Proc. NatL Acad. Sci. USAVol. 79, pp. 6047-6051, October 1982Medical Sciences

Reconstitution after transplantation with T-lymphocyte-depletedHLA haplotype-mismatched bone marrow for severecombined immunodeficiency

(monoclonal antibody/chimerism/tolerance/alloreactivity/differentiation)

ELLIS L. REINHERZ*, RAIF GEHAt, JOEL M. RAPPEPORTt, MARJORIE WILSONt, ANN C. PENTA*,REBECCA E. HUSSEY*, KATHLEEN A. FITZGERALD*, JOHN F. DALEY*, HERBERT LEVINE*,FRED S. ROSENt, AND STUART F. SCHLOSSMAN**Department of Medicine, Harvard Medical School Division ofTumor Immunology, Sidney Farber Cancer Institute; *Hematology, Brigham and Women's Hospital;tDepartment of Pediatrics and Division of Immunology, Children's Hospital Medical Center, and Harvard Medical School, Boston, Massachusetts 02115

Communicated by Eugene Braunwald, June 30, 1982

ABSTRACT Severe combined immunodeficiency (SCID) ispotentially correctable by bone marrow transplantation if a pa-tient has a suitable histocompatible donor. In the absence of anHLA-matched donor, lethal graft-versus-host disease (GVHD),which is mediated by alloreactive donor T cells, may occur. In anattempt to prevent GVHD in one SCID patient lacking a matcheddonor, we treated maternal haplomismatched bone marrow witha unique nonmitogenic T-cell-specific monoclonal antibody (anti-T12) and complement to remove mature T cells. Despite the re-moval of >99% mature T cells, the child developed significantlife-threatening GVHD, which was terminated by a 5-day courseof intravenous anti-T12. Subsequently, immune reconstitution oc-curred by 6 wk: the mature circulating T cells proliferated in re-sponse to soluble and allo-antigens in vitro and provided help forB-cell immunoglobulin synthesis. The patient was removed froma protective environment and discharged without evidence of fur-ther infection. Both HLA and chromosomal analyses showed thatthe circulating cells in the patient were of maternal origin. Moreimportantly, the maternal T cells were no longer reactive withrecipient cells. Mixing experiments indicated that the state of tol-erance that resulted in this chimera was not due to active suppres-sion. We conclude that HLA-mismatched transplantation forSCID can be undertaken if mature alloreactive donor T lympho-cytes are depleted before and after bone marrow grafting.

Patients with severe combined immunodeficiency (SCID) havemajor defects in immune function because they generally lackboth T and B lymphocytes (1-3). Clinical manifestations of thisdefective immunity include early onset of life-threatening pul-monary infection, moniliasis, chronic diarrhea, and wasting thatprogressively worsens despite conservative therapies. Althoughthe precise etiology ofSCID has not been determined, previousstudies indicated that the circulating lymphoid cells of SCIDpatients are of two major phenotypes as defined by monoclonalantibody analysis: either TlO+T3-T4-T8- or TlO+T3+T4+T8+(4). The former population is derived from a percursor bonemarrow or early thymocyte compartment whereas the latterrepresents a late stage of thymocyte differentiation (5). Onlythose SCID patients with circulating cells of the more matureTlO+T3+T4+T8+ phenotype demonstrated immunologic func-tion.

Corrective therapy for SCID currently requires bone mar-row transplantation (6). Unfortunately, the invariably lethalgraft-versus-host disease (GVHD) accompanying transplanta-tion with histoincompatible cells has necessitated donor recip-

ient HLA identity as a prerequisite for transplantation. This has,in effect, denied potentially corrective therapy to the majorityof patients (=60%). Moreover, even among the 40% of individ-uals who are transplanted with donor bone marrow cellsmatched at HLA-A, -B, and -DR loci, there is often some degreeofGVHD or post-transplant infection or both.

