1080

the nursing and clerical staff of the accident and emergencyservice who took on extra work in this trial on top of the dailyround of the department.

Requests for reprints should be addressed to W. H. R., RoyalVictoria Hospital, Grosvenor Road, Belfast BT12 6BA.

REFERENCES

1. Armitage, P. Sequential Medical Trials. Oxford, 1960.2. Rains, A. J. H., Capper, H. M. Bailey & Love’s Short Practice of

Surgery. London, 1965.3. Lowden, T. G. in The Hand (edited by R. G. Pulvertaft); chap 12.

London, 1966.4. McNair, Y. J. Hamilton Bailey’s Emergency Surgery. Bristol, 1967.5. Ellis M. The Casualty Officer’s Handbook. London, 1970.6. Logan, C. J. H. Ulster med. J. 1960, 29, 22.7. Price, D. J. E., O’Grady, F. W., Shooter, R. A., Weaver, P. C.

Br. med. J. 1968, iii, 407.8. Harris, D. M., Wise, P. J. Practitioner, 1969, 203, 207.9. Bauer, A. W., Perry, D. M., Kirby, W. M. J. Am. med. Ass. 1960,

173, 475.10. Burns, J. I., Curwen, M. P., Huntsman, R. G., Shooter, R. A.

Br. med. J. 1957, ii, 193.11. Anderson, J. ibid. 1958, ii, 1569.12. Ellis, M. ibid. 1959, i, 299.

ESTABLISHMENT OF IMMUNOLOGICAL

COMPETENCE IN A CHILD WITH

CONGENITAL THYMIC APLASIA BY A

GRAFT OF FETAL THYMUS

C. S. AUGUST R. H. LEVEY

A. I. BERKEL F. S. ROSEN

Departments of Medicine and Surgery, Children’s HospitalMedical Center, and Departments of Pediatrics and Surgery,

Harvard Medical School, Boston, Mass. 02115

H. E. M. KAY

Royal Marsden Hospital, London S.W.3

Summary Fetal thymus tissue was implanted intoa 21-month-old patient with congenital

aplasia of the thymus gland (DiGeorge’s syndrome).Clinical and immunological studies carried out for 16months thereafter revealed prompt and long-lastingimprovement in previously defective cellular immunefunctions including dermal sensitivity to monilia anti-gen and dinitrofluorobenzene, skin allograft rejection,and the responses of peripheral blood leucocytes invitro to phytohæmagglutenin and to monilia antigen.It is suggested that implanting fetal thymus tissues intopatients with DiGeorge’s syndrome offers at present thebest hope of improving their deficient cellular immunefunction.

Introduction

IN 1965 DiGeorge described four patients with

hypoparathyroidism and absent thymus glands whichhe attributed to an error in the development of struc-tures derived from the third and fourth pharyngealpouches during embryonic life.1,2 The immunologicalconsequences which were subsequently found in theseand similar children involved a profound inability tomount cellular immune responses.3-6 It was speculatedthat these patients would be ideal recipients for trans-plants of fetal thymus tissue,5 and indeed two

apparently successful attempts to establish cellularimmune function in this way have been reported 6,8

Descriptions of the two immunologically correctedpatients have stimulated many questions as to whetherthe recovery was in fact spontaneous or was mediated

by the implanted fetal thymus tissue.9 9 This report

provides further details about one of the previouslyreported patients and describes the immunologicaltests that have been performed over an 18-month periodsince the fetal thymus tissue was implanted. Webelieve that the data strengthen the previous conclusionthat the rapid acquisition of immunological competencein our patient was due to the implanted fetal thymusand not to spontaneous recovery.

