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Clinical ImmunologyVol. 98, No. 1, January, pp. 54–61, 2001doi:10.1006/clim.2000.4948, available online at http://www.idealibrary.com on

Correction of DiGeorge Anomaly with EBV-Induced Lymphomaby Transplantation of Organ-Cultured Thymus and

Epstein–Barr-Specific Cytotoxic T Lymphocytes

Richard Hong,* Violet Shen,† Cliona Rooney,‡ Dennis P. M. Hughes,§Colton Smith,¶ Patricia Comoli,\ and Linqi Zhang**

*Department of Pediatrics, University of Vermont Medical School, Burlington, Vermont 05401; †Section of Hematology/Oncology,Children’s Hospital of Orange County, Orange, California 92868; ‡Center for Cell and Gene Therapy, Baylor College of Medicine,

Texas Children’s and Methodist Hospitals, Houston, Texas 77030; §University of Michigan Hospitals, Ann Arbor, Michigan 48109;¶University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; \IRCCS Policlinico San Matteo,

D. le Camillo Golgi 2, 27100 Pavia, Italy; and **Aaron Diamond AIDS Research Center, New York, New York 10016

lrm

A young woman with DiGeorge anomaly showednormal immune tests as a child and did not experiencethe symptoms of profound T cell immunodeficiency.However, she had chronic pulmonary infections whichled to bronchiectasis. At age 14, she developed an Ep-stein–Barr virus-induced lymphoma and her T cellfunction tests were markedly abnormal. After inten-sive chemotherapy, she received an organ-culturedthymus transplant but because of an abnormally highEBV DNA titer was also given autologous EBV-specificcytotoxic T cells, prepared prior to transplant. Titersfell from 80,000 genome copies/mg DNA to 2000 within6 weeks. She was clinically well and her T cell testsimproved. Sixteen months after the transplant, how-ever, her tumor returned; EBV DNA levels had risen to40,000 copies/mg DNA. She again received autologousEBV-specific cytotoxic T lymphocytes and valcyclovirand Cytogam as well. Her tumor resolved on this ther-apy and she has remained well to this date, 29 monthsafter the recurrence. T cell tests, which had deterio-rated with the recurrence of the tumor, now shownormal responses. This experience records a numberof unique features of thymus transplantation. This isthe first recorded successful thymus transplant in apatient with partial T cell immunity and thus expandsthe potential of this treatment modality to patientsother than infants with complete DiGeorge anomaly.The patient demonstrates cytotoxic activity againstmouse cells, demonstrating the ability to respond to anew antigen which requires host antigen presentingcells. Measurement of a 1 TRECs (T cell receptor exci-ion circles) shows evidence of increasing and sus-ained thymopoiesis since the transplant at a levelonsistent with the age of the transplant donor ratherhan that of the recipient. © 2000 Academic Press

Key Words: DiGeorge anomaly; thymus transplant;a 1 TREC; EBV lymphoma; anti-EBV CTLs.

m

1521-6616/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

54

INTRODUCTION

T cell deficiency in DiGeorge anomaly (DGA) is vari-able in its incidence and severity (1). Most patientswith this disorder have normal T cell immunity andrequire neither correction nor constant monitoring oftheir immune systems. A few, approximately 5–10%,need restoration of their T cells by thymus transplanta-tion (2, 3), bone marrow transplantation (4, 5), or infusionof peripheral blood (6). In these cases, T cell immunity isnil and transplant rejection is not a problem.

We encountered a young woman with DiGeorgeanomaly who manifested normal immunologic testingin childhood and did not experience a clinical coursesuggestive of profound T cell deficiency in her earlyyears. However, at age 14, she developed B cell lym-phoma induced by Epstein–Barr virus, a complicationindicative of impaired T cell immunity. After chemo-therapy, she was transplanted with organ-cultured al-logeneic human thymus and with autologous EBV-specific cytotoxic T lymphocytes (CTLs). This reportdetails the results of the transplant and subsequentclinical course.

