Immunology, 1973, 25, 343.

Studies on Thymus Products

I. MODIFICATION OF ROSETTE-FORMING CELLS BY THYMICEXTRACTS. DETERMINATION OF THE TARGET RFC SUB-

POPULATION

MIREILLE DARDENNE AND J.-F. BACH

Clinique Niphrologique, INSERM U 25, Hopital Necker, 161, rue de Sevres, 75730Paris, Cedex 15, France

(Received 4th January 1973; acceptedfor publication 6th March 1973)

Summary. Thymic extracts confer on normal bone marrow rosette-formingcells (RFC) in vitro a high sensitivity to anti-theta serum (AOS) and azathioprine(AZ) which they usually lack. Thymic extracts can also confer high sensitivity toAOS and AZ to spleen RFC from adult thymectomized, neonatally thymectomized'thymus-deprived' and nude mice. However, the amount of thymic extracts neces-sary to get the effect is significantly higheron thymus-deprived and nude mouse spleenRFC than on RFC from spleens of adult thymectomized mice or normal mousebone marrow. Thymic extracts are also active in vivo and there is a good correlationbetween the in vitro minimum concentration giving AOS and AZ sensitivity to spleenRFC from adult thymectomized mice and the in vivo minimum dose giving such aneffect after intravenous injection. The effect of extract in vivo does not appear beforethe fourth hour after the injection and is transient, disappearing after 48 hours.Injection of thymic extracts induces the appearance of a 'thymic activity' (TA)in the serum with a short half-life (2 hours). Injection ofthymic extracts into normalmice does not modify sRFC characteristics in spleen, lymph nodes and bonemarrow. It is suggested that thymic extracts act reversibly on a population ofT-RFC precursors.

INTRODUCTION

It has been reported that the injection of thymic cell-free extracts can restore theimmunological competence of neonatally thymectomized mice for the rejection of skingrafts and the graft versus host reaction (Goldstein, Guha, Zatz, Hardy and White, 1972;Goldstein, Asanuma, Battisto, Hardy, Quint and White, 1970; Trainin and Small, 1970).These data and those of Osoba, showing partial reconstitution of neonatally thymectom-ized mice by thymus grafts in a Millipore chamber (Osoba and Miller, 1963), suggest thatthe thymus may act as an endocrine gland. However, some criticism has been made ofthese experiments (Davies, 1969a; Kruger, Goldstein and Waksman, 1970) and theexistence and biological significance of thymic hormones is still controversial.We have previously reported (Bach and Dardenne, 1972a, 1973a) that in mice a large

portion of spontaneous rosette-forming cells (sRFC) are dependent on the presence of thethymus. There is in the spleen and in the thymus a population of sRFC, the rosette-

343

344 Mireille Dardenne and J.-F. Bach

forming capacity of which is very sensitive to inhibition in vitro by azathioprine (AZ) andanti-theta serum(AOS ) (Bach and Dardenne, 1972b, 1973a). These sRFC are absent fromthe spleens of mice thymectomized at birth (Bach and Dardenne, 1972b) or as adults(Bach and Dardenne, 1973a; Bach, Dardenne and Davies, 1971). We report here experi-ments showing the action, in vitro and in vivo, of thymic extracts on an sRFC populationpresent in normal bone marrow and spleen of adult thymectomized mice. Preliminaryresults ofsome of the in vitro experiments have already been published (Bach, Dardenne,Goldstein, Guha and White, 1971).

MATERIALS AND METHODSMice

C57/B16, Swiss and C3H mice were provided by the Centre d'Elevage des Animaux deLaboratoire du C.N.R.S. (45 Orleans, La Source). CBA-Harwell mice came from theChester Beatty Research Institute (London). These mice were always used at the age of6-8 weeks. C3H germ-free mice and nude mice were given by J. C. Salomon. Nude micewere used at the age of 3-5 weeks, when their weight was over 10 g.

Thymic extractsCalf, mouse and human thymic extracts were donated by A. L. Goldstein and N.

