Severe combined immunodeficiency with natural killer-cell predominance: Abrogation of graft-versus-host disease and immunologic reconstitution with HLA-identical bone marrow cells

reconstitution rnerrow cells

with

Lawrence J. Sindel, M.D., Rebewa W. y, l&A. Franc-es E. Ward, Ph.D., George H. IWkkq, Ph.D., Charks &spitz, M.D., and Hiltel Karen, Ph.D. Durham,

N. C., and Sao Paula,

Sksrsse

E.

Brazil

A 3 %-month-old infant with severe combined immunodeficiency was found to have an unusuaf blood lymphocyte phenotype. Thirty percent of her cells formed rosettes with sheep erythrocytes, but only 7.9% reacted with the pan T monoclonal antibody OKT3, and 5% reacted with an antibody (OKT4)-recognizing T-helper cells. Surprisingly 19.4% of her cells reacted with an antibody (OKT8)-recognizing T-suppressor cells and 94% reacted with OKTIO. Few reacted with other monoclonal antibodies detecting cellular activation antigens. Despite absence of T or B cell function, her monocyte-depleted blood lymphocytes caused a high degree of speci$c lysis of “‘Cr-labeled K562 erythromyeloid cells in a natural killer-ceil assay. Most of her Iymphocytes were large and had azurophilic granules and a monocytoid nucleus. Because she had received a nonirradiated, unrelated red-cell transfusion 3 days earlier, 4.8 X IO9 nucleated bone-marrow cells from her HLA-identical brother were given shortly after admission. Two days later a graft-versus-host reaction began but subsided completely within 3 days. On day 15 posttransplantation, a profuse secretory diarrhea began, uccompanied by a rise in her sermn IgE from 4 to 3000 IL’. With engraftment, the number of TlO+ tells and natural killer-cell function fell to normal, and full immunologic reconstitution was eachieved. (J ALLERGY CLIN

IMMJNOL73:829-36, 1984.)

It is now recognized that the syndrome of SCID includes severaldifferent disorders.1-4Some patients have low numbersof both T and B cells.* othershave Prom the Departmentsof Pediatrics, Microbiology and Immunology, Medicine and Surgery, Duke University School of Medicine. Durham, N. C., and the Departmentof Pediatrics, Escola Paulh de Medicina, Sao Paula, Brazil. Suppo@& by Rrants from the National Institute of Allergy and Intk&ious DiseasesAI18613, National Institute of General Medical Sciences GMIO356, National Cancer Institute CA23354, and the General Clinical ResearchCenters RR-30. Received for publication July 19, 1983. Accepted for publication Dec. 29, 1983. Reprint reqteats: Rebecca H. Buckley, M.D., P.O. Box 2898, Duke University Medical Center, Durham, NC 27710. Rebecca H. Buckley is the recipient of an Allergic Diseases Academic Award A170830. Hillel I&en is the recipient of a ResearchCareer Development Award CAOO581.

Abbreviations used

SCID: Severecombinedim~~~j~y ~ ADCC: Antibody-dependent, ceil-medi&?rI cycotoxicity NK: NaturalMing I G-V-H: Graft-versus-host PHA: ~~o~m~~in LGLs: Largegram&xIymphocyfes Con A: Concanavatin A HSV- I : Herpessimplexvirus ape I

I

elevated numbers of B cells,‘-” a&l a fq+ l&ayehad normal numbersof both T and B @lk.’ ;kxrrsetit& the autosomal recessive type lack the * pathway enzyme adenvainedea genetic, molecular, and ceBuiizlt~t~~~~~, i&ants