METHODS

Clinical procedures were performed under informedconsent on protocols filed with the relevant institu-tions.

The thymus for transplant was obtained from a6-month-old female who underwent corrective cardiacsurgery for tetralogy of Fallot. The thymus was pre-pared for transplant by organ culture (7). In this tech-nique, thin (;1 mm thick) slices are maintained at theiquid–air interface of a nutrient medium. The organetains its original three-dimensional structure andost of the lymphocytes leave the slice, leaving pri-

arily a thymus stromal matrix. After implantation,

CCD

CCCCC

%E%E%E%E

55EBV LYMPHOMA AND DiGEORGE ANOMALY

the transplant becomes filled with host stem cellswhich mature and populate the peripheral T cell com-partment (3). The thymus HLA types were -A 2,3/-B7,15 and the patient was HLA -A 2,32/-B 60,62.

Cytotoxic T cell lines from the patient to Epstein–Barr virus (EBV) were generated prior to transplant bycoculture of peripheral blood mononuclear cells with anEBV-transformed autologous B cell line (lymphoblas-toid cell line, LCL). After 9 days and weekly thereafter,the responder cells were restimulated with irradiatedautologous LCLs. The CTLs were expanded by twiceweekly stimulation with 40 units/ml of interleukin 2from day 14 of culture. We prepared a cytotoxic T cellline which could be given should a reactivation of herEBV-associated lymphoma occur in association withthe transplant (8). Although the usual response in pa-tients with intact T cell immunity consists primarily ofT cells bearing the ab T cell receptor (TCR), the pa-tient’s response consisted of mostly T cells bearing gd Tcell receptors (82.5%, Table 1). These cells were har-vested and cryopreserved.

EBV DNA levels in the peripheral blood were mea-sured as previously described (9).

T cell receptor b-chain usage in her blood mononu-clear cells was measured by a quantitative polymerasechain reaction (10) and the degree of heterogeneity ofthe T cell repertoire was assessed by amplification of Tcells of the BV 22JB1s6 subfamily. The PCR productwas cloned using a TOPO TA cloning kit (Invitrogen,Carlsbad, CA 92008), and colonies were selected ran-domly and sequenced.

Cytotoxicity against murine cells was assessed es-sentially as described by Lucas et al. (11), except thatstimulation was accomplished with a murine cell line(P815 from American Type Culture Collection, Rock-ville, MD) and autologous plasma was substituted forhuman AB serum. The stimulating cell line served astarget in the assay. EL4 mouse leukemia cell lineswere used as irrelevant targets.

To assess the extent of thymopoiesis, we measuredthe amount of a major T cell receptor excision circle,termed a 1 TREC, using a method recently described(12). Briefly, genomic DNA was extracted from freshlydrawn peripheral blood or cryopreserved peripheralblood mononuclear cells of the patient. The copy num-bers of a 1 TRECs per millions of PBMC was thenmeasured by a real-time PCR/molecular beacon assay.To normalize for cell equivalents of the input DNA, weemployed a separate real-time PCR/molecular beaconassay to quantify the CCR5 coding sequences, knownto be present at two copies per cell.

CLINICAL HISTORY

The patient was thought to be normal until the age of

13 months (3/81), when she manifested tetany due to

hypocalcemia. Parathyroid hormone levels were unde-tectable. She was successfully maintained on Hytak-erol. Thereafter, she experienced recurrent bouts ofotitis media and sinopulmonary infections, and at age2, had pneumococcal sepsis. DiGeorge anomaly wassuspected, and a search for chromosome 22 deletion ledto equivocal results. Immune system evaluation, in-cluding serum immunoglobulin levels, and titers ofspecific antibodies to tetanus toxoid, pertussis, andpolio antigens were normal at this time.

