer, pli 6-6, containing 0-02% saponin was placed into each microcuvette. 20 .litres of hsemolysate was added, and an o.D. reading was made against a water blank at 630 mµ (reading A). 5 {litres of neutralised 5% sodium-cyanide solution was added to the cuvette; it was mixed, and a second reading was taken at 630 mµ (reading B). 5 .litres of 5% potassium-ferricyanide solution was then added to the same cuvette, and the optical density at 540 mµ was determined (reading C). The methxmoglobin and haemoglobin concentrations were computed as follows, on the basis of factors derived from methacmoglobin solutions of known concentrations: B

Total

v

/

haemoglobin (mg. per ml.) =23-3 C.

The factor 0-98 was applied to the A reading to compensate for the fall in o.D. produced by a dilution of the 0-320 ml. haemoglobin solution by the 5 µlitres of the cyanide solution.

Experiments The reduction of methaemoglobin in the red cells from the five subjects in the Nile-blue-containing incubation system is presented in the figure. The cells from both subjects with intermediate G.-6-P.D. activity reduced methsemoglobin in two components, as we have previously shown to be typical for subjects heterozygous for G.-6-P.D. deficiency (Beutler et al. 1962, 1963). At the end of 3 hours, an aliquot was removed from the incubation system containing cells from heterozygotes for G.-6-P.D. deficiency; in the other cases the aliquot was taken when the methamioglobin level had fallen to between 35 % and 45 %. The aliquot was immediately chilled in ice and washed twice in cold 0-9% sodium-chloride solution. Slightly more than 13 ml. of a 0 - 15 % red-cell suspension in plasma was exposed to a mixture of water-saturated 10% carbon dioxide/ 90% nitrogen for 20 minutes, by rotating at room temperature in a sealed separatory funnel which was repeatedly flushed with the gas mixture. 10 ml. of the cell suspension was then transferred anaerobically to the millipore-filter assembly; the upper chamber had been equilibrated with 10% CO2/90% N. The suspension was then filtered at 82 mm. Hg, the filter was changed, and a second 13 ml. aliquot was treated in the same manner.

The red-cell suspension passing the millipore filter was centrifuged, and the small button of red cells obtained was washed once in 0-9% NaCl solution. Simultaneously, the red cells from an accurately measured 3-4 ml. aliquot of the gassed but unfiltered 0-15% red-cell suspension were separated by centrifugation and washed. Each sample of cells was hsemolysed by freezing and thawing twice. Using micropipettes, the hasmolysate was transferred to another iced test-tube, and the total volume was accurately adjusted to between 110 and 150

The reduction of methaemoglobin in the nitrite-treated red cells of the five subjects studied.

The incubation system, described in detail previously (Beutler and Baluda 1963), contained glucose, buffer, and Nile-blue sulphate in addition to the erythrocytes. The two-component nature of the methæmoglobin-reduction curves obtained from the heterozygous subjects is clearly evident.

.litres. The haemoglobin and methaemoglobin content and G.-6-P.D. activity of all samples was then determined. Results The results of these studies are shown in the table. When cell suspensions were prepared from the double heterozygotes (no. 1 and 2) the non-sickled forms passing the filter had a very high methaemoglobin content and a low enzyme activity. The filtered cells contained 2-34 and 2-56 units of activity per gramme haemoglobin. This is less enzyme activity than is found in the red cells of the average affected male hemizygote-i.e., 2-7 units per g. haemoglobin (Marks and Gross 1959). The low enzyme activity found in the cells passing the filter is not a result of their manipulation, since normal erythrocytes (no. 5 and 6) lost no enzyme activity when carried through the same

THE RESULTS

N = Negro, C = Caucasian, MHb=methæmoglobin.