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with SCID have profound defects in cellular and humoral immunity, and death occurs uniformly before age 2 yr unless such infants are kept in complete sterile isolation, or immunologic reconstitution is achieved through transplantation of immunocompetent tissue.* The cellular defects have involved not only antigen-specific responses but also ADCC” and NK.6 Although Lopez et al.’ have reported detectable NK activity against HS V-l -infected fibroblasts in two infants with SCID, previous efforts by that group,8 others,s and us6 to detect NK activity in patients with SCID against the widely used erythromyeloid cell line, K562, have been unsuccessful. In this article, we describe a new phenotype of SCID in which virtually all of the blood lymphocytes had morphologic and functional characteristics of NK cells. Our patient had received a transfusion of nonirradiated packed red blood cells from an unrelated donor shortly before referral. We were able to abrogate the expected G-V-H reaction that developed by infusion of bone marrow cells from a sibling with identical HLA antigens; moreover, complete immunologic reconstitution was achieved with an eventual decline in blood NK cells to a normal percentage.

PATIENT AN0 METHODS Patient The patient was a 3%month-old Brazilian fe@e infant referredto Duke University Medical Center with a diagnosis of SCID. As a normally grown, healthy, 3-month-old breast-fed infant, she had been observed for routine wellbaby care and had been found to have a borderline low hemoglobin. She was started on oral iron supplementation, after which she developed diarrhea. She was then placed on a lactose-free soy formula with no improvement. Two days later a small, soft palate ulcer was noted. A biopsy specimen demonstrated no fungi or bacteria but a mononuclear cellular infiltrate. Serum concentrations of IgG, IgA, and IgM were reported as nondetectable. A blood count revealed a hemoglobin of 6.9 gm/dl and an absolute lymphocyte count of 1615/mm” with 17.5% “atypical” lymphocytes. A sheep erythrocyte rosetting study found only 20% of her blood lymphocytes to be T cells, and her blood mononuclear cells failed to proliferate in vitro when the cells were stimulated with PHA. Three alternate-daydoses of transfer factor were administered along with two 20 ml/kg infusions of frozen plasma with no clinical improvement. Three days before transfer, lOO-milliliter packed, repeatedly washed, nonirradiated red cells from an unrelated donor were administered in treatment of her anemia. On arrival at Duke, the infant was found to be well hydiated and in no distress but febrile and irritable. Her height and weight were between the tenth and twenty-fifth percentiles. She had a 2 X 2 cm shallow, soft palate ulcer with necrotic edges entending to the tonsillar arches. No tonsiilar tissue was visible. She had an apljarent irritant diaper der-

CLIN. IMMUNOL. JUNE 1984

matitis caused by intermittent loose stools. Initial blood work revealed an absolute lymphocyte count of 107 1 /mm:!.

HLA typing of the patient, her parents, and siblings identified a healthy 6-year-old brother with identical HLA antigens. On the fourth hospital day, 4.8 x lo” nucleated marrow cells from the HLA-identical brother were administered to the patient intravenously (8.7 x 108 cells/kg recipient body weight). Two days after the marrow-cell infusion, the patient developed a morbilliform rash behind the ears and on the face that progressed to involve the entire trunk and proximal extremities, a high spiking fever, and severe diarrhea. Without any specific therapy, these signs and symptoms of G-V-H resolved spontaneously and completely 3 days later. For the next 10 days the patient appeared completely well. On day 15 after marrow-cell infusion, she developed a profuse watery diarrhea necessitating more than 200 ml/kg per day of intravenous fluids to maintain hydration. She had no fever or skin rash with this diarrhea. Diarrhea and formula intolerance persisted, necessitating total parenteral nutrition for 112 days. No immunosuppressive agents or steroids were administered. Refeeding was started with elemental formulas and slowly advanced in strength. Malabsorption and mild to moderate diarrhea prompted frequent formula changes until day 98 after marrow-cell infusion when a soy formula was introduced. That was tolerated well for 3 days, but then diarrhea increased. The soy formula was discontinued and then tried again 2 days later in a test dose of 30 ml at one-fourth strength. Within 30 min the patient vomited and developed profound lethargy and profuse watery diarrhea. Supportive therapy was accompanied by resolution of these symptoms within 4 hr. After several days of gastrointestinal rest, an evaporated cow’s-milk formula was started. This was tolerated well, and feeding progressed until she was able to take a normal infant diet (free of all soy products) by day 139 posttransplantation. Diarrhea ceased, and her stools became entirely normal. She was discharged home to Brazil at 8 mo of age. Subsequently, she has tolerated viral infections and live virus vaccines well and has required only routine well-child care for the past 20 mo.