Recent studies in animal model systems, however, suggesta strategy to circumvent these difficulties (7, 8). In the mouse,for example, removal of immunocompetentT lymphocytes fromdonor marrow before transplantation allows successful haplo-type-mismatched engraftment without GVHD. To explore theutility of this approach in the human, we used a monoclonalantibody to eliminate virtually all mature T cells from a hap-lotype-mismatched donor bone marrow innoculum beforetransplantation of a patient with SCID. The transient GVHDthat followed was terminated by intravenous administration ofthe same antibody with reconstitution of mature T- and B-cellfunction by 6wk after transplantation. The recipient is currentlya stable chimera with circulating donor T lymphocytes that havebecome tolerant to recipient HLA antigens.

MATERIALS AND METHODSProduction of Monoclonal Antibodies and Cell Sorter Anal-

ysis. A series of monoclonal antibodies was used to define cellsurface antigens on human lymphocytes of T- and B-cell lineageisolated by density centrifugation (9). The production and char-acterization of monoclonal antibodies anti-T3, anti-T4, anti-T8,anti-T6, anti-T10, anti-Tll, anti-Ia, and anti-Bl have been de-scribed (10-14), and their specificities are further defined inTable 1.

In addition, we used a T-cell-specific monoclonal antibodytermed anti-T12, developed through a previously reported hy-bridization and screening strategy (5). Although similar to anti-T3 in cellular expression (present on all peripheral T cells andmature medullary thymocytes and absent from cells of non-Tlineage and immature thymocytes), it defines a unique cell sur-face glycoprotein of 120,000 daltons, in contrast to anti-T3,which defines a 19,000-dalton cell surface glycoprotein (15). Theanti-T12 antibody was chosen for its selective binding patternon mature T cells and medullary thymocytes and lack of mito-genicity onTlymphocytes. Anti-T12 was shown to be of the IgMisotype by virtue of its reactivity with a goat anti-mouse IgMantibody and lack of reactivity with a goat anti-mouse IgG an-tibody (Meloy Laboratories, Springfield, VA).

Abbreviations: MHC, major histocompatibility complex; SCID, severecombined immunodeficiency; GVHD, graft-versus-host disease; MLC,mixed lymphocyte culture.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Preparation of Anti-T12 for Intravenous Use. The anti-T12monoclonal antibody was obtained in ascites form from BALB/cJ mice that had been innoculated previously with the T12 hy-bridoma. This preparation contained >50% specific monoclonalantibody but was contaminated by albumin and normal mouse

immunoglobulin. To develop a purified anti-T12 preparation,the monoclonal antibody was separated from the other asciteselements by a standard Sephadex G-200.(Pharmacia) sizing col-umn. With this technique, the immunoglobulin ofthe IgM iso-type was rapidly resolved into a single protein peak. Anti-T12was obtained at =20 mg/ml of ascites. This material was testedfor endotoxin contamination and found to be negative by limulusassay (Microbiological Associates, Walkersville, MD). Routinemicrobiological cultures did not yield bacteria or fungal con-

tamination. The antibody preparation was stored at -700C insterile 2-ml freezing vials. It was thawed, centrifuged at 100,000X g for 20 min to remove immunoglobulin aggregates, and fil-tered through a 0.22-,.Lm filter prior to clinical use. A dose of100 ,g of anti-T12/kg was administered consecutively for 5days.