Although the recurrent pulmonary infections led tobronchiectasis, her T cell immunity, at age 4 years(1984), was sufficiently intact for her to recover fromvaricella infection without any complications. At age 9(1989), an angiomatoid malignant fibrous histiocytomawas removed from her back. At age 12 (1992), an en-larged preauricular lymph node was biopsied and thepossibility of a clonal B cell proliferation was raised.However, the lymphadenopathy resolved without ther-apy, so no treatment or further investigation was in-stituted. Testing of her T cells at this time showed alow percentage (26% CD41 cells—absolute number un-

TABLE 1Anti-EBV T Cell Lines Developed by Patient

at Various Times

Marker

Date (%)

2/96a 11/96b 9/98c

CD3 93.32 94 93.87CD31CD41 0.11 1 3.21

D31CD81 17.42 68 67.79D31DR1 92.76R1 99.03

ab 8.98 84.9 57.14gd 82.49 9.8 26.49

D16 5.23D161CD561 4.43 1.2 10.5D561CD162 15.87 22 5.91D81CD561 6.22 3.5D19 0 ,1 ,1

Lysis (auto LCL)d 89 62.6 68.4:T 5 20:1Lysis (auto LCL)d 77 73 72.1

:T 5 5:1Lysis (mismatch LCL)e 80 42.2 22

:T 5 20:1Lysis (mismatch LCL)e 77 25.4 11

:T 5 5:1

a Three months before thymus transplant.b Six months after thymus transplant.c Twenty-eight months after thymus transplant.d Percentage of lysis of an autologous EBV line used to generate

CTLs. E:T, effector to target ratio; lysis demonstrated by chromiumrelease.

e Percentage of lysis of the EBV line of another individual, not HLAmatched.

determined) but a vigorous response to stimulation in

be

56 HONG ET AL.

vitro with candida, tetanus, and herpes antigens (Ta-le 2). At age 14 (1994), she had generalized lymphad-nopathy and sputum culture grew Mycobacterium

gordonae. She received isoniazid, rifampin, ethambu-tol, and biaxin. Nevertheless, her adenopathy per-sisted and a biopsy of an axillary node revealed Blineage malignant lymphoma of the large cell type. Thetumor showed k-chain monotypy and EBV was de-tected by in situ hybridization (EBV/LMP1). She wastreated with a modified French LMB 89 regimen, con-sisting of one cycle of cyclophosphamide, vincristine,and prednisone; two cycles of cyclophosphamide, vin-cristine, prednisone, doxorubicin, high-dose metho-trexate, and intrathecal methotrexate and hydrocorti-sone; two cycles of cytarabine, high-dose methotrexate,and intrathecal methotrexate and hydrocortisone.Eight months after completion of chemotherapy, shereceived an organ-cultured thymus transplant (5/1/96).The tissue was placed in the omentum and a few pieceswere also placed in the right forearm for subsequentbiopsy. She received antithymocyte globulin (ATGAM,Pharmacia-Upjohn, Kalamazoo, MI), 30 mg/kg, on theevening of surgery and the following day to aid inengraftment. The procedure was tolerated without in-cident.

EBV DNA levels were monitored in the blood aspreviously described (9). On the day of the transplant,prior to administration of antithymocyte globulin, herlevels were 80,000 genome copies of EBV/mg DNA (Fig.1). This level is 200 times higher than the upper limitof normal and is indicative of incipient overt EBV lym-phoma (9). Her EBV DNA remained at this level for 2weeks, at which time she received EBV-specific CTLs(1.5 3 107 cells/M2) generated as described previously(Table 1). She received another infusion of the samenumber of cells 6 days later. One week after the firstinfusion, the EBV DNA level had fallen to 4000 cop-ies/mg DNA and to 2000 copies by week 6. She wasclinically well throughout this time and had no adversereactions to the infusions.

Four months after the transplant, a biopsy of thethymus transplanted into the muscle was performed.Normal thymus histology, including a well demarcatedcorticomedullary junction and Hassall corpuscles, wasfound. Staining of the section with monoclonal anti-body to CD1a confirmed that the lymphocytes withinthe tissue were developing thymocytes and not an in-filtrate of inflammatory or rejecting cells (Fig. 2).