Methods Serum IgG, IgM, and IgA quantifications were done by single radial diffusion with use of isotype-specific goat and rabbit anti-human immunoglobulin anti-sera, reference standards, and age-appropriate normal values developed in this laboratory. ” Serum IgD and IgE were measured by double antibody radioimmunoassay. I1 Anti-diphtheria and anti-tetanus antibody titers were measured by tanned cell hemagglutination.‘* T and B cell enumerations and lymphocyte-stimulation studies were carried out as described previously I3 Cytofluorographic analyses of lymphocyte sub-

Populationswere done on an Or&hoCytofluorograf System (Ortho Diagnostic Systems, Inc., Westwood, Mass.) 50 H with use of mouse monoclonal antibodies to human cellsurface

antigens’4.‘5 and affinity-purified

fluoresceinated

goat anti-mouse. IgG (Tago Inc., Burlingame, Calif.). NK activity was assayed by use of peripheral blood mononu-

VOLUME NUMBER

!Severe combined immunodeficiency

73 6

831

clear cells (isolated from heparinized venous blood by Ficoll-Hypaque density gradient sedimentation) used as effector cells against “Q-labeled K562-cell line targets.‘. ” In some experiments, effector cells adjusted to 106/ml in RPM1 1640 medium supplemented with 1 0 % fetal calf serum (RPMI-FCS 10) were depleted of monocytes by allowing them to adhere to plastic at 37” for 1 hr before the assay. The plastic nonadherent lymphocytes, containing 1 % to 2 % peroxidase-positive cells, were used as effector cells. Assays were done in triplicate in volumes of 0.2 ml of RPMI-FCS 10 in wells of round-bottom microtiter plates (lS-MRC-96, Linbro Chemical Co., Inc., McLean, Va.). Effector cell to target cell ratios ranged from 2.5 : 1 to 40 : 1 with 1 x lo4 targets per well. The plates were incubated for 120 min at 37’ C in a humified incubator with 5 % CO,.

TABLE I. Comparisons of lymphocyte phenotypes of patient with those of normal LGLs

RESULTS

*Slg = surface i;mmunoglohulin;ERFC = sk@p arytbrcqte rosetting cells; OKT3 = anti$en identified by mono&& antibody reacting with 8 5 % to 9 @ %T cells; 3&l‘ = antigenidentif&i by a monoclonaia&b&y reactingwith to 9 0 % T cells and 4 4 % to 6 8 % NK cells; OKT4 = a&&n recognizedby monoclinal antibodyreactingwith helperT c&a; OKTB = antigen recognizedby monoclonalantibodyreactingwith suppressor T cells andwith 1 9 %to 2 3 % NK cells; OKTtO or VEPlO = antigen recognizedby two diBere& monoel+xxaI S&W&S taut& with activatedlymphocytesor 6 0 % to 6 4 % NK cells; QKII = Ia antigen recognizedby monoclonala~~~~ reactive with B cells, activatedT cells, and 1 8 % to 6 7 % MK celfs; 5EQ = antigen now known to be the transferrin DX+W, which is found on activatedlymphocytesbut not on NK cells. tHighly enriched by gradient isolation. Data from reference17.