Trainin. Most studies used calf thymic extracts but some experiments were performedwith mouse extracts (fraction 3). Details of the preparations of these extracts have alreadybeen published (Goldstein et al., 1972; Trainin and Small, 1970). Goldstein's fractionsused in our experiments were the acetone precipitate (fraction 3) and the Ecteola separa-ted fraction (fraction 6). We have added a letter when several batches of one fraction wereused. Amounts of thymic extracts are expressed in protein weight, evaluated according toLowry's technique (Lowry, Rosebrough, Farr and Randall, 1951). Control preparationswere made from spleen, muscle and brain, according to the same method as that used forA. Goldstein's fraction 3 and N. Trainin's crude fraction. Endotoxin (Salmonella enteritidis)was also used in some experiments.

Anti-theta serum (AOS)AOS was prepared by injection of CBA thymocytes into AKR mice (Raff, 1971). The

injections (20 x 106 thymocytes intraperitoneally (i.p.)) were performed once a week,six times. The mice were bled before the two last injections and 7 days after the last one.Sera were cytotoxic for CBA thymocytes at concentrations higher than 1/200. No serumhad cytotoxic autoantibody active against AKR thymocytes.

ChemicalsAzathioprine (AZ) (Burroughs Wellcome) was .used as its sodium salt (preparation

for clinical i.v. use).

Ultrafiltration membranesAmicon Centriflo membranes CF 50 A (mol. wt cut-off: 50,000) and UM 10, UM 2

membranes (mol. wt cut-off: 10,000 and 1,000 respectively) were used.

Thymic Extracts and Rosette-forming Cells. I

Cytotoxicity testsThese were performed using 5tCr-labelled thymocytes, as previously described (Bach

and Dardenne, 1972b).

Rosette testThe method used for rosette formation has already been described (Bach and Dardenne,

1972a). Briefly, this method consists of mixing 3 x 106 nucleated cells (from spleen, bonemarrow, thymus or lymph nodes) with 12 x 106 sheep red blood cells (SRBC) in a plastichaemolysis tube. Cells are then centrifuged for 5 minutes at 200 g at 40 and resuspendedusing a roller (10 cm diameter) rotating at 10 rev/mm in a vertical plane; more vigorousagitation tends to destroy 0-positive RFC (Charreire, Dardenne and Bach, 1973).

Rosette inhibition by AZ and AOS (Bach and Dardenne, 1972b)Lymphoid cells were incubated for 90 minutes at 370 with doubling dilutions of AZ

and AOS and the rosette test was performed. The AZ minimal inhibiting concentration(MIC) and AOS rosette inhibition titres were defined as the concentrations giving 50 per centrosette inhibition compared to control tubes done without AZ and AOS.

ThymectomiesAdult thymectomy was performed by suction (Miller, 1960) after nembutal anaes-

thesia. Neonatal thymectomy was performed in C3H germ-free neonates anaesthetized byrefrigeration. In all cases, absence of thymic remnants was verified when the mice werekilled. 'Thymus-deprived' C57/B16 and C3H mice were prepared by injection of adultthymectomized and irradiated (850 R) mice with 1 x 107 bone marrow cells (Davies,1969b).

RESULTS

Thymic extracts were incubated with mouse lymphoid cells or injected i.v. into thy-mectomized mice and changes in sRFC were studied. The results were similar for allthymic extracts: we will first detail the action of two extracts, one slightly purified (A.Goldstein's fraction 3), the other one highly purified (A. Goldstein's fraction 6). We willthen compare the activities of all fractions studied on a quantitative basis, in vivo andin vitro.

A. In vitro ACTIVITIES OF THYMIC EXTRACTS

When bone marrow cells from normal or adult thymectomized mice, or spleen cellsfrom adult thymectomized mice were incubated with low concentrations of thymicextracts for 30 minutes at 370, the initially low sRFC sensitivity to AZ and AOS wasincreased to the level of normal thymus and spleen sRFC. In no instance did thymicextracts modify the number of sRFC.