She remained clinically well, and follow up assess-ment by axial computed tomograms showed continuingdecrease in lymphadenopathy during the first yearposttransplant (Fig. 4). However, 16 months after thetransplant (9/30/97), her tumor returned, manifestedby fever, pleural effusions, and mediastinal lymphad-enopathy. Her EBV DNA levels had risen to 40,000

genome copies/mg DNA (Fig. 1). She was given two

more infusions of anti-EBV CTLs and started on vala-cyclovir and Cytogam. She improved and became afe-brile, her pleural effusions resolved, and her adenopa-thy decreased. Her EBV DNA levels fell progressivelyand 3 months after the T cell infusions, the level was200 genome copies/mg DNA. Now, 48 months aftertransplant (29 months after recurrence), she remainsasymptomatic. Valacyclovir and Cytogam have beendiscontinued. Tests of T cell function taken at the timeof the recurrence showed loss of antigen reactivity, butthis function has been regained (Table 2). Also, whennew autologous CTLs were generated 6 and 28 monthsafter the transplant, she responded with a vigorousresponse in vitro, but the cells were now predominantlyab, instead of the original majority gd response (Table1). These cells also showed greater specificity for au-tologous EBV cell lines compared to the first CTL linedeveloped prior to thymus transplantation. Magneticresonance imaging of the transplant site in her forearmshows a 2 3 2 cm hypodense area, representing theimplanted thymus.

CT scans taken at various intervals over the courseof her illness are shown in Fig. 4. Pulmonary nodulesand lymphadenopathy are now absent.

Analysis of her T cell receptor b-chain usage in amass culture of her peripheral blood mononuclear cellsstimulated by phytohemagglutinin for 48 h revealed allBV families to be present (Fig. 3). To demonstrate theheterogeneity of her T cell repertoire in a more detailedfashion, the PCR product obtained using primers forthe T cell receptor family BV22BJ1s6 was cloned and17 individual colonies were randomly chosen and se-quenced. The BV22 T cell receptor family was chosenas it represents only a small percentage of the T cellsand heterogeneity of a very minor population wouldimply a high degree of overall BV diversity. Each of thesequences was different (Table 3).

HLA typing of her peripheral blood mononuclearcells, 6 and 24 months after the transplant, revealedonly a single population of cells, all of the host type.

Stimulation of her peripheral blood mononuclearcells resulted in a vigorous cytotoxic response againstmouse P815 lymphoma cells (Table 4).

Quantitation of a 1 TREC (Table 5) showed that atthe time of her tumor recurrence, thymopoiesis wasminimal, but since then has shown progressive in-crease to levels more consistent with the age of thethymus donor (6 months) than that of the patient (20years) (reference 12).

DISCUSSION

In this young woman with a modest amount of T cellimmunity at the time of transplant, we have achieveda successful engraftment of organ-cultured thymus.

The indication for the transplant was the loss of T cell

mot

m

57EBV LYMPHOMA AND DiGEORGE ANOMALY

function to a degree that permitted the development ofan EBV-induced B cell lymphoma. In most patientswith DiGeorge anomaly (approximately 90–95%), Tcell function is adequate for normal health. Previousstudies have suggested that levels of CD41 T cellsgreater than 400/mm3 are all that are necessary forfreedom from infection susceptibility (1). However, re-ports of autoimmune disorders in older patients withDGA who have not had problems with infections indi-cates that those with numbers of CD41 cells in anambiguous range (e.g., from 400 to 540/mm3) could

FIG. 1. EBV DNA levels at various times in response to treat-ents. The black arrow indicates the time of thymus transplant. The

pen arrows show the times of the T cell infusions (details in the

TABLymphoc

Date CD4a CD8a PHAb Con Ab

11/11/93 ?(26%) ?(41%) 3 NDc

11/14/95 204 844 27 104/18/96d 232 974 35 127/19/96e 400 920 ND ND11/22/96 420 560 217 166/2/97 ND ND 18 29/30/97e,f 107 157 25 81/12/98 ND ND 75 39/1/98 392 312 105 36/15/99 379 364 239 ND3/20/00 500 300 138 NDNormal: .800 .400 .100 .15

a CD4 and CD8 numbers are absolute numbers of cells/mm3 of bloononuclear cells.b PHA, phytohemagglutinin; Con A, concanavalin A; PWM, pokew

cytomegalovirus antigen; HSV, herpes simplex antigen. All prolifeincorporated tritium counts by stimulated cells divided by counts ishown in the bottom row.