In Vitro Lysis of Bone Marrow Cells. After obtaining in-formed written .consent by procedures approved by the appro-

priate ethics review committee, we took multiple bone marrowaspirates from the patient's mother under general anesthesia.Subsequently, bone marrow mononuclear cells were obtainedby Ficoll-Hypaque density centrifugation and washed fourtimes to remove residual heparin. The mononuclear cells werethen placed in 15-ml sterile plastic tubes (Falcon) and centri-fuged at 200 x g for 5 min. To the mononuclear pellets (20 x

106 cells per tube) was added 1 ml ofa 1:500 dilution ofpurifiedanti-T12. The cells and antibody were spun gently in a Vortexevery 10 min for 1 hr and then treated with 0.4 ml of rabbitcomplement (Pel-Freez), and the mixture was placed at 37°Cfor 1 hr. After antibody/complement treatment, cells were

washed three times to remove dead cells, and the entire pro-

cedure was repeated two times. After the third antibody/com-plement treatment, cell viability was >99%. Total cell loss didnot exceed 30% during this procedure. Anti-T12/comple-ment-lysed cells were placed in (final vol, 50 ml) RPMI 1640medium (GIBCO) and reinfused over a 2-hr time period.

Functional and Phenotypic Analyses of Lymphoid Cells. Invitro T-cell proliferative responses to mitogens and antigens andB-cell immunoglobulin production after pokeweed mitogenstimulation were determined as described (16, 17). Epics V cellsorter analysis of lymphoid cells with monoclonal antibodies,indirect immunofluorescence and microcytotoxicity assays, andchromosome banding used standard techniques (10, 18, 19).

Patient. The patient was a 4-month-old first female child ofsecond cousin parents from the Cape Verde Islands with SCID(3). Erythrocytes from the child had normal activity for aden-osine deaminase and nucleoside phosphorylase. Because of thelack of a related HLA-matched donor, conventional bone mar-row transplantation could not be performed. Therefore, thepatientwas conditioned for transplantation with busulfan (8 mg/kg), cytoxan (200 mg/kg), and antilymphocytic serum (0.2 mg/kg) over 8 days. This conditioning regimen was used becausetwo prior attempts at reconstitution with T-cell-depleted mar-row without conditioning resulted in no evidence of engraft-ment. Maternal bone marrow was depleted of mature T cellswith a monoclonal antibody, anti-T12, and complement andcells at 160 x 106/kg were infused into the patient 1 day aftercessation of conditioning.

RESULTSBefore transplantation, 10% of the patient's circulating lym-phoid cells were reactive with anti-T10 and anti-Til (12). Thelatter monoclonal antibody defines a sheep erythrocyte recep-tor-associated antigen (15). In contrast, there was no reactivitywith anti-T3, anti-T4, anti-T8, or anti-T12 (Table 1). These find-ings suggest that all circulating lymphoid cells ofT lineage wereimmature. Moreover, no B cells were detectable in peripheralblood with anti-Bi, which defines a B-cell-specific antigen pres-ent throughout B-cell ontogeny (14). Expression of the HLA-D-related Ia antigen on 5% of circulating blood lymphoid cellslikely correlates with a population of monocytes, precursorcells, or both (13).The patient received a bone marrow transplant from her hap-

lotype-mismatched mother. Before transplantation, the bonemarrow specimen contained =10% mature T lymphocyteswhereas, after three cycles of anti-T12 monoclonal antibodyand rabbit complement treatment in vitro, <0.1% mature Tcells were detectable by indirect immunofluorescence witheither anti-T12 or anti-T3. Given that this 10-kg patient re-ceived nucleated bone marrow cells at 160 x 106/kg, <2 x 106mature maternal T cells contaminated the transplant innoculum.

Despite elimination of the majority of T cells from the ma-ternal bone marrow, acute GVHD developed at day 10 aftertransplantation. The clinical signs of rash, hepatitis, diarrhea,and ascites were associated with detection of mature T cellsbetween wk 1 and 2. At that time, 15-25% of lymphocytes werereactive with anti-T3, anti-T12, or both. Because ofthe severityof acute GVHD, the patient received anti-T12 [(0.1 mg/kg)/day] intravenously during the second week. An Epics V cellsorter analysis of circulating lymphoid cells from the patient

Table 1. Development of lymphoid populations after bone marrow transplantation% cells reactive with monoclonal antibody after transplantation.