191 process. Further, this difference could not merely be a manifestation of red-cell age. Because younger red cells have a greater capacity to reduce methæmoglobin than have older cells (Jung 1949, Lohr et al. 1958, Jalavisto and Solantera 1959), filtration exerted some selective influence on the red cells of the subject with a sickle trait but with normal G.-6-P.D. (no. 3). This selective influence, however, was quite small. The difference between the methsmoglobin content of the mixed cells and of the filtered cells, as well as the difference in enzyme activity between the mixed cells and the filtered cells was less than 20%. Undoubtedly, this is a function of red-cell age. In each case the methasmoglobin concentration in the mixed plasma-suspended cells was somewhat lower than the concentration of methaemoglobin at the time an aliquot was removed from the Nile-blue-containing incubation mixture (see table). This can undoubtedly be accounted for by the non-specific methsmoglobin-reducing activity of plasma on red-cell suspensions (Beutler and Baluda 1963). In previous studies we found that after 3 hours’ incubation of red cells of hemizygous G.-6-P.D.dencient males in the system used, methaemoglobin levels were 65 to 80% (Beutler et al. 1963). The slightly lower levels encountered in the filtered cells from female heterozygotes (63% and 60%) are readily accounted for by the non-specific methssmoglobin-reducing effect of plasma. It is quite clear, therefore, that the cells obtained through filtration of the treated blood of double heterozygotes resembled in every way the cells of hemizygous males, and it is quite important that the cells were obtained in relatively high yield. This excludes the possibility that these were a relatively few cells representing one tail of a normal distribution curve.

Discussion

The evidence for inactivation of an X-chromosome in the normal human female is quite incomplete. Yet, the problems of dosage effect involving the X-chromosome and the greatly varied expression of certain X-linked traits in the female are very nicely explained by this hypothesis. Some evidence in favour of inactivation of at least some of the loci of the human X-chromosome has been presented. The locus for G.-6-P.D. deficiency is one of those most suitable for investigation. We originally demonstrated that the rate of methaemoglobin reduction and the rate of destruction of reduced glutathione (G.S.H.) in erythrocytes from heterozygotes for G.-6-P.D. deficiency follow a two-component curve, closely resembling that found in artificial mixture of normal and enzymedeficient cells (Beutler et al. 1962). We recognised that the evidence for the existence of two red-cell populations was indirect, and that other explanations were possible. For example, a two-component curve might have resulted from an abrupt change in enzyme activity with age, occurring at approximately the midpoint of the life-span of the cell. This seems unlikely, especially since one would then expect two component curves with cells from normal subjects. It might have been supposed that the capacity of red cells to reduce methaemoglobin would decrease considerably after 3 hours. The ability of red cells to maintain glutathione levels, however, remains unimpaired for at least 6 hours, and the same metabolic pathways are involved. Further, there is no decrease in the rate of methaemoglobin reduction in enzyme-deficient cells incubated in the same system. A " thresholdeffect " may also be postulated, wherein both the rate of methxmoglobin reduction and G.S.H. preservation does not bear a continuous linear relationship to G.-6-P.D. activity. This would represent a most extraordinary biochemical situation. We therefore considered the interpretation of two redcell populations to be by far the most likely. However, studies of the disappearance of diisopropylfluorophos-

phate32 (D.F.P. 32)-labelled. red cells during drug-induced haemolysis in heterozygotes for G.-6-P.D. deficiency were interpreted as showing that no normal red cells were present (Brewer et al. 1962). Such a conclusion seems unwarranted because: 1. The calculations made failed to take into account the fact that only half of the enzyme-deficient cells are destroyed by drug administration (Beutler et al. 1954), and, therefore, that the cells remaining after the episode of drug-induced haemolysis would represent a mixture of normal and enzymedeficient cells. 2. The assumption was made that massive haemolysis of abnormal cells would exert no influence on the survival of circulating normal cells. In point of fact, it has been demonstrated that destruction of non-viable cells affects the survival of circulating red cells (Jacob et al. 1962). 3. The destruction of enzyme-deficient red cells through the administration of drugs is very complex and poorly understood (Beutler 1960). In attempting to define gene action, it is important to measure a parameter as close to the gene product as possible. Destruction of red cells through the effect of drug administration is a relatively remote consequence of enzyme