c ND, not done.d Transplant performed on 5/1/96.e Anti-EBV CTLs given in June 1996 and September 1997.f Recurrence of lymphoma.

ext).

actually have some thymus compromise. They mightsuffer from complications of deficient T cell functionother than infection, such as autoimmunity (13, Hongunpublished). Although this patient had normal T cellfunction tests as a child, the recurrent sinopulmonaryinfections resulting in bronchiectasis and the culture ofatypical mycobacterium species from her sputum indi-cate that even though her clinical course was not typ-ical of profound T cell deficiency (difficulty with mo-nilia, cytomegalovirus, pneumocystis, or varicella) herresistance to infectious agents was not entirely normal.It may be prudent to monitor older DGA patients moreclosely.

EBV DNA levels were extremely high at the time ofsurgery and remained as elevated for the next 2 weeks.Previous studies of immunocompromised patients withsuch elevated levels of viral DNA have invariably dem-onstrated rapid progression to clinically overt lym-phoma (9). We have shown, in bone marrow transplantpatients, that progression can be prevented, and viralDNA levels reduced to normal, by infusion of EBV-specific CTLs (8, 15). This patient showed an equiva-lent response. Of interest, however, is that the infusedcells consisted primarily (83%) of gd T cells, ratherthan the classic ab T cell receptor cells present in linesinfused into earlier patients (14). The gd T cells form aminor (2–5%) component of the circulating T cells andtheir exact function in body defenses is unclear. How-ever, certain B cell lymphomas will stimulate their

2Studies

PWMb Candb Tetb CMVb HSVb

ND 42 23 ND ND14 ,1 ,1 ,1 ,119 141 3 1 1

ND 29 3 15 624 24 1 ND ND2 ,1 ,1 ND ND1 1 1 ND ND1 ,1 2 ,1 4.8

29 ND ND ND NDND 4.7 ND ND NDND 12.3 15 ND ND.20 .3 .3 .3 .3

except row 1, which shows CD41 and CD81 cells as a percentage of

mitogen; cand, candida antigen; tet, tetanus toxoid antigen; CMV,ion responses are reported as stimulation indices, the number ofrporated by nonstimulated cells. Normal values and responses are

LEyte

od,

eedratnco

growth in vitro and infusions of such lines into severe

id

58 HONG ET AL.

combined immunodeficiency mice will prevent thespread of human Burkitt lymphoma cells (16). Thepreferential in vitro gd T cell receptor response to EBVn this patient was probably a manifestation of herefective ab T cell reactivity and another indication of

FIG. 2. Biopsy of the thymus transplant 4 months after surgerydemarcation and Hassall corpuscle. (B) Stained with antibody to CD1both views.

FIG. 3. BV chain repertoire of patient demonstrated by quanti-tative PCR analysis; results from a normal individual are shown for

comparison.

her altered T cell immunity at the time of her acquisi-tion of the tumor. More recently, as T cell immunityhas returned more toward normal, her in vitro T cellresponse to EBV cell lines has been predominantly ofab CTLs (Table 1).

The cytotoxicity characteristics of EBV-inducedCTLs changed after the transplant. The original lineshowed equal cytotoxic reactivity against a heterolo-gous LCL and the patient’s own LCL. The nonspecific-ity may be a manifestation of lymphokine-activatedkiller (LAK) cell activity as the CTL line showed 22%CD561 cells. Alternatively, the control of her tumormay have been brought about by the ab CTLs, which,although present in only a small percentage, areknown to expand up to 3 logs in vivo (15). Thus, theexact mechanism by which the expanding EBV cellswere controlled by the first CTL infusions is unclear.