Case-reportAn infant developed hypocalcasmic tetany on his 3rd day

of life. Although seizures disappeared with calcium-

gluconate therapy, hypocalcxmia persisted for a longertime. During his lst year, he had oral moniliasis, otitismedia, recurrent upper and lower respiratory-tract infec-tions, and a deep-seated abcess of the skin. During thatyear he was admitted to hospital six times; growth anddevelopment were retarded. Immunological studies whenhe was 10 months old revealed mild lymphopenia, defectivedelayed hypersensitivity, poor responses of his lymphocytesto phytohaemagglutinin (P.H.A.) in vitro, and normal levelsof serum-immunoglobulins. He had primary and secondaryantibody responses to typhoid vaccine and to tetanus anddiphtheria toxoids, respectively. A skin-graft from anunrelated donor apparently was accepted.When studied again at 21 months of age, the patient’s

immunological deficiency was still marked,8 although anambiguous response to dinitrofluorobenzene (D.N.F.B.) at

the highest concentration tested-500 jig. per ml.-sug-gested that immunological function might have improvedslightly. The serum-calcium concentration was 9-3 mg. per100 ml. Implantation of thymus fragments from a femalefetus estimated to be 16 weeks’ gestation was carried out asdescribed previously. 8 When tested 4 days later, the

peripheral blood leucocytes responded normally to P.H.A.,whereas 2 days before the implant they had been virtuallyunresponsive. When tested 2 weeks later, the patientresponded normally to challenge doses of D.N.F.B. andmonilia. He promptly rejected a skin-graft from an unrelateddonor.

Since discharge from the hospital in September, 1968, hehas had occasional upper-respiratory-tract infections andthree bouts of uncomplicated otitis media. Although he hasgrown at a normal rate since the implantation, both heightand weight remain below the third percentile. Intellectualand motor development have been rapid. Although speechdevelopment has been slower, he is no longer thought to beglobally retarded. No masses have ever been palpable at thesite of the implanted thymic fragments.

Immunological studies consisting of complete blood-

counts, a number of skin-tests, and in-vitro studies oflymphocyte function were carried out 2, 4, 8, 12, and 14months after thymic implantation. During the last studythe patient received a skin-graft from an unrelated donor. 14days later, when the graft appeared to be undergoing rejec-tion, it was excised and a regional lymph-node was biopsied.

Methods

Immunological testing was performed by methodsdescribed previously. 10 Skin tests with monilia antigen(Hollister-Stier), streptokinase-streptodornase (S.K.S.D.,Lederle), diphtheria toxin and toxoid (MassachusettsBiological Laboratories) were injected intradermally on theforearm in 0.1 ml. sterile saline solution and read 48 hourslater. Only induration greater than 5 mm. in diameter wasconsidered to be unequivocally positive. Contact sensitivityto D.N.F.B. and dinitrochlorobenzene (D.N.C.B.) were patch-tested by applying the appropriately diluted chemical to thepatient’s skin for 48 hours. The tests were read when thepatches were removed and again 24 hours later. Onlyredness associated with vesiculation was considered to

represent a specific immune response.

1081

TABLE I-IMMUNOLOGICAL STUDIES IN A PATIENT WITH CONGENITAL

ABSENCE OF THE THYMUS FOLLOWING IMPLANTATION OF FETAL

THYMUS

Peripheral blood leucocytes were cultured by standardmethods 11 at concentrations of 0-75 to 1.5 x 106 mono-nuclear cells per ml.: P.H.A., monilia antigen,12 and S.K.S.D.(10, 50, and 100 (J-g. per ml.) were added to cultures tostimulate blast-cell transformation. Cultures with P.H.A.added were incubated for 3 days; those with antigen wereincubated for 7 days. On the final day of culture, 3H-thymidine (new England Nuclear Co., sp. act. 2-0 C permillimole) was added to each culture to achieve a finalconcentration of 1-0 (J-C per ml. 3 hours later colchicine wasadded to a final concentration of 0-04 (J-g. per ml.; 3 hoursthereafter, the cells were resuspended and 0-1 ml. cellsuspension was pipetted in triplicate on to filter-paper discs(Whatman no. 3, diameter 2-3 cm.). Incorporation of

3H-thymidine into D.N.A. was determined as radioactivityprecipitable by 10% trichloroacetic acid (T.C.A.) by a modi-fication of the method of Mans and Novellï.I3 The discswere immersed in cold 10% T.C.A., washed sequentially in5% T.C.A., ethanol-acetone (1/1), and acetone. Radio-activity bound to the discs was estimated by liquid scintil-lation spectrometry. Total radioactivity was calculatedby multiplying the counts per minute (c.p.m.) per disc(representing 0-1 ml.) by the total culture volume. Theremaining cells were harvested, smeared on microscopeslides, and stained with acetic acid/orcein. Thus, the appear-ance of the cells and the number of mitotic figures could becorrelated with the c.p.m. found on the discs.