deficiency. Nonetheless, the D.F.P.32 survival data have caused be cast upon the presence of two red-cell in G.-6-P.D. deficiency (McKusick 1962, populations Harris 1963). It was apparent further that more direct evidence was required to determine whether or not there were two red-cell populations in heterozygotes for G.-6-P.D. deficiency. In the studies reported here, it has been possible to demonstrate directly, for the first time, that a sizeable population of grossly enzyme-deficient red cells can be isolated from the blood of subjects who are heterozygous for G.-6-P.D. deficiency. If a correction for nonspecific cell loss is made on the filter, then this accounts for approximately half of the cells. By simple subtraction it is evident that a population of red cells with normal enzyme activity must also be present. We interpret the course of events in the double heterozygote for G.-6-P.D. deficiency and sickle-cell trait as follows. First, approximately 90% of the haemoglobin in the red cells was converted to methxmoglobin with sodium nitrite, which was then removed by washing. During incubation with Nile-blue sulphate, cells containing normal amounts of enzyme rapidly reduced their methaemoglobin so that they were virtually methæmoglobin-free at the end of 3 hours. At this time, however, enzyme-deficient red cells still contained a relatively high proportion of methæmoglobin. This was reduced slightly during suspension in plasma, but it remained at levels well over 50%. When the oxygen tension was reduced, the cells containing primarily oxyhaemoglobin underwent sickling. Cells containing a high proportion of methsemoglobin failed to sickle. When the suspension was subjected to millipore filtration, the sickled forms containing a normal amount of enzyme were held back by the filter. Undoubtedly, some unsickled forms were trapped together with the sickled forms in the filter. A high proportion of the unsickled methsemoglobincontaining cells passed the filter, however, and as would be expected if two red-cell populations were present, the cells passing the filter contained a high average proportion of methxmoglobin and contained very little G.-6-P.D. activity: they resembled entirely the red cells of the fully

some

doubt

to

affected male. Other data bearing on the inactivation of the G.-6-P.D. locus have been presented. Gartler et al. (1962) have demonstrated that tissue cultures from the skin of

192

subjects show decreased enzyme In Gartler obtained skin-biopsy specimens 1961, activity. from some of our patients and from other patients who were heterozygous for G.-6-P.D. deficiency; long-term culture of these cells was carried out, and the growth curves were interpreted as being compatible with the growth of a mixture of two populations of cells (Gartler 1962). Cloning was not accomplished by Gartler, but it has recently been performed by other investigators who confirmed that two types of cells were present-those with low and those with normal enzyme activity (Davidson et al. 1963). G.-6-P.D.-deficient

The evidence for inactivation of loci of other sex-linked compelling. It has been pointed out that the fundus of heterozygotes for the sex-linked ocular abnormalities, choroideraemia and ocular albinism, demonstrate mosaicism (Lyon 1962). But examination of such fundi does not reveal clear-cut mosaicism showing alternation between normal and abnormal patches. Rather there is a fine mottling of the fundus which might represent mosaicism but which cannot clearly be interpreted as doing so. It has also been pointed out that the sex-linked anaemia described by Rundles and Falls (1946) fits the inactivated-X hypothesis. Female heterozygotes demonstrate small numbers of abnormal cells and large numbers of normal cells. What is disturbing is the fact that the number of abnormal cells comprise perhaps less than 5% of the total cells in the smears. This is true both in the original illustrations and in examination of smears from heterozygotes which we have had an opportunity to see, through the courtesy of Dr. Ronald Bishop. Furthermore, some female heterozygotes had normal blood but splenomegaly. These findings could be explained through much shortened survival of defective erythrocytes in the peripheral blood. However, no evidence of excessive hxmolysis is apparent in the hemizygotes, all of whose cells should bear the abnormality. It is also possible that the abnormal cells in the marrow have a reduced survival value or proliferate more slowly, thus leading to gradual elimination of the mutant clone. In the case of colour blindness, another sex-linked trait, we have been unable to demonstrate mosaicism (Krill and Beutler 1964). This could have been owing to the presence of a very fine mosaic, smaller than the half-degree targets which were used in testing the retina; nonetheless, it must be conceded that the results were negative. In the case of ectodermal dysplasia, Motulsky (1962) has demonstrated alternating areas of involved and uninvolved skin. The interpretation of this data is not entirely clear, however, since hemizygous males also demonstrated some patchiness. Histological study of muscles from heterozygotes for sexlinked dystrophy has shown patches of normal and abnormal muscle (Pearson et al. 1963). Examination of the red cells of subjects heterozygous for the X-gA blood-group has failed to demonstrate mosaicism (Gorman et al. 1963). This may indicate that the entire X-chromosome is not inactivated or that the X-gA antigen is not produced by the erythroblast, or it could be due to technical factors. Nonetheless, the reported result is negative, and this has recently been confirmed (Reed et al. genes is less

1963). In view of the somewhat equivocal results obtained when other sex-linked characteristics of man have been studied, further confirmation of inactivation of the locus for G.-6-P.D. deficiency in one of the two X-chromosomes of the female is of particular interest.