Table I presentsthe results of blood lymphocyte surface-markerstudies in the patient before bone marrow transplantation.IgM- and IgD-bearinglymphocyteswere not detectable,and only 3 0 % of the patient’s lymphocytes formed rosettes with sheep erythrocytes.Cytofluorographicstudieswith the pan T reagent,OKT3, revealedan evenlower percentage of positive lymphocytes, and only 5 % of the cells reactedwith a monoclonalantibody(OKT4) to lymphocytesof the helper phenotype.Surprisingly, the percentageof cells reacting with a monoclonal antibody(OKT8)-recognizinglymphocytesof the suppressor/cytotoxic phenotype was normal (19.4%) and was higher than the percentagereacting with OKT3 (7.9%). In further contrast,9 4 % of her blood lymphocyteswere found to react with OKTlO and VEPlO monoclonalantibodies,probablyrecognizing the sameantigenon peripheralblood NK cells” and on very immature thymocytes as well as on many rapidly dividing or activatedcells.‘” However, only 6 % of the patient’s cells reactedwith a monoclonal antibody to Ia antigens,and none reactedwith the monoclonal antibody 5E9, which recognizes the transferrinreceptor.‘”Sinceboth of theseantigensare also associatedwith activatedlymphocytesbut were presenton few or none of the patient’s cells, one would have to assumethat the higher number of T lO-positive cells did not reflect an activatedstateof the patient’scells. As NK cells are alsoknown to bear T lO and VEZPlOand to react with OKTS,” the possibility that most of the patient’slymphocyteswere NK cells was raised.This was borneout in NK assaysof the patient’s monocyte-depleted blood lymphocytes wherean exceedinglyhigh degreeof specific lysis of K562 cells was found, even at an effector-to-target cell ratio of 1.2: 1 (TableII). In addition, most of the patient’s cells resembledLGLs, presumedto represent most

of blood

NK

cells

by the criteria

of

% of Total lymphocytes posith for:* Slg ERFC OKT3 3Al OK-l-4 OK-l-8 OKTIO VEPI 0 OK111 SE9

Patient 0.0 30.0 7 9 35.6 5.0 lY.4 94.0 93.4 6.0 0 0

Nwrnal LGLst 0 h to 0 to ‘$4 to 1 to 19 to

23 17 68 9 23 60 to 64 60 to 64 I8 to 67 0

azurophilicgranules,cell size, monocyroidnucleus, cytoplasmic/nuclearratio and myeIop&roxidase neg ativity . I* TableI alsodemonstmtes the sir&larity of the patient’s lymphocyte phenotypic characteristicsto those of a population of cells highly enriched for LGLs.‘; F ig. 1 displays graphically the changing percentagesof tlhe patient’s lymphocytes~~~uI~~~s after infusion of bone marrow ce& from ‘her HLAidenticalbrother.Over a 4m~th , the $mmxttlid to norage of T lO-positive cells gradual m a l, whereasTS-positive cells the normal range by 2 m o pas percentageof T8-positive lyrn~~~~s slowly increasedto becomeand remain b&her than normal. The percentage of T4-positivecells alsoincreasedbut still hadnot reachedthe lower lim it of normalafter 10 mo, and the ratio of T4- to TS-positivecells was the inverseof normalthroughoutthe entim posttransplantation period. Despite the predominanceof lymphocytesof the suppressorphenotypehowever,the p&em’s lymphocyte proliferativereepon~es to PHA a& Con A roseto normal by day 12 and remainedhigh thronghoutthe posttransplantation period (Fig. 2). Iaitiafiy, the re-

832

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CLIN. IMMUNOL. JUNE 1994

Dars Past-TransplantaIm

f+G. 1. Results of sequential cytofluorogrephic analyses of blood lymphocyte subpopulations before and for 10 mo sfter bone marrow transplantation of identical HLA by use of monoclonal antibodies OKTlO,~ OKT3 (pan T), OKT4 (recognizing helper phenotype), and OKT8 (recognizing suppressor phenotype). Decline to normal of percentage of TlO+ cells was accompanied by decrease to normal of natural killer cell activity. BM, bone marrow transplant.