(1) DescriptionWhen normal bone marrow or spleen cells from adult thymectomized mice were

incubated with thymic extracts (10 pg/ml fraction 3 and 0 01 pg/ml fraction 6a) for 30minutes at 37°, washed twice and incubated again for 60 minutes at 370 with AZ or AOS

345

Mireille Dardenne and J.-F. Bach

TABLE 1

DECREASE OF AZ MIC AFTER INCUBATION WITH THYMIC EXTRACT (FRACTION 6a) IN C57/B16 AND NUDEMICE. (MEANS+ 2 SE., SG. = SIGNIFICANT)

Amount ofAZ MIC AZ MIC thymic extract

RFC without with (fraction 6b)Number thymic extract thymic extract lowering

(per 106 cells) (jg/ml) (pg/ml) AZ MIC-.3 pg/ml

Normal mice (eight mice)Bone marrow 410+100 95+ 10 1-3+0-3 1-2 pg±0-2Spleen 1-200+ 120 1-2+0-1 No sg. changeLymph nodes 250+ 70 62 ± 8 No sg. change

Adult thymectomized mice(twelve)

Bone marrow 380+ 160 105+ 12 1-4+0-3Spleen 1P150+ 140 80+9 0-9+0-4 1-9 yg±0-8

Neonatally thymectomizedmice (six mice)

Bone marrow 370+ 180 105+ 15 6+4Spleen 1P080+ 100 65 + 20 0-9+ 0-3 Not done

'Thymus-deprived mice'(eight mice)

Bone marrow 410+ 150 110+20 3-5 +2Spleen 1-120+ 140 90+ 15 1-2+0-3 3-6 pg± 1-2

Nude mice (five mice)Bone marrow 310 + 60 105+ 15 4-1+0-8Spleen 940+ 120 80+ 15 1-2+0-3 10-2 pg±3

TABLE 2

INCREASE OF AOS ROSETTE INHIBITION TITRE AFTER INCUBATION WITHTHYMIC EXTRACT (FRACTION 6a, 10 pg/ml) IN C57/B16 AND NUDE MICE

(RANGE OF EXPERIMENTAL RESULTS)

AOS titrewithout

thymic extract

AOS titrewith

thymic extract

Normal mice (six mice)Bone marrow 1/10-1/20 1/80-1/320Spleen 1/160-1/320 1/160-1/320

Adult thymectomized mice(eight mice)

Bone marrow 1/20 1/160Spleen 1/20-1/40 1/160-1/230

Neonatally thymectomizedmice (six mice)

Bone marrow 1/20 1/160Spleen 1/20-1/40 1/160

Nude mice (five mice)Bone marrow 1/20-1/40 1/160Spleen 1/20-1/40 1/160-1/320

346

Thymic Extracts and Rosette-forming Cells. I

at variable concentrations, AZ and AOS MIC were decreased to the level of that observedfor thymus and normal spleen RFC (Tables 1 and 2). At low concentrations of extractthe effect was only partial (Fig. 1). Thymic extracts had no effect on those RFC which aretotally resistant to AOS and AZ, i.e. the population of RFC, which is normally inhibitedby high concentrations of AOS (1/40) and AZ (100 pg/ml) is the only one sensitive tothymic extracts. Spleen, muscle and brain fractions, prepared as fraction 3 and endotoxinwere inactive in the same protocol. The thymic extract, prepared from the mouse, provedto be active at 1 ug/ml both for AZ and AOS tests.

100 _ S a50

~2512

N'< 6

3 0-@

15

i7

0 0 15 03 06 1-2 25 5 10

Extract concentration (pg)

FIG. 1. Increase of AZ sensitivity of bone marrow RFC after in vitro treatment with spleen andthymus extracts (fraction 6b). (AZ MIC azathioprine rosette minimal inhibiting concentration inpg/ml). (0) Spleen extract. (e) Thymic extract.