2 wk (treatment)Antibody Population defined 0 wk 1 wk Before After 4 wk 6 wk 8 wk 24 wkAnti-T3 Mature thymocytes. and T cells 0 15 28 <1 21 49 50' 75Anti-T12 Mature thymocytes and T cells 0 15 27 <1 20 50 52 74

Anti-T4 HelperT cells 0 - 11 <1 10 22 30 41Anti-T8 Suppressor T cells 0 18 <1 14 16 18 29

Anti-T6 Cortical thymocytes (TL like) 0 - 0 - 5 8 0 0Anti-T10 Thymocytes and precursor cells 10 20 35 - 25 45 22 5Anti-T11 All thymocytes and T cells 10 25 32 0 26 42 54 80

Anti-Ia HLA-D-related antigen 5 - 0 - 5 24 28 21Anti-Bl B cells 0 0 - 3 12 14 16

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A B

D

FIG. 1. Selective depletion of matureTlymphocytesafteranti-T12treatment in vivo. Peripheral blood lymphocytes were analyzed on anEpics V cell sorter by indirect immunofluorescence with anti-T3 andfluorescein isothiocyanate-labeled goat anti-mouse IgG/IgM. At theonset of acute GVHD, 28% of circulating lymphocytes were reactivewith anti-T3: (A) The logarithm of the integrated fluorescence(LIGFL) is represented on the x axis, cell size is represented on the yaxis, and cell number is represented on the unmarked z axis; (B) dot-plot analysis of the same sample showing LIGFL and forward anglelight scatter (FALS), a determinant of cell size. (C and D) Correspond-ing plots for circulating lymphocytes 4 hr after in vivo anti-T12 ther-apy; virtually all T3+ T cells have been depleted (<0.1% T3-reactivecells) from the patient's peripheral blood.

before and 4 hr after anti-T12 treatment is shown in Fig. 1.Approximately 28% of T cells were reactive with anti-T3 beforetreatment whereas, after treatment, virtually no T cells (T3+)were detected. Analysis of circulating mononuclear cells withanti-T3 ruled out the possibility that modulation was accountingfor the decrease in fluorescence reactivity after treatment andsuggested that the anti-T12 antibody was efficient in depletingmature peripheral blood T cells. Within 2 days of treatment,the acute GVHD improved and after 5 consecutive days ofther-apy, all signs of GVHD had terminated. Consequently, intra-venous monoclonal antibody therapy was stopped. It should benoted that no steroids, methotrexate, or other cytotoxic drugswere administered during this period.

By the fourth week after transplantation, mature T cells com-prised >20% of peripheral blood lymphocytes, although thesecells were not associated with any GVHD. The expression ofanti-T10- and anti-Tll-reactive cells, as well as reactivity of5%of cells with anti-T6, a marker unique to cortical thymocytes,suggested that some immature cells were circulating (10). Inaddition, an imbalance in T4 helper to T8 suppressor T lym-phocytes existed since the T4/T8 ratio was <1 whereas, in thenormal circumstance, it is usually .2: 1.

Between the fourth and sixth week, the percentage ofmatureT cells doubled (from 21% to 49%) and the T4/T8 ratio returnedto normal. A B-lymphocyte population was detectable for thefirst time (3-12% Bl-reactive cells). Again, the reactivity ofanti-T1O and anti-T6 with 45% and 8% of blood lymphocytes, re-

spectively, suggested the existence ofprecursor cells. Betweenwk 6 and 8, anti-T10 reactivity diminished (45% to 22%) and acirculating T6 population was no longer detectable. A normal

Table 2. Functional evidence for immunologic reconstitutionafter transplantation

Transplantation statusBefore After

T-cell proliferation,* cpmPhytohemagglutinin 237 ± 41 126,151 ± 9,863MLC 67 ± 8 62,995 ± 7,648Tetanus toxoid 359 ± 46 52,954 ± 1,739

B-cell IgG production, ng/ml <100 4,600Results are mean ± SEM.