TABLE II. NK-cell activity

in a patient

with SCID % Specific lysis (SW) of K662*

Effector-totarga ratio 4O:l

2O:l IO:1 5:l 2.5:1 1.2:1

8 TlO+ lymphocytes

Pre-BMTt

19.3 k 78.4 k 57.3 + 49.8 k 33.0 5 26.7 k 92

4.2 6.3 3.7 4.8 2.3 3.0

1 -month Post-BMT

60.6 " 1.8 53.0 r 1.8 37.6 + 2.5 75

6-woaks Postt-BoyIT

57.4 + 2.0 44.7 " 3.0 38.3 2 2.0 78

emon#s Post-BMT

Nomlel mean-+sD

9.8 + 0.9 4.6 -+-0.5 1.5 IfI 0.4 1.4 + 0.3 0.8 + 0.3 0.7 * 0.3 4

41.1 k 6.4 20.7 " 3.2 10.4 r 1.6 8~2

*2-hour assay. tBMT = bone marrow transplantation.

sponse to PI-IA was higher. After day 60 posttransplantation,the predominantresponsewas to Con A; however, at her most recent evaluation, the PHA responsewas again better than the Con A response. T4+ cells are known to respondwell to PHA, whereas T8+ cells respondpoorly to PHA but well to Con A.14 Her lymphocyte responseto pokeweedm itogen did not becomenormal until after day 60. The proliferative responseto tetanusrosesharplyafter eachof the first two immunizationsbut failed to rise after the third immunization when responsivenessto Con A and$be percentageof T8+ cells were highest; more recently, however, her tetanus responsewas again excellent. Serumconcentrationsof IgG, IgA, and IgM rose to normal by day 12 posttransplantation.IgG and IgA have remainednormal, but IgM has recently fallen

below normal for her age (Fig. 3). The decline noted at day 18 after grafting followed the onset of profuse secretory diarrhea and was believed likely to have been due to protein loss. In contrast, the serum-IgE concentration rose rapidly and steadily from the psetransplantation value of 4 IU to reach a peak more than 3000 IU/ml at day 40 postransplantation(Fig. 4). A slow decline followed with a late resurgenceafter the immediatesystemic reaction to soy formula. Marked peripheral blood eosinophilia developedwith the first upsurge noted during the early G-V-H reactioncausedby the nonirradiatedallogeneic lymphocytes in the packed red blood cells administered3 days beforeadmission. The secondpeak of eosinophilia occurred after the height of the I@ rise but sim~usly with the markedrise in serumglutamic-pyruvictransamina&,