(2) KineticsThymic extracts were effective when AZ was added to spleen cells simultaneously with

the extract. Conversely, the effect of AOS on RFC was obtained only when the extractwas added 5-10 minutes before adding AOS. Cell washing after 5-30 minutes incubationwith the extract, before adding AZ or AOS, did not suppress the effect. No effect wasobserved if cells were incubated with the extract at 40 and washed before adding AZ orAoS.

(3) Consumption studiesThymic extracts (fraction 6b) was incubated with spleen cells from adult thymectomized

mice, as described above, for 30 minutes at 370. The cells were then centrifuged and thesupernatant tested on new spleen cells from adult thymectomized mice. No significantincrease in the minimal active concentration of the extract was noted.

(4) AbsorptionsOne batch of AOS was incubated with thymic extract (fraction 6a) (2 mg/ml), for 120

minutes at 37°. No precipitate was visible, even after centrifugation at 20,000 rev/min.Absorbed AOS was then compared with initial AOS for inhibition of normal spleen sRFCand cytotoxicity against 51Cr-labelled CBA thymocytes. No difference of activity in thesetwo tests was observed between the absorbed and the initial AOS.

347

Mireille Dardenne and J.-F. Bach

(5) Diafiltration of thymic extractsFraction 6b and N. Trainin's crude extract and dialized fraction (Trainin and Small,

1970) were filtered on Amicon UM 2 and UM 10 membranes. The activity was stillfound after UM 10 filtration (at the same concentration as in the control preparation)but it was retained by UM 2 membranes.

(6) Action on lymphoid cells other than normal bone marrow and spleen cellsform adult thymectomizedmice

Incubation of spleen and lymph node cells from normal mice and lymph node cellsfrom adult thymectomized mice with thymic extracts did not modify RFC sensitivity to AZand AOS, even at high concentrations ofthymic extracts (50 pg/ml, fraction 6a).

->50-

25

E 12 2ON

N 3

<-15 --

0 12 3 6 12

Thymic extract concentration (jig)FIG. 2. Comparison of in vitro effects of thymic extract (fraction 6b) on normal bone marrow andspleen RFC from adult thymectomized 'thymus-deprived' C57/B16 and nude mice. The latter aresignificantly less sensitive to thymic extracts than the former (P< 01) (means+2 SE). (A) Nudespleen. (0) Deprived spleen. (-) Adult Tx Spleen. (in) Normal bone marrow.

Thymic extracts restored AZ and AOS sensitivity of bone marrow and spleen RFC ofneonatally thymectomized, 'thymus-deprived' and nude mice, making it equal to that ofnormal spleen RFC (Tables 1 and 2). However, they did not modify the lymph node RFCsensitivity to AZ and AOS.

(7) The effect of variation ofconcentration of thymic extractThe dose-dependence of thymic and spleen extracts on bone-marrow RFC from adult

thymectomized C57/B16 mice is represented in Fig. 1. The dose-dependence curve wasalso studied for fraction 6b on normal bone marrow cells and spleen cells from 'thymus-deprived' C57/B16 and C3H mice and from nude mice. As shown in Fig. 2, the amount ofthymic extract necessary to lower AZ MIC down to 10 ug/ml was significantly higher inspleen cells from nude mice than in normal bone marrow cells and adult thymectomizedmice. Cells from 'thymus-deprived' mice were sensitive to intermediate concentrations.

348

Thymic Extracts and Rosette-forming Cells. I

B. In vivo ACTIVITIES OF THYMIC EXTRACTS

(1) Injection into normal miceThe RFC characteristics (number and AZ-sensitivity) of bone marrow and spleen RFC

examined 24 hours after injection of 100 jug of fraction 6b into normal C57/B16 mice wereunchanged (five mice examined).

125A

0B InA-B C

30

6N

< 12

_>25 D- -

_10 1 10 10-2 10-3 10-4

tl9

FIG. 3. Increase ofAZ sensitivity of spleen RFC from adult thymectomized C57/B16 mice after injectionof thymic extracts. Goldstein's fraction (A) 6a, (B) 4a, and (D) spleen extract, and (C) Trainin's crudethymic fraction. (Means + 2 SE.)