* Determined as [3H]thymidine incorporation.

distribution of T and B cells has persisted for >8 months. Al-though not shown, full hematologic reconstitution appeared bywk 6 after transplantation.

The concomitant emergence of peripheral blood cells withmature T- and B-cell markers and disappearance of immaturelymphoid cells suggested that the patient may have undergonenormal lymphoid differentiation and acquisition of immunefunction. To test this possibility, T-cell proliferative responsesto mitogens, antigens, and alloantigens were measured. In ad-dition, the capacity of B cells to differentiate into immunoglob-ulin-secreting plasma cells in the presence of pokeweed mito-gen and autologous T lymphocytes was determined. As shownin Table 2, before treatment the patient made no significantproliferative responses to the mitogen phytohemagglutinin, al-loantigen.. or soluble antigen (medium alone, -200 cpm). Herlymphoid cells did not respond to pokeweed mitogen in vitrowith production of Ig. Bywk 6 (Table 2), one could see excellentT-cell responses and B-cell function was evident. Taken to-gether, our results indicate that transplantation of bone marrowafter initial in vitro and then in vivo T-cell depletion can besubsequently associated with development oflymphocyte pop-ulations of mature phenotype and function.To determine whether the circulating blood and bone mar-

row population after transplantation were ofthe maternal donorgenotype, HLA and erythrocyte typing and chromosomal band-ing studies were performed 5 wk after transplantation. As shownin Table 3, the maternal peripheral blood HLA lymphocyte ge-notype was distinct from the patient's HLA genotype since theformer expressed Aw3O, Bw22, and Dr5 and lacked Aw23,Bw44, and Dr4, whereas the converse was true for the latter.After transplantation, the patient's peripheral blood and bonemarrow were of the maternal HLA genotype. Further supportfor this idea was the finding that karyotyping of the patient'slymphocytes after transplantation showed bright satellites onboth chromosomes 22 by banding analysis that were identicalto the mother's pattern (19). Erythrocyte group analysis, like-wise, demonstrated that erythroid cells were of maternal originafter transplantation.

Given the pretransplant in vitro data showing that the pa-tient's lymphocytes were stimulatory for maternal T cells in amixed lymphocyte culture (MLC) as well as the developmentof GVHD, it was important to determine whether or not thematernal T lymphocytes in the recipient after transplantationwere reactive with original recipient cells obtained and cryo-preserved before transplantation. As shown in Table 4 (Exp. 1),maternal lymphocytes taken from the mother proliferated inMLC against irradiated recipient bone marrow obtained beforetransplantation. In contrast, lymphocytes of maternal genotypepresent in the recipient after transplantation (Exp. 2) were nolonger responsive to the same pretransplant bone marrow.These studies indicated that the maternal T lymphocytes cir-culating in the child were no longer reactive with the child'scells.

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Table 3. Parental lymphoid and hematopoietic cell engraftment in a haplomismatchedchild recipient

HLA type ChromosomalCell origin Peripheral blood Bone marrow banding* Erythrocyte type

Maternal Aw3O, 32 ND + B+C+E-S+Fyb+Xga+B14, Bw22Dr3, 5

PatientBefore transplantation Aw23,32 Aw23,32 - B+C-E+S-Fyb-Xge

B14, Bw44 B14, Bw44Dr3, 4

After transplantation Aw3O, 32 Aw3O, 32 + B+C+ES+Fyb+Xga+B14, Bw22 B14, Bw22Dr3,5

ND, not done.* +, Presence of bright satellites on both chromosomes 22; -, absence of bright satellites on both chro-mosomes 22.