VOLUME 73 NUMBER 6

Severe combined

immunodeficiency

833

consistentwith therebeing a secondG-V-H reaction. The latter was consideredmost likely to be caused by the HLA-identical donor’s cells. The third and fourth late peaksof eosinophiliacoincided with attemptsto feedher andwith the adversereactionto soy formula. Diphtheriaand tetanusantibodiescould not be detectedin the patient’s serumon the day of transplantation (TableIII). After a diphtheria-pertussis-tetanus immunization on day 27 posttransplantation,her tetanustiter rose to 1: 243, and her diphtheriatiter roseto 1: 6561.A repeatimmunizationon day 61 was followed by a rise in her tetanustiter to 1: 6561, anda rise in her diphtheria titer to 1: 19,685. The titers remainedhigh for 25 days. Slowing declinesafter the third boosterto a tetanustiter of 1: 243 and a diphtheria titer of 1: 9 at 10 m o posttransplantation. Isohem- FIG. 2. Results of serial ~~~ $tWk$ agglutininswere nondetectableon the day of trans- before and during 10-month plantationandroseto 1: 16 (anti-A) and 1: 32 (anti-B) vigorous fes~rtsee to rtl by day 40 posttransplantationand then declined baan praaent since day 12. f&l, bow mwrc~w; DT, diphtheria-tetanus booster. slightly. Evidenceof T-lymphocytechimerismwas detected by day 12 posttransplantation; all lymphocytemeta- lineageof origin of thesecells. The previousf&ding phasesgeneratedafter PHA stimulationwere of m a le of a lack of NK activity in SCW was karyotype. B-cell chimerism was demonstratedlater dencesupportinga T ceil Iisle by rigorously depletingthe patient’s blood mononu- activity has beennormalin alI cell defects studiedexceptx-h clear cells of T-lymphocytesand coculturing her B cells with purified T cells from a female donor in tive disease.6, *alQFIowever,the pokeweedm itogen-stimulatedcultures;cells having tion in patients with IX&or@ the m a le karyotype were detectedreadily. F inally, presentpatient, and in six other completered-cell typing at 5 m o postransplantation T-cell deficiency2o(and revealedantigensS and JKb that were absenton her suggeststhe pssiwity cells before transplantationbut were presenton her from a different lineagethan that for T c&ls. Further brother’s cells. In addition, she had lost the Rh e study of mononu&earcells;fmws with antigenthat her brotherlacked. Unfortunately,com- cellular i~~~~i~~y plete red-cell typing of the original blood donor in monoclonal &b&es known may provide valuableinform& Brazil was not available.Thus while red-cell chime- cells21-23 rism is proved; loss of the recipient’s own red-cell ing this question. Becauseof her small size, a lim ited n-r of b lineageis not. The finding of most lymphocyteswith NK phethat Wre not av@Me notype and function in this infant was unexpected, them with the newerreage+Wa since our previousstudiesof NK activity againstthe at the tim e of our initial s&d&s. A particularly instructive feat@%of t&is ir+fant’s K562 target in a variety of forms of primary immunodeficiencyhadrevealedSCID to be the only defect clinical coursewas the studied in which NK activity was totally lacking.6 transfusionof norii@adi donor. Fatal G-V-W has occu This finding was confirmedby others,Sp QalthoughNK SCID who were antsy as few activity against a different type of target (HSV-linfected fibroblaats)had been observed.’NK cells, non-HLA-identicallyrn~c~~~.~~ first describedin 1975, are LGLs known to have the patient had liiely received between capacity to kill certain targets such as tumor or 3 x 1OeviabIe unrelated~1~~~~~ virally-infected cells in a nonantibody-dependentdaysbeforereferral, we p @ icted th fashion.6Therehasbeenconsiderabledebateover the velop severeG-V-H diseaseshortly

834

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Sindel et al. IqA

IaG

;; ‘, E

CLIN. IMMUNOL. JUNE 1984

&lqM

4120

600-

DaysPost-Tronsplonlollon FIG. 3. Serum concentrations of the three major immunoglobulin classes before and during lo-month period after bone marrow cell infusion. Normal concentrations were detected by day 12. BM, bone marrow: D, diarrhea. leoo~3oQ-

1500 -

- 3000

12OQ~200-

-2400 E . - ieoo % n .Z - 1200~

FIG. 4. Sequential values for serum IgE, blood eosinophil counts, and serum glutamic pyruvate transaminase (SGPT) before and after bone marrow transplantation. Peaks of eosinophilia correlated with two episodes of G-V-H with attempts to reintroduce oral feedings and with probable anaphylactic reaction to soy formula. Rises in IgE coincided with second episode of G-V-H and with reaction to soy, and peak SGPT level with second episode of G-V-H. BM, bone marrow: D, diarrhea; soy, soy formula.

The finding that she did havea healthy HLA-identical brother offered an opportunity to m itigate the expected reaction that did indeed develop on day 2 posttransplantation,far too soon for it to have been causedby the marrow-donor’scells. On the basis of HLA A, B, C, and DR loci typing alone, and without waiting for resultsof m ixed leukocyte-culturestudies, we proceededwith an infusion of an unusually large number of unfractionated marrow cells from her brother in the hope that any mature T-lymphocytes presentwould recognizethe unrelatedallogeneiccells and eliminate them. Although direct proof for this is obviously lacking, the early G-V-H reaction that would have been inevitably fatal subsidedspontaneously after 3 days, strongly suggestingthat a graftversus-graftreactiondid occur. Touraineet alz5 were also able to abrogatean ongoingG-V-H reactionin an

infant with SCID who had received an unrelated nonirradiated blood transfusion by giving HLAidentical bone marrow cells. However, in contrastto the outcome in our patient, they did not achieveimmunologicreconstitutionuntil a secondmarrowtransplant from the samesibling was given. An unusual feature of our patient’s posttransplantation coursewas the profuse extendedsecretory diarrhea that developedon day 15 without the two most common signs of G-V-H, e.g., rash and fever. Despite the absenceof these signs, there were other features to suggestthat this was a manifestationof G-V-H, including the time of development,striking hyperimmunoglobulinemiaE, eosinophilia, and elevation of liver enzymes (Fig. 4). G-V-H reactions havebeenobservedin many other patientswith SCID who have received HLA-identical bone marrow