(2) Injection into adult thymectomized miceFraction 6b was injected i.v. at two different dose levels into C57/B16 mice thymecto-

mized 7-10 days before. Spleen, bone marrow and serum were collected 1, 2, 3 and 4 hours,and 1, 2, 3 and 4 days after the injection. A spleen extract was injected into control mice.

E

E6 E 1/32 e t\

25 - 1/8 _

_50 - 1/2 -

0 1 2 3 4 12 24 48 72 96

HoursFIG. 4. Eflects on spleen RFC andserum'thymic activity' (TA) of i.v. injection of 100 pg of thymic extract(fraction 6b). (-) Serum TA. (0) AZ MIC.

(a) Efect on RFC. The low AZ sensitivity ofspleen RFC from thymectomized mice wasrestored to normal 24 hours after the injection of 10 ug of fraction 6a (Fig. 3), whereas nocorrection was observed after the injection of 5 mg of the spleen extract. The in vitro dose-effect curve of three fractions is shown in Fig. 3. For lowest doses offraction 6a, one may notesensitivities to AZ intermediate between RFC sensitivity in normal and thymectomizedmice. The effect of thymic extracts on spleen RFC was not seen until the 24th hour andwas then at its maximum (normal spleen AZ sensitivity for optimal doses). This effect

349

350 Mireille Dardenne and J.-F. Bach

was rapidly lost since AZ MIC had come back again to the level in adult thymectomizedmice 72 hours after the thymic extract injection (Fig. 4). No change in bone marrow RFCsensitivity was noted, even after highest doses. Fraction 6b was also tested in the sameprotocol in nude mice. No effect on spleen RFC was noted, even after injection of 500 ug(three mice examined).

(b) Appearance of 'thymic activity' (TA) in the serum (Figs 4-5). The serum of mice injectedwith 30 and 100 jug of fraction 6b was filtered through CF 50 Amicon membranes andincubated with adult thymectomized spleen cells, as described above for thymic extracts.Whereas no TA is usually found in the serum of adult thymectomized mice, it appearedwithin 30 minutes after the injection of extract. Although the injection was performedi.v., no TA was detectable after 15 minutes. The maximum activity was observed after2 hours and all activity had disappeared within 48 hours.

1/512 -

1-1/128 -

E1/32-

1/80

1/2 F W| WX| 'I| I I0 1 2 3 4 1224 48

Hours

FIG. 5. Serum 'thymic activity' (TA) after injection of 30 and 100 jig of thymic extract (fraction 6b).(0) 60pg. (A) 30pg.

C. CORRELATION BETWEEN in vivo AND in vitro ACTIVITIES OF THYMIC EXTRACTS

Fifteen thymic extract preparations were tested in vitro, both on normal bone marrowand spleen cells from adult thymectomized mice. All thymus extracts were active both onbone marrow and spleen RFC. The in vivo activity was tested by injecting eight of theminto adult thymectomized mice. A significant correlation was found between extractconcentrations active in vitro (Table 3) and doses active in vivo. Control preparations fromorgans other than the thymic did not show any in vivo or in vitro activity on RFC.

DISCUSSION

Thymic extracts did not modify the number of sRFC in lymphoid organs from normaland thymectomized mice. However, thymic extracts did modify RFC sensitivity to AZand AOS in normal bone marrow cells or spleen cells from adult thymectomized mice,increasing it to the level of that found in normal thymus and spleen cells. Spleen extractsdid not induce the same effects. The use ofAZ and AOS as markers ofT-RFC has alreadybeen extensively discussed (Bach and Dardenne, 1972b, 1973a). In brief, it was shown thatfour classes ofsRFC could be distinguished in terms of sensitivity to inhibition by AOS andAZ: class I RFC, present in thymus and normal spleen, inhibited by very low AZ and AOSconcentrations, depleted by adult or neonatal thymectomy; class II RFC, present in normal

Thymic Extracts and Rosette-forming Cells. I

lymph nodes, inhibited by 'intermediate' AZ and AOS concentrations, depleted by neonatalbut not by adult thymectomy; class III RFC, present in normal bone marrow and spleenof adult thymectomized mice, inhibited by high AZ and AOS concentrations, unalteredby neonatal thymectomy and present in the spleens of nude mice, and class IV RFC,present in normal bone marrow and normal spleen, also unaltered by neonatal thymectomyand present in the spleens of nude mice, not inhibited by AZ nor AOS, even at highconcentrations. Our studies indicate that class III RFC represent the target RFC popu-lation of thymic extracts. Their origin will be discussed in the following paper.

TABLE 3In vitro AND in vivo MINIMUM ACTIVE DOSES OF THYMIC EXTRACTS (MEANS + SE). THYMICEXTRACTS HAVE BEEN INCUBATED WITH NORMAL BONE MARROW CELLS OR SPLEEN CELLS FROM

ADULT THYMECTOMIZED MICE AND THE RFC CHANGES STUDIED

Adult thymectomized spleen

Normal bone marrow In vitro In vivo(pg/ml) (jg/ml) (pg)

A. Goldstein's fractionsThymus Fraction 4a 12+5 10+2 Not done

Fraction 4b 8+4 7+2 100 jugFraction 6a 0 05+0 03 0 07 + 0 03 1 pgFraction 6b 0 5+003 0-6+0-1 50 pg

Spleen Fraction 4 >100 >100 >1 mgBrain Fraction 4 >50 >50 Not doneSalmonella enteritidis endotoxin > 100 > 100 Not done

N. Trainin's fractionsThymus crude extract 45+12 120+ 70 1b5 mgThymus dialized fractions 5 + 4 7 + 5 0 7 mgSpleen crude extract 75 + 15 350+ 50 >5 mg

It has been suggested that reconstitution of immunological competence of neonatallythymectomized mice by thymic extracts could be due to an adjuvant effect, for examplebecause of endotoxin contamination or the antigenicity of heterologous proteins (Krugeret al., 1970). Endotoxin was inactive in our system. The antigenicity of calf extracts didnot interfere since mouse extracts showed similar activity. It is unlikely that an adjuvanteffect operated in our short-term in vivo and in vitro experiments, which do not involve thedevelopment of an immune response. Cell toxicity of the extracts could explain increasedsensitivity to AZ and AOS but this is unlikely as low doses of extract in vivo (1 ug/ml) andin vitro (0-01 ,ug/ml), were effective. Moreover, the correlation found between activitiesin vivo and in vitro is reassuring since non-specific effects are likely to be different in vivoand in vitro.The hypothesis that 0-antigen contained in thymic extracts sticks to RFC can be

excluded because: (1) AOS activities are not decreased after absorption with thymicextracts; (2) brain extracts have no activity; (3) the effect on AZ-sensitivity is not easilyexplained by such an hypothesis. Finally, the demonstration of 'thymic activity' in normalserum, disappearing after thymectomy and reappearing after thymus grafting (Bach andDardenne, 1973b) is probably the best argument in favour of specificity.

It is possible that thymic extracts need some metabolic transformation in vivo, sincethere is a lag time of more than 15 minutes before seeing TA in the serum after i.v.injection of thymic extract. A similar metabolic transformation may occur in vitro.

351

352 Mireille Dardenne and J.-F. BachOur studies do not provide any precise evaluation of the molecular weight of products

contained in thymic extracts active on RFC. However, we have found the active productsin Goldstein's and Trainin's extracts retained by UM 2 membrane but not by UM 10,which would indicate mol. wt between 1000 and 10,000. This is, however, a roughevaluation compatible with Goldstein's latest evaluation of 12,500 (Goldstein et al., 1972).Our data do not give any resolution of the discrepancy between Trainin's (Trainin andSmall, 1970) and Goldstein's (Goldstein et al., 1972) results. In fact, we have no proof thatthe factor(s) detected in our experiments is (are) the same as those described by Goldsteinand Trainin. In other words, we cannot exclude the possibility that the factor modifyingRFC is present in Goldstein's and Trainin's thymic extracts, but is not the factor active intheir own system.The mechanism of thymic extract effects on RFC and their biological significance with

regard to thymus humoral function will be discussed in the accompanying article dealingwith serum thymic factors (Bach and Dardenne, 1973) since so far we have not foundany difference between the effects of thymic extracts on RFC and those of serum thymicfactors.

REFERENCESBACH, J. F. and DARDENNE, M. (1972a). 'Antigen

recognition by T-lymphocytes. I. Thymus and bonemarrow dependence of rosette forming cells in themouse.' Cell. Immunol., 3, 1.

BACH, J. F. and DARDENNE, M. (1972b). 'Antigenrecognition by T-lymphocytes. II. Similar effectsof azathioprine, ALS and antitheta serum on rosetteforming lymphocytes in normal and neonatallythymectomized mice.' Cell. Immunol., 3, 11.

BACH, J. F. and DARDENNE, M. (1973a). 'Antigenrecognition by T-lymphocytes. III. Evidence fortwo populations of thymus-dependent rosetteforming cells.' Cell. Immunol. 6, 394.

BACH, J. F. and DARDENNE, M. (1973b). 'Studies onthymus products. II. Demonstration and charac-terization of a circulating thymic hormone.' Immuno-logy, 25, 353.

BACH, J. F. and DARDENNE, M. and DAVIES, A. J. S.(1971). 'Early effect of adult thymectomy.' Nature:New Biology, 231, 110.

BACH, J. F., DARDENNE, M., GoLDSTEIN, A.L., GuHA,A. and WHITE, A. (1971). 'Appearance of T-cellmarkers in bone marrow rosette forming cells afterincubation with thymosin, a thymic hormone.' Proc.nat. Acad. Sci. (Wash.), 68, 2734.

CHARREIRE, J., DARDENNE, M. and BACH, J. F. (1973).'Antigen recognition by T-lymphocytes. IV. Diff-erences in antigen binding characteristics of T-andB-RFC: a cause for variation in the evaluation ofT-RFC.' Cell. Immunol. (In press.)

DAVIES, A. J. S. (1969a). 'The thymus and cellularbasis of immunity.' Transpl. Rev., 1, 43.

DAVIES, A. J. S. (1969b). 'The thymus humoral factorunder scrutiny.' Agents and Actions, 1, 1.

GOLDSTEIN, A. L., ASANUMA, Y., BATTISTO,J.R., HARDY,M. A., QuINT, A. and WHITE, A. (1970). 'Influenceof thymosin on cell-mediated and humoral immuneresponses in normal and immunologically deficientmice.' J. Immunol., 104, 359.

GOLDSTEIN, A. L., GUHA, A. ZATZ, M. M., HARDY,M. A. and WHITE, A. (1972). 'Purification and bio-logical activity ofthymosin, a hormone ofthe thymusgland.' Proc. nat. Acad Sci. (Wash.), 69, 1800.

KRUGER,J., GOLDSTEIN, A. L. and WAKSMAN, B. (1970).'Immunologic and anatomic consequences of calfthymosin injection in rats.' Cell. Immunol., 1, 51.

LowRY, O.H., ROSENBROUGH, N. G. FARRi, A. L. andRANDALL, R. J. (1951). 'Protein measurement withthe folin phenol reagent.' J. biol. Chem., 193, 265.

MILLER, J. F. A. P. (1960). 'Studies on leukaemicmice. The role of the thymus in leukaemic genesis bycell-free leukaemic filtrates.' Brit. J. Cancer, 14, 93.

OSOBA, D. and MILLER, J. F. A. P. (1963). 'Evidencefor a humoral thymus factor responsible for thematuration of immunological faculty.' Nature (Lond.),199, 359.

RMFF, M. C. (1971). 'Surface antigenic markers fordistinguishing T and B lymphocytes in mice.'Transpl. Rev., 6, 52.

TRININ, N. and SMALL, M. (1970). 'Studies on somephysicochemical properties of a thymus hormonalfactor confering immunocompetance on lymphoidcells.' J. exp. Med., 132, 885.