To determine whether this inability to respond to recipientmajor histocompatibility complex (MHC) was secondary tosuppression or tolerance, a mixing study was performed inwhich maternal lymphocytes were combined (1:1) with recip-ient lymphocytes of maternal genotype and added to the pre-transplant irradiated cryopreserved bone marrow specimenfrom the recipient in a MLC (Exp. 3). As shown, with this mix-ture, significant proliferation was obtained. This degree ofpro-liferation represented slightly more than the average prolifer-ation of experiments 1 and 2 and strongly argues against activesuppression by maternal lymphocytes in the recipient as themajor mechanism ofproliferation inhibition. Rather, it suggeststhat the circulating maternal lymphocytes had lost alloreactivityor become tolerant to recipient HLA antigens.

DISCUSSIONIn the present study, we performed a bone marrow transplan-tation on a patient with SCID using maternal HLA-haplotypemismatched bone marrow that had been depleted of mature Tlymphocytes. T lymphocytes were eliminated from the bonemarrow before infusion by using anti-T12/rabbit complement.Three cycles of in vitro anti-T12/complement treatment re-duced the percentage ofT cells in the innoculum by a factor of0.001 as determined by indirect immunofluorescence. Furtherreduction of T cells in the donor marrow was not attemptedbecause of the possible adverse effects in putative donor stemcells. Nevertheless, significant clinical GVHD, manifested byskin rash, hepatitis, diarrhea, and ascites, developed 10 daysafter transplantation. The patient was then treated by intrave-

Table 4. After parental-. F1 bone marrow transplantation, thechimera's T lymphocytes of maternal genotype are tolerant torecipient (F1) HLA antigens in MLC

Proliferation to F1 bonemarrow cellst

[3H]Thymidine,Exp. Responder cells* cpm SI1 Maternal lymphocytes (P) 18,046 ± 2,670 29

in mother2 Maternal lymphocytes (P) 1,120 ± 101 1

in recipient (F1)3 Maternal lymphocytes in mother 13,462 ± 1,660 19

and in recipient (1:1 mixture)Results are mean ± SEM. SI, stimulation index.

*HLA: Aw3O, 32; B14, Bw22; Dr3, 5.tHLA: Aw23, 32; B14, Bw44; Dr3, 4.

nous infusion ofanti-T12, which resulted in termination ofsignsofGVHD. By 6 wk after transplantation, the patient was notedto have mature T and B lymphocytes in peripheral blood thatwere shown to respond normally to alloantigens in a MLC, thesoluble antigen tetanus toxoid, and the mitogen phytohemag-glutinin in vitro. In addition, her B lymphocytes were able todifferentiate into plasma cells and secrete IgG in the presenceof autologous T cells and pokeweed mitogen. Thus, it appearsthat normal immune reconstitution occurred in this child withSCID despite the use of haplotype-mismatched bone marrow.Currently, 8 months after discharge from the hospital, she con-tinues to do well in growth, development, and psychomotorareas without any evidence of infection.The hypothesis that T-cell depletion of donor bone marrow

innoculum can prevent GVHD has been supported by work inseveral animal systems. In the mouse, anti-Thy 1/complementtreatment ofbone marrow before transplantation into irradiatedH-2-incompatible hosts results in hematopoietic reconstitutionand the absence of detectable GVHD (7). Similarly, in the pri-mate, agglutination of bone marrow cells with plant lectin canbe used to differentially separate T- from non-T-lineage ele-ments (8). Bone marrow depleted ofT lymphocytes in this fash-ion has been used to reconstitute haplotype-mismatched ani-mals without GVHD.

Although the removal of mature T lymphocytes was nearlycomplete in our patient, significant acute GVHD was observed.Since abrogation of GVHD followed the in vivo removal ofT3+T12' T cells, the alloreactive lymphocytes responsible forGVHD were almost certainly derived from this residual ma-ternal T-cell population, which expanded after transplantation.The presence ofGVHD in our patient suggests that there wasa greater histocompatibility difference in this parental into F1transplant situation than in the murine and primate systems orthat transient GVHD may have existed in the animal systemsbut gone undetected. In addition, the maternal cells used forthe donor innoculum in this clinical circumstance may well haveincluded cells previously sensitized to recipient HLA antigensduring pregnancy. Furthermore, the human marrow was ob-tained by repeated aspirations, which results in a greater like-lihood of contaminating peripheral T cells. The use of bonemarrow curettage from virgin animals of parental strain in theexperimental systems likely transferred fewer sensitized T lym-phocytes to the recipient.The present results suggest that, to abrogate human GVHD,

it may be necessary to remove alloreactive T lymphocytes fromdonor bone marrow innoculum both before and after trans-plantation. Alternatively, it is possible that in vivo removal alone