We elected to give two doses of anti-thymocyte glob-ulin to aid in engraftment, hoping that given periop-eratively, it might provide sufficient obtundation of therejection response but not compromise her immunesystem to a point where return of tumor or unaccept-able susceptibility to infection would result. It washoped that when the thymus graft was established, itwould tolerize the patient for the donor histocompati-bility antigens by negative selection of developing thy-mocytes (17, 18), an advantage not available to otherorgan grafts. In this way, chronic immunosuppressioncould be avoided. The appearance of the biopsy 4

) Normal histology is apparent with well-defined corticomedullarya marker of developing thymocytes. Original magnification, 3100 in

. (Aa,

months after the transplant revealed no evidence of

y.

59EBV LYMPHOMA AND DiGEORGE ANOMALY

rejection and was histologically normal, showing briskthymopoiesis (Fig. 2). There is evidence, provided bymagnetic resonance imaging, of persistence of the graftto this date (19). Furthermore, ongoing thymopoiesis isshown by the a 1 TREC analyses (Table 5).

Measurement of a 1 TREC has been shown to be asensitive and accurate assessment of thymopoietic ca-pability in a number of clinical situations, post thymustransplantation (20), during the course of HIV infec-tion (12, 21), and following bone marrow transplant forimmunodeficiency (22) and stem cell transplant formyeloma (23). In bone marrow and stem cell trans-plants, the thymus output is the result of heterologous

FIG. 4. (A) CT scan through the midthorax at the time of diagnbilateral pulmonary nodular infiltrate. (B) Six months following cBibasilar nodular lesions recurring 4 months after the thymus transp(D) 1.5 years after repeat cell infusions and start of antiviral therap

or autologous stem cells differentiating from the pa-

tient thymus. In our case, we measure thymopoiesis inan organ-cultured non-HLA-matched thymus trans-plant ultimately populated by autologous stem cells.Infants undergoing cardiac surgery provide the thy-muses for transplant; hence, the age of the thymusdonor can be quite disparate from that of the recipientwhen adults are transplanted. In stem cell-trans-planted patients over age 50, there was a clear differ-ence in thymic output when compared to patients un-der age 50. The numbers of a 1 TREC presentlyrecorded in our patient (age 20) are more in line withthose of small infants the age of the thymus donor (6months) (12). Based on this single observation, one can

s showing extensive mediastinal and hilar adenopathy with diffuseotherapy; significant decrease in adenopathy and infiltrates. (C)

t and associated with return of elevation in EBV DNA copy number.

osihemlan

infer that the thymus operates according to its age-

(a

P

N

60 HONG ET AL.

related capacity, irrespective of the age of the environ-ment. More observations will be needed to validate thisconclusion. An alternative explanation, that the pe-ripheral blood total T cell level is still low and the highoutput is an attempt to raise the T cell number, is notexcluded.

Testing the ability of human T cells to acquire cyto-toxicity to mouse cells was developed by Lucas et al.11) as a unique test of T cell immunity, of value inssessing CD41 T helper cells and their genetic restric-

tion. Since it has been shown that the genetic restric-tion of T cells is determined by thymic elements andnot influenced by the bone marrow cells which fill thetransplant posttransplant, the use of non-HLA-matched thymuses should be clinically ineffective sincethe antigen presenting cells of the host would not havethe same HLA as the thymus donor (24, 25). However,in actual practice, adequate protective T cell responseswhich maintain the clinical well being of the host canbe obtained without histocompatibility matching of thetransplanted thymus tissue and the host (20, 26). This