We are grateful to Dr. D. Powars, Dr. R. Day, and Dr. S. Wright for making blood samples available to use for study. This work was supported in part by a U.S. Public Health Service

grant. REFERENCES

Beutler, E. (1960) in Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson); p. 1037. New York.

(1961) J. clin. Invest. 40, 1856. (1962) in Mechanisms of Anemia (edited by I. Weinstein and E. Beutler); p. 195. New York. Baluda, M. C. (1963) Blood, 22, 323. Dern, R. J., Alving, A. S. (1954) J. Lab. clin. Med. 44, 439. Baluda, M. C. (1963) Proceedings of the IXth European Congress of Hæmatology, Lisbon (1961) (in the press). Fairbanks, V. F. (1962) Proceedings of the IXth International Society of Hæmatology, Mexico City (in the press). Yeh, M., Fairbanks, V. F. (1962) Proc. nat. Acad. Sci. 48, 9. Brewer, G. J., Tarlov, A. R., Powell, R. D. (1962) J. clin. Invest. 41, 1348 Davidson, R. G., Nitowsky, H. M., Childs, B. (1963) Proc. nat. Acad. Sci., 50, 481. Evelyn, K. A., Malloy, H. T. (1938) J. biol. Chem. 126, 655. Gartler, S. M. (1962) at the American Society of Human Genetics, Corvallis, Oregon. Gandini, E., Ceppellini, R. (1962) Nature, Lond. 193, 602. Gorman, J. G., Dire, J., Treacy, A. M., Cahan, A. (1963) J. Lab. clin. Med. 61, 642. Harris, J. W. (1963) The Red Cell; p. 268. Cambridge, Mass. Itano, H. A. (1950) PH.D. thesis for the California Institute of Technology. Jacob, H. S., MacDonald, R. A., Jandl, J. H. (1962) J. clin. Invest. 41, 1367. Jalavisto, E., Solantera, L. (1959) Acta physiol. scand. 46, 274. Jandl, J. H., Simmons, R. L., Castle, W. B. (1961) Blood, 268, 525. Jung, F. (1949) Dtsch. Arch. klin. Med. 195, 454. Krill, A. E., Beutler, E. (1964) Invest. Ophthal. (in the press). Löhr, G. W., Waller, H. D., Karges, O., Schlegel, B., Müller, A. A. (1958) Klin. Wschr. 36, 1008. Lyon, M. F. (1961) Nature, Lond. 190, 372. (1962) Amer. J. hum. Genet. 14, 135. Marks, P. A. (1958) Science, 127, 1338. Gross, R. T. (1959) J. clin. Invest. 38, 2253. McKusick, V. A. (1962) Ann. intern. Med. 56, 991. Motulsky, A. (1962) at the American Society of Human Genetics, Corvallis, -

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EXPERIMENTAL LUNG TRANSPLANTATION M.D.

IAN W. MACPHEE Glasg., M.Sc. Lpool, F.R.C.S.E., F.R.C.S. SENIOR LECTURER

E. S. WRIGHT M.D., M.Sc. McGill, F.R.C.S. RESEARCH ASSOCIATE

UNIVERSITY DEPARTMENT OF

SURGERY, LIVERPOOL

THE first experiments in pulmonary transplantation carried out in 1947 by the Russian surgeon Demikhov (1962), who transplanted the right lower lobe of the lung of one dog into the lung of another. The recipient survived for 7 days. When Staudacher et al. (1950) removed the right lower lobe and reimplanted it into the same dog, the longest survival was 12 days, whereas when the lobe was transplanted into another dog each recipient died by the 8th postoperative day. Later work demonstrated

were

clearly that, although a lung autograft could survive indefinitely with unimpaired function, homografts were always rejected and the donor animals usually died by the 10th postoperative day. The next phase consisted in attempts to suppress the immune response of the recipient against the donor lung. Neptune et al. (1953) administered corticotrophin to four dogs with lung homografts. The average survival-time was 25 days; one dog survived 42 days. In a control group the average survival-time was only 6 days. Hardin and Kittle (1954) tried to prolong survival-times in dogs with pulmonary homografts by means of anti-histamines, cortisone, total-body irradiation, and splenectomy; but no significant beneficial effect was observed with any of these measures. The discovery that anti-metabolites and alkylating