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may be sufficient to eliminate the alloreactive population.T-cell-specific monoclonal antibodies offer advantages over

conventional antisera or lectin-depletion techniques (20, 21).First, they can be tailored specifically to individual cell lineagesand furthermore restricted to antigens expressed during eithermature or immature stages of differentiation (5). Second, theycan be selected for various functional virtues, including the in-ability to activate the immune response or induce modulationofsurface antigens that they define (20). The latter is a necessaryprerequisite to ensure successful complement-mediated lysis.Third, they can be derived from the appropriate immunoglob-ulin isotype to optimally fix complement and engage the reticu-loendothelial system. In this regard, anti-T12 is a monoclonalantibody of the IgM isotype that fixes complement, detects a120,000-dalton glycoprotein without initiating its modulation,does not induce lymphocyte proliferation or activation, and isrestricted in its reactivity to mature immunocompetent T lym-phocytes. Last, the quantity ofmonoclonal reagents is unlimitedand, unlike heteroantisera, their specificity is constant.

It is clear that, after transplantation, all hematopoietic cellswere of the maternal phenotype. Of note, transiently, there wasthe appearance of immature cells in the peripheral circulation.Nevertheless, after termination ofGVHD, the subsequent ma-ture T cells that appeared in the circulation did not mediateGVHD. These cells responded normally in a MLC, whereas,before transplantation, the child had no response. Despite thisalloreactive capacity, the T lymphocytes ofmaternal HLA geno-type did not react with the child's cryopreserved haplotype-mismatched bone marrow. This state of tolerance within thechimera was not due to demonstrable suppressor cell mecha-nisms. Rather, mixing studies supported the hypothesis thatcells that differentiated in the child had become tolerant to thechild and were not immunosuppressed. These results are con-sistent with murine studies indicating that the development ofrecognition of "self MHC" is dependent not on the genotypeof the T cell itself but rather on the MHC antigens expressedby thymic epithelial cells (22, 23).

Previous studies indicated that patients with SCID had im-mature cells circulating in peripheral blood (4). Whether thiswas due to the absence of normal precursor cells or the failureofa microenvironment to facilitate differentiation was not clear.The development of immunocompetent T lymphocytes afterremoval ofmature maternal T lymphocytes suggests that an in-tracellular defect rather than a microenvironmental defect isresponsible for disease in our case.The present study represents only one experience in hap-

lotype-mismatched transplantation. In this regard, it is evidentthat a series of studies will be needed to determine the generalapplicability of such an approach to transplantation in situationsin which HLA identical donors are lacking. However, the de-velopment of functional donor T lymphocytes that are tolerantto recipient HLA antigens in our patient and a successful HLA-A-, -B-mismatched transplant reported by others (24) suggestthat, after initial mature donor T-cell depletion from bone mar-

row, HLA-mismatched transplantation is biologically rationaland technically feasible. The potential for extending correctivebone marrow transplantation to individuals without previouslysuitable donors appears promising.Note Added in Proof. Since the preparation ofthis manuscript, a secondpatient has been given a transplant in a similar fashion with paternalT lymphocyte-depleted HLA haplotype-mismatched bone marrow forWiskott-Aldrich syndrome. At 3 wk after transplantation, hematologicreconstitution is evident.

This work was supported by National Institutes of Health Grants CA19589, RO1 NS17182, and RR 128.

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