VOLUME NUMBER

8evere combined immunodsficiency

73 6

cells. However, such reactionshave all been transient, and most have been m ild, involving only the skin. The severity of our patient’s reactionwas due probably to the large number of cells we had felt necessaryto a d m inister. An alternatepossibility is that it could have been a chronic G-V-H reaction causedby the unrelatedblood-donor’scells. In view of the rapidity and completenessof resolutionof the initial G-V-H reaction, however,we feel that this is unlikely. Elevatedserum-IgEconcentrationshave been observed previously during G-V-H,26 and this is believed to be causedby abnormalIgE imrnunoregulation during that reaction. Although we do not know the specificity of her IgE, our patient experienceda severeimmediatereactionon ingestionof a soy form u la that she had been drinking at the onset of her secondG-V-H reaction.The episode,broughton by only I ounce of %-strengthsoy formula, was followed also by a secondupswing in her serum-IgE concentrationandanotherrise in her bloodeosinophil count. Despitethe problemswith G-V-H, therewas early and progressiveevidenceof immune reconstitution. The appearance by day 12 posttransplantation of both normal serum immunoglobulinsand vigorous proliferative lymphocyteresponsesis earlier than the appearanceof thesefunctions in many successfulbone marrow transplantsin patientswith SCID.24The high numberof cells a d m inisteredmost likely accountsfor this. Our patient is one of the few patientswith SCID whose lymphocyte subpopulationshave beenevaluatedphenotypicallyas well as functionallythroughout the posttransplantation course. O f particular note is the fact that there was an early rise and sustained predominanceof cells of the suppressor/cytotoxic phenotypethroughoutthe entireperiod.This contrasts with findings in other types of patients undergoing bone marrow transplantationswho were reportedto demonstratea deficiency of cells of the suppressor phenotypein acute G-V-H diseasewith an increase during chronic G-V-H reactionsin somepatientsand a deficiencyin others.27Sinceour patientm a d eabundant immunogiobulinsand antibodiesand her lymphocytes proliferated vigorously when they were stimulatedwith m itogensthroughoutthis time, there was obviously poor correlationbetweenresultsof the phenotypicand many of the functional studies. The bestcorrelationswereobservedin the higherresponse of the patient’s lymphocytesto Con A at the tim e of predominance of T8+ cells and in the declinein NKcell activity in associationwith the decreasein percentageof T lO-positive cells.

TABLE III. Serum antibody transplantation

titers*

after .-

Days after martww-w#I A*-

Anti-diphtherua toxoid Anti-tetanus toxoid Anti-A Anti-B

016

835

54

i&b&an

75

746

314

0

27

729

6,561

243

9

0

27

6,561

19,h33

243

243

0 0

16 32

32 32

x 33

8 I6

4 16

*Reciprocal of last dilution resulting in Positive maction.