TABLE 3BV22JB1s6 T Cell Receptor Amino Acid Sequences

V D J

CASS ETGTNWG SPLHFGNFCAS RARD SPLHFGNFCASS GARVD SPLHFGNFCAS NP NSPLHFGNFCAS TDRAK NSPLHFGNFCASS RQGV NSPLHFGNFCAS GENRGR SPLHFGNFCAS RRQ NSPLHFGNFCAS RFFK SPLHFGNFCAS RTGQG NSPLHFGNFCASS PPGQG NSPLHFGNFCASS DGPQH SPLHFGNFCASS GTGVT SPLHFGNFCASS EGN SPLHFGNFCASS LAARTES NSPLHFGNFCASS VTTQGRWD SPLHFGNFCAS GRR NSPLHFGNF

TABLE 4Cytotoxicity against Mouse Cells

Target

Effector:target ratio(percent killing)a

50:1 25:1 12.5:1

atient P815 21.1 13.7 11.9EL4 4.3 4.3 2.6

ormal P815 32.6 11.3 1.1EL4 4.5 3.1 1.0

a T cells sensitized only to P815.

has been strikingly demonstrated even when the his-tocompatibility differs across species. Thymectomizedmice transplanted with pig thymus demonstratemouse T cells restricted to murine H-2, respond withantibody to T-dependent antigens, and clear Pneumo-cystis carinii infection (27). The anti-mouse CTL re-sults shown here represent laboratory confirmationthat the T cells of this patient show appropriate geneticrestriction and can mount an effective T cell immuneresponse, in keeping with the clinical well being ofDGA patients reconstituted with organ-cultured thy-mus. Also, the development of cytotoxic T cells againstmouse cells shows that this patient can respond to anew antigen.

Her improved T cell immunity is documented by herincreasing T cell numbers, improved proliferative re-sponses, and continued clinical well being. The breadthof her T cell repertoire is indicated by the representa-tion of all variable b-chain families and the diversity ofthe T cell repertoire when examined at a very discretelevel (the BV6JB1s6 subfamily). Although early aftertransplant her T cell immunity was not sufficient toprevent a return of the tumor, the ultimate response ofthe tumor without further chemotherapy and her abil-ity to remain in remission for over 2 years suggest thatnow her immune system is making significant contri-bution to tumor control. Anti-viral therapy is effectiveonly in stopping the replicative cycle of EBV, but can-not stop the lymphoproliferative process which is in-duced by latent virus.

Transplantation of organ-cultured thymus in pa-tients with essentially no T cell immunity has beenaccomplished in infants and small children with con-genital primary immunodeficiency disorders (2, 3, 20,26). In those cases, complete engraftment is seen andthe ultimate benefit depends upon the underlying dis-ease and the quality of the tissue transplanted. Thy-mus transplants in adult patients, in AIDS, and inbone marrow transplant patients have yielded essen-tially negative results (28–30). In AIDS, immunosup-pression was not employed. In bone marrow transplan-tation, specific immunosuppression for the transplantwas not given, and the residual immunodeficiency fol-lowing transplant conditioning and that associated

TABLE 5a 1 TREC Values

Date a 1 TRECs/million cells

9/23/97 1566/29/99 89,4933/20/00 227,769Normala (age 20) 30,000–50,000

a Values from reference 12.

with graft-versus-host disease prophylaxis was utilized

wGC

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

2

2

3

R

61EBV LYMPHOMA AND DiGEORGE ANOMALY

to aid engraftment. In both of these situations, tissuehas not been found at the graft site. The gratifyingresults seen in this case suggest that renewed effortsmay be indicated for thymus transplantation in prop-erly selected adult patients if suitable immunosuppres-sion can be devised.

ACKNOWLEDGMENTS

The thymus transplant was performed by Dr. Jeffrey Reese, De-partment of Surgery, University of Vermont Medical School. Thecontinuing generosity of Greg and Tulley Raetz is gratefully ac-knowledged. We thank Dr. Malcolm Brenner for helpful discussionsand Ms. Eva Skulsky for performance of a 1 TREC quantitation. This

ork was supported in part by NIH Cancer Center Support (CORE)rant PO CA2175, AI 46964, the American Lebanese Syrian Associatedharities, and the University of Vermont Immunomodulation Fund.

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eceived July 19, 2000; accepted with revision September 6, 2000; published online November 28, 2000