Fig. 1-Biopsy of first skin allograft taken2 months after application.Arrow indicates the junction of graft (to

left) and patient’s skin. (Trypan-blue andeosin. Reduced by a third from x 80.)

Fig. 2-Biopsy of second skin allografttaken during rejection reaction a monthafter implanting fetal thymus tissue.Arrow indicates junction of the graft (to

right) and patient’s epidermis. (Trypan-blue and eosin. Reduced by a third fromx 80.)

Fig. 3-Biopsy of third skin allografttaken during the rejection reaction 14months after implanting fetal thymustissue.

Arrow indicates junction of graft (toleft) and patient’s epidermis. (Trypan-blueand eosin. Reduced by a third from X 32.)

ResultsTable i presents most of the absolute lymphocyte-

counts, as well as the results of skin tests to a variety ofantigens carried out over a 14-month period beginningimmediately before the implantation of fetal thymustissue. The patient’s absolute lymphocyte-counts,which had averaged 2660 per c.mm. before the attemptat reconstitution,6 averaged 2400 per c.mm. (range1240-3207) during the first 8 months afterwards and3475 per c.mm. (range 2068-4860) in the 6 monthsafter that. When the patient’s peripheral leucocyteswere examined under the electron microscope,14relatively more small lymphocytes and correspondinglyfewer primitive forms were found than at the earlierexamination.16The results of the skin tests indicate that our patient

showed normal delayed dermal hypersensitivity to

monilia and D.N.F.B. 2 weeks after receiving fetal

thymus and subsequently. The onset of cutaneous

sensitivity to S.K.S.D. could not be detected at all until1 year after implantation but could be demonstratedunequivocally by testing with a more concentratedantigen. In addition, on one occasion our patientdemonstrated delayed skin hypersensitivity to diph-theria toxoid.The concentrations of immunoglobulins in the

patient’s serum were found to be normal wheneverthey were determined by immunoelectrophoresis or byradial immunodiffusion. Schick tests on two occasionsindicated the presence of neutralising antibody to

diphtheria toxin. The patient had isoh2emagglutininsin a titre of 1/8 a year after implantation.At the time the immunological studies which estab-

lished the diagnosis were performed, skin from anunrelated donor was grafted to the patient’s thigh and14 days later both the graft and a regional lymph-nodewere biopsied. These procedures were repeated 2weeks and 14 months after the implantation of fetalthymus. Grossly, the first allograft appeared to havebeen accepted indefinitely. Histologically, rare mono-

1082

Fig. 4-Lymph-node obtained when patient was 10 months oldNote cortical germinal centres. Deeper cortex, however, is

hypocellular. (Periodic acid/Schiff. Reduced by a third fromx 32.)

nuclear cells were visible in the graft biopsy at 14 days,and a few mononuclear cells were present in the speci-mens obtained 2 (fig. 1) and 4 months later. Carefulexamination of the infiltrating cells revealed a paucityof small lymphocytes at all times. However, theabsence of skin appendages in the allograft at the laterbiopsies (fig. 1) may indicate that the graft underwentprolonged and mild rejection.The skin which was grafted 2 weeks after the patient

received fetal thymus tissue was rejected promptly,and, when biopsied, was almost completely necrotic(fig. 2). The skin which was grafted 14 months laterappeared grossly to be undergoing rejection moreslowly. Fig. 3 shows that a dense cellular infiltrate ispresent at the margin of the graft (arrow) and in thedermis at 14 days.

Figs. 4-6 show the appearance of the three lymph-nodes which were biopsied at the same time as the skingrafts. Germinal centres and plasma-cells were present

Fig. 5-Lymph-node obtained a month after implanting fetalthymus tissue.

Deeper cortex appears to be densely populated with smalllymphocytes in contrast to earlier specimen. (Periodic acid/Schiff. Reduced by a third from x 32.)

Fig. 6-Deep cortical germinal centre in lymph-node obtained 14months after implantation of fetal thymus tissue.Mitotic figures (arrows) are numerous. (Trypan-blue and

eosin. Reduced by a third from x 344.)

in all three. In the first specimen, however (fig. 4),the deep cortex-the area designated by Parrot" thymus-dependent "-contains reduced numbers ofsmall lymphocytes, particularly in the area surroundingpostcapillary venules. In the second (fig. 5) and thirdspecimens, cellularity in the deep cortex has increasedand small lymphocytes cuffing postcapillary venulesare prominent. Germinal centres became evident inthe thymus-dependent areas of the specimen obtained14 months after the patient’s implant (fig. 6).Our previous report described the improved P.H.A.

response of the patient’s leucocytes after implantationof the fetal thymus tissue. Furthermore, cytogeneticdata suggested that the responding cells originated inthe recipient and not in the graft. The results ofadditional in-vitro studies carried out with the patient’speripheral blood leucocytes are summarised intable 11. During the past 14 months, the patient’sleucocytes have continued to respond normally to

P.H.A. (table II). In accordance with the results of skintests, the patient’s leucocytes began to respond in vitroto monilia antigen. In fact, the patient’s cells have

usually responded better than the accompanyingcontrols. Chromosomal analysis of the patient’s cellsrevealed that 15/15 mitotic figures were XY, suggestingthat they too originated in the patient and not in thegraft. Additional studies have shown that, when dermalreactivity to S.K.S.D. was manifest for the first time, thepatient’s leucocytes also responded in vitro to that

antigen.Discussion

This report describes the development of immuno-logical function in a boy with congenital aplasia of thethymus and parathyroid glands that followed the

1083

TABLB II-UPTAKE OF "H-THYMIDINE STIMULATED BY P.H.A. IN THELEUCOCYTES OF A PATIENT WITH CONGENITAL APLASIA OF THE

THYMUS, FOLLOWING A FETAL THYMUS GRAFT

*‘ Unstimulated control average 1.9 (n=16), range 0-45.

implantation of thymus fragments from a 16-week-oldfemale fetus. There can be no doubt that the patient’simmunological competence changed during the periodencompassed by the study, and that his newly acquiredimmunological abilities have persisted. In this respect,this patient parallels the one described by Clevelandet al. 6

The possibility exists that the patient may haveacquired full immunological competence spontaneously,and that the dramatic changes in dermal hyper-sensitivity were the result of primary immunisation bythe challenge doses of D.N.F.B. and monilia (but notS.K.S.D.) which were administered immediately beforeimplanting the fetal thymus tissue.16 We observedspontaneous improvement in immunological functionin a patient with DiGeorge syndrome (C. P. in reference5). The child, however, had a milder immune deficiencythan the present case at the outset, and his immunefunctions have returned very gradually in marked con-trast to the rapidity with which the present patientgained immunological competence after implantationof fetal thymus fragments.The survival of the transplanted thymus in the

patient was not ascertained, in order to avoid anyunnecessary surgical procedures. Kay and Soothill 17have evidence from a patient described by Cottier,18and Gitlin has presented evidence,19 that implantedfetal thymus fragments are capable of long-term survi-val when implanted in muscle. Moreover, human fetalthymus tissue remains viable for at least 24 hours ifstored in tissue-culture medium at refrigeratortemperatures 20-treatment which resembles that givento the fetal thymus between its removal and implanta-tion. In addition, it has been shown in experimentalanimals that thymus epithelial reticular cells survive inmillipore diffusion chambers under conditions wherecortical lymphocytes do not.21,22 Moreover, grafts ofsuch surviving cells were able to restore immunologicalfunction to neonatally thymectomised mice, even whengenetic differences existed between donor and recipientstrains.The chromosomal studies reported by Cleveland et

al. and the karyotypic analyses of mitotic figures in thepresent case suggest that the cells which respondednormally in vitro to mitogenic stimuli after implantationwere derived from the patient rather than from the

grafted fetal thymus. A considerable body of experi-mental evidence suggests that these cells may originatein the bone-marrow,23 and finding normal numbers oflymphocytes in the marrows of both of our patients 6and DiGeorge’s 2 is consistent with this possibility.Our studies do not indicate whether the grafted fetalthymus served as a source of so-called " thymushormone" , 23 or whether its reticular epithelialelements directly influenced the patient’s lymphoidcells. In either case, the results of the in-vitro studiesof the patient’s leucocytes, his prompt rejection of skinallografts, and the appearance of his lymph-nodes onsequential biopsies suggest that a population of thymus-dependent lymphocytes endowed with the capacity tomediate cellular immunity expanded rapidly.Both this patient and the child described by Cleve-

land et al. appear to have shown no signs of graft-versus-host reactions. 6, 8 Moreover, both patients’newly acquired immunological competence has per-sisted for at least 16 and 18 months, respectively.These facts indicate that, until purified preparations of" thymus hormone " or mediators of cellular immunitybecome available, implanting fetal thymus tissue intopatients with congenital absence of the thymus offersthe best hope for ameliorating their profound immuno-logical deficiency.We acknowledge the generous cooperation and assistance of

Dr. Park Gerald and Mrs. Kathleen Dale, of the Division ofClinical Genetics, in carrying out the initial in-vitro leucocytestudies, and the assistance of Mr. Robert McEnany, of theDepartment of Visual Education, Children’s Hospital MedicalCenter, in preparing photographs. This work was supported bygrants AI-05877, AI-09230, and FR-00128 of the U.S. PublicHealth Service. The fetal tissue bank of the Royal MarsdenHospital is supported by a grant from the Medical ResearchCouncil. C. S. A. was a trainee in paediatric hasmatology undergrant TO-1-AM-05581.

Requests for reprints should be addressed to F. S. R.

REFERENCES

1. DiGeorge, A. M. J. Pediat. 1965, 67, 907.2. DiGeorge, A. M., Lischner, H. W., Dacou, C., Arey, J. B. Lancet,

1967, ii, 1387.3. DiGeorge, A. M. in Immunological Deficiency Diseases in Man

(edited by D. Bergsma); p. 116. New York, 1968.4. Lischner, H. W., Dacou, C., DiGeorge, A. M. Transplantation,

1967, 5, 555.5. Kretschmer, R., Say, B., Brown, D., Rosen, F. S. New Engl. J. Med.

1968, 274, 1295.6. Cleveland, W. W., Fogel, B. J., Brown, W. T., Kay, H. E. M.

Lancet, 1968, ii, 1211.7. Kay, H. E. M. in Immunologic Deficiency Diseases in Man (edited

by D. Bergsma); p. 168. New York, 1968.8. August, C., Rosen, F. S., Filler, R. W., Janeway, C. A., Man-

howelin, B., Kay, H. E. M. Lancet, 1968, ii, 1210.9. Dempster, W. J. ibid. 1969, i, 468.

10. Kretschmer, R., Janeway, C. A., Rosen, F. S. Pediat. Res. 1968,2, 7.

11. Moorehead, P. S., Nowell, P. C., Mellman, W. S., Battipe, D. M.,Hungerford, D. Exp. Cell. Res. 1960, 20, 613.

12. Shannon, D. C., Johnson, G., Rosen, F. S., Austen, K. F. New

Engl. J. Med. 1966, 275, 690.13. Mans, R. J., Novelli, G. D. Arch. Biochem. Biophys. 1961, 94, 48.14. Kretschmer, K., Kretschmer, R., Rosen, F. S. Unpublished.15. Kretschmer, R., Kretschmer, K., Rosen, F. (abst.) Pediat. Res.

1969, 3, 370.16. Lischner, H. W., Huff, D. S., Dacou, C., DiGeorge, A. M. Pro-

gramme and abstracts, 79th annual meeting of the AmericanPediatric Society, Atlantic City, April 30 to May 3, 1969, p. 69.

17. Kay, H. E. M., Soothill, J. F. Lancet, 1969, i, 571.18. Cottier, H., Burki, K., Hess, M. W., Hassig, A. Birth Defects Orig.

Art. Ser. 1968, 4, 152.19. Gitlin, D., Rosen, F. S., Janeway, C. A. Pediatrics, Springfield, 1964,

33, 711.20. August, C. S., Rosen, F. S. Unpublished.21. Hays, E. F. Blood, 1967, 29, 29.22. Levey, R. H., Trainin, N., Law, L. E. J. natn. Cancer Inst. 1963,

31, 199.23. Miller, J. F. A. P., Osoba, D. Physiol. Rev. 1967, 47, 437.