F inaIly, there is little to be learnedfrom our case concerningthe physiolagic roie of NK cells. Since infections had aheadydevelopedby 3 % m o of age, the NK cells presenthad not providede o m p ieteprotection; however, no viral agentswem ever grown from her nasopharyngeal or stool cuftu~es.Whether the NK-cell predominancein our patient contributed to the G-V-H reactionssheexperiencedcan only be speculated.Our patient’s G-V-H diseasedid gradually clear as the percentage of NK cells de&i, and thoseof the suppressorphenotypeexceededthose of other lymphocytes&populations.Lopezet al.’ found an associationof normal ~e~~s~a~t HSV-I-NK activity with the later developmentof G-V-H disease in a group of 13 patientswith a variety of diseases transplantedwith dlogeneicbonemarrow or fetal tissue; those with low HSV-l-NK activity had no G-V-H disease. W e are indebted to Mrs. Ruby Johnson and Mrs. Leslie Derr for their skilled technical assistance, Dr. Barton F. Haynes for his helpful advice, and Mrs. Lara B. Whitfield for her excellent secretarial assistance in the preparation of this paper. W e also acknowledge with deep appreciation the skilled and dedicated care of the nurses and technical staff of the Rankin Clinical Research Center. REFERENCES SO, 1. Buckley RH, GilbettsenRB, Schiff RI, Fern&a E, Sam11 Waldmann‘IA: Heterogeneityof lymphocyte ~~~fa~ns in severe combined immunodeficiency:Evidence against a stem cell defect. .I Clin Invest 58: 130, 1976 2. Bortin MM.. Rimm AA: Severecombinedij~~~~ieney disease.Characterizationof the diseaseand resuIts of transplantation. JAMA 238591, 1977 3. PahwaSG, PahwaRN, Good RA: IIetemget@y crfB lym#ocyte differentiation in severecombinedimmunodefrciencydisease.J Clin Invest 66543, 1980 4. Hirschhorn R: Defects of purine metabohsm in immunodeficiencydiseases.Prog Clin lmmunol 3~67,1977 ceIlulnr cyto5. Sanal SO, Buckley RH: Ant~~y-aunt toxicity in Primary immunodeficiencydiseaeesand with different normal leukocyte subpopulatians:Importanceof the ty&?e of target. J Clin Invest 61:1, 1978

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6. Koren HS, Amos DB, Buckley RH; Natural killing in immunodeficient patients. J Immunol 120:796, 1978 7. Lopez C, Kirkpatrick D, Sore11M, O’Reilly RJ: Association betweenpretransplantnaturalkill and graft-versus-hostdisease after stem cell transplantation.Lancet 2:1103, 1979 8. Lipinski M, Virelizier J-L, Tursz T, Griscelli C: Natural killer and killer cell activities in patients with primary immunodeficienciesor defects in immune interferon production. Em J Immunol l&246, 1980 9. Vossen JM, Astaldi A, van de Griend RJ, Dooren LJ: T cell subsets identified by monoclonal antibodies during immune reconstitution of a patient with severe combined immunodeficiency and a patient with DiGeorge’s syndrome. In Touraine IL, Gluckman E, Griscelli C, editors: Bone marrow transplantation in Europe. II. Amsterdam-OxfordPrinceton, 1981, Excerpta Medica, vol II, p 218 10. Buckley RH, Dees SC, O’Fallon WM: Serum immunoglobulins. I. Levels in normal children and in uncomplicatedchildhood allergy. Pediatrics 41:6OO,1968 11. Buckley RH, Fiscus SA: Serum IgD and IgE concentrationsin immunodetlciency. J Clin Invest 55:157, 1975 12. Buckley RH, Dees SC: Serum immunoglobulins. III. Abnormalities associatedwith chronic u&aria in children. J ALLERGY‘t&294, 1968 13. Schiff RI, Buckley RH, Gilbertsen RB, Mctzgar RS; Membrane receptorsand in vitro lymphocyte responsivenessin immunodeficiency. J Immunol 112:376, 1974 14. Reinherz EL, Schlossman SF: Regulation of the immune response-Inducer and suppressorT lymphocyte subsets in human beings. N Engl J Med 303:370, 1980 15. Haynes BF: Human T lymphocyte antigens as defined by manoclonal antibodies. Immunol Rev 57:127, 1981 16. Lozzio CB, Lozzio BB: Human chronic myelogenousleukemia cell line with positive Philadelphia chromosome. Blood 45:321, 1975 17. Ortaldo JR, Sharrow SO, Timonen T, HerbermanRB: Determination of surface antigenson highly purified human natural killer cells by flow cytometry with monoclonal antibodies. J Immunol 127:2401, 1981

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