ol,

ft.,

37:

tol.

¡nd

,J.

y, P.

)80,

Eur. J. lmmunof . 1y)0. 20: 24ll-2416

José R. Regueiroo,

Paloma Perez-Aciego.Pcdm Apsricio+.C¡dor Mslinez-A.+'P¡blo Morales ¡ndAntonio Arnaiz-Villel|au

Inmunología, Hospilal lj¿ de

Octubre' DePsrtme¡tt of PedialryUn¡versitY of Valladolido,I)eDrlmena of Medicine,University of Alcalá de Henaresurnd Centro de Biologia Molecularr,M¡drid

I Introduction

B lymphocytes are activated and synthesize lg in responseto most antigenic challengcs only in the prescnce of Tlymphocytes. This so-called Tcell "help" is excrted mainlythrough soluble lymphokines, secreted after the engage-ment of spccific TcR by antigen [1].

A familial TcR immunodeficiency (TcRID) which consistsof a selective expression defect of TcR structures on thesurface of otherwise normal T lymphocytes has bcenpreviously descr¡bed by us [21. Functional analyses of thesecells revealed an imoaired immunc rcsDonse to alloanti-geos. tetanus toxoid and mitogens [3].'However. normalr¡actination schedules, including immunization with attc-nuated measles. mumps, rubella and poliomyeliris virus.x¡ere followed without any incidenr l4l. This suggests thatother mechanisms for immunitv mav sufficc in these cascs.Í.e. nonspecific and Ig, and/ór oiher. This ohscrvarionFompted us to pursuc a more dctailed study of Blymphocyte phenotype and function. both l,l vivo and r¿vnto, itr thcse ID which lack normal TcR exorcssion. Our¡Bults indicate rhat normal B lymphocytés capablc of¡pccific antibody responses are presenr in TcRlD. alrhoughtlre synthesis oi IgG2 ana of ipecific anribodics agai;r

lr 83431

|r¡ pan by FIS, CICYT and NATO grants. TheI to this article by J. R. R. and P P A. is equal and

ordet of authorshiD is árbitrarv.

¡deDc.: Jose R. Reguciro, Inmunología. Hospiral 12 dcE-28041 Madrid. Sóain

TcL: IL2-dep€ndent T ccll linc TCRIDT TcR

¿3 JUL 200+

function in Tc€ll receptor immunodcficiency 2411

Low IgG2 and polysaccharide response in a T cellreceptor expression defect*

B lymphocytcs require appropriate T lymphocyte cooperation to synthcsizeimmunoglobulins (lg). Such interaction presumably takes place after engagementoftheTcell receptor (TcR) by antigen.The present work addresses B lymphocytefunction (and phcnotype) in a novel type of ¡mmunodeficie ncy which ischaracterized by a TcR expression defect. In contrast to expcctat¡ons. the twoaffected siblings that were studied displayed normal irr vlvo antibody responses toboth endogenous and exogenous protein antigens. However, they showedimpaired responscs to ccrtain polysaccharidc antigens together with a selectiveIgG2 deficicncy.l'hese results suggest that some polysaccharide responses may bcmo¡e Tccll dcpendcnt than previously suspectcd, and support the notion that 'fcell dysfunctions (of this or other kind), rather than Ig gene deletions, may be themolecular basis of certain lgG2 deficicncies.To rule out a concomitant gross B celldysfunction in these individuals, B lymphocyte phenotype and function wereassayed in vitro, and found to be normal. A T cell linc dcrived from one of thesiblings displayed an abnormal TcR on the cell surlace, but it showed sevcralnormal TcR-mediatcd functions. This suggesls that the low number of peripheralT lymphocytcs that have been found to express low .IbR lcvels in theseimmunodeficiencies may be operatiotral, and supplying sufficient "help" for thcobse¡vcci normal antibody rcsponscs to all testcd protein, but not polysaccharide.antlpens.

polysaccharide antigens seems to be severely impaired. lncontrast to freshly isolatcd peripheral blood T lympho-cytes, an lL2-dependent Tcell line (TcL) derived from oncof the affected individuals was found to bc functionallynormal, supporting the notion that thc low TcR expressionin this kind of TcRID may, under certain circumstanccs,sufficc for protcin but not Polysaccharidc B cell res-ponses.

2 Materials and methods

2.1 TcRID palients

A 3-ycar-old hoy wilh severe TcRID (V) and h¡s 7-year-oldbrothe¡ with a mild form of TcRID (D) were studied. Theirclinicaldata havc bccn prcviously described [2-4]. ln brief,sib V was a sevcrc combincd lD variant with normalnumber, but abnormal function, ofT lymphocytes. HisTcRexo¡cssion defect has been shown to correlate with animpai¡ed response to antigens (including skin tests withtuberculin) and mitogens [3]. A lack of CD3l (andprobably CD3y; unpublished) may be involved in thedevelopment of the defective phcnotype fzl. The boy diedin Junc 1986 (aged 32 months) aftcr a bronchopneumoniaand a concurrent scvcre autoimmune hemolytic anemia.Sib D is essentially healthy (although rarc respiratorydistress episodes have been recorded), and he is normal inother respects cxcept in hisTcell function and phenotyPe,which is similar to that of his brother. No Ig substitutionthcrapy was uscd in cither of the sibs.

2.2 B lymphocyfe fünctional asssys

Total scrum IgG, lgA and IgM levels were determined byrate nephelometry (Beckman, Brea, CA). lgc subclasses

Ve¡hgssesellschafr mbH. Dó94O Weinheim. l99t) @r4-2980t9t1 | | Ll-241 t$3.50 + .2510

¿4r¿ J. K, ¡(eguetro, tí terez-Acrego, E Apanclo et al.

dnd total IgE levels were measured by ELISA using specificmAb as described previously [5].

Vaccination with bacterial polysaccharides and poliomyeli-tis antigens was performed following the manufacturer'sinstructions. Briefly, an s.c. dose of either pneumococcal(Pneumovax 23; Merck,West Point, PA) or Haemophilus 6(Prohibit, Connaught Labs., Swiftwater, PA) or Salk'spolio (Inst. Berna, Madrid) vaccine was injected to bothpatients and age-matched controls. Serum was obtain€dbefore and 15 days (Salk's), I month (Prohibit) or4 monrhs (Pneumovax) afler vaccinat¡on.

Specific serum lg titers against dilferenr virus were derer-mined by C fixation or by neutralization. Specific IgG titersfo¡ rubella were tested by commercial fluoro-immunoas-says (Whittaker, Walke¡sville, MD). Specific Ig titersagainst polysaccharide antigens we¡e performed by stan-da¡d ELISA using the vaccines as a source of antigen.

2.3 lsolation of lymphoid cells, generation of B and Tcell lines ¡nd FCM analysis

Téchniques for the isolation and FCM analysis of PBMChave been previously described [4]. B lymphocytes wereidentified by immunofluorescence on PBMC with FITC-conjugated F(ab')2 goat anti-human Ig (Kallestad, Ausrin,TX) and also with va¡ious FlTC-conjugated mAb. EBV-transformed B cell lines [6] and IL2-dependent Tcell lines[4] were obtained f¡om fresh PBMC, and their surfacephenotype was analyzcd by FCM and/or cytotoxicity (seebelow).

2.4 C-mediated cttoloxicity assays

The surface expression of various T (and B) lymphocyteantigens was also evaluated by a standard two-step micro-lymphocytotoxicity technique using monomorphic mAbdiluted to their end-point [7].

2,5 mAb

B1 (aDti-CD2O) and 12 (anti-HLA-DR) were purchasectfrom Coulrer (Hialeah. FL): OK-series mAb fróm Ortho

Thble l. Srrccific scrum antibodv titersa)

Eur. J. Immunol. 19 . 20:24111416

(Raritan, NJ); Leu-16 (anti,CD20) from Becton Dickinso¡(Erembodegem, Belgium); ts pan (anti-CD19) fror¡Eurodiagnostic. Apeldoorn, 'Ihe Nethe¡lands); Ib-seriismAh from lmmunolech (Marseille. France); TSt/2 (anti_HLA-DR) was obtained from F Sanchez-Madrid (Hospiratde la Princesa, Madrid); BMAO3I (anti-Ticr/p) from R_Kurrle. Marburg. FRG; X-63 murine myeloma SN (nesa_rive se¡1.r¡¡ from M. Lopez-Borer (Hospital de la prinie_sa)iT3bsvó5 (anti-CD3) f¡om J. E. De Vries (NetherlandCancer Institute. Amsterdam,The Netherlands).

2.6 f lymphocyte functional ¡ssays

Techniques for measuring proliferation and synthesis ofIL2 by PBMC or lL2-dependent Tcell lines (TcL) havebeen described [4]. An anti-CD25 rnAb (Mar 108, kindlvprovided by M. Lopez-Botet) was added to the cultures tóavoid autologous use of secreted IL2. Serum soluble CD25and CD8levels were measured by ELISA (T-Cetl Sciences,Cambridge, MA).

In addition. the specific cytoroxicity of a TcR-defective TcLagainst a mouse plasmacytoma (P815) or against allogeneicEBVtransformed lymphoblastoid feeder cells (GúS-1)was measured by 5lC¡ release after incubating .l h at 37 "i,To this end,TcR-defective TcL cultured either alone or withirradiated GUS-l cells and allogencic PBMC were mixed atdifferent cef l ratios (Eft : l{Yl,2lI) with P8l5 or cUS-1 inthe presence or absence of anti-CD3 mAb (T3bsv65, 1/10)or of PHA as cross-linking reagenr.

3 Resuhs

3.1 B lymphocyte function and phenoaype

The highest specific antibody titers againsr different exo-genous and endogenous antigens io both TCRID sibs areshown in Table 1. All the virus-sDecific antibodv titersanalyzed were normal in both siblings. and seroconversionwas observed upon infection (sib V) or vaccination (D). Incontrasl. serum iso-hemagglutinins wcre absent in sibVand sib D showed very low levels of specific antibodiesagainst bacterial polysaccharides (from pneumococcus orI{aemophilus) both before and after vacclnatlon.

i: ,: sibv, :' p¡a .Pa6! ,

Normal rarge 'F¿ posl

= v16> vl6 (= lznt*Uló E4 x pre

= VI6 ¿4 x p{e

>11 2 >4xpft>1/ 2 >4xpre¿1/ 2 ¿4xpre

>11 g

¿ 3000 =4

x pre>6 >4 x prE

a) When scroconve¡sion occurreddue to recognized infection (Pa-rainfluenza, Adenovirus) orvaccinalion (Polio, Pne¡rmo-coccus, llacmophilus). titersbefore (prc) and after (post)conv€¡sion are shown. Highestvalues are indicated in allothercases.

b) The blood group of bothsiblings was Al.

c) Titer expresscd as 7o of a con-¡rol positive normal serum.

j Eur J. fmmunol. lgql.20: 24ll-2416 B cell function inTcell receptor immunodeficicncy 2413

Prompted us to look more closely into the surface exp¡es-s¡on of different TcR epitopes on T c€lls using mAb andC-mediated cytotoxicity (Thble 4).Very low levels of cyto-toxicity (comparable to the low cell surface expressionobserved by FCM [4]) were apparent when sib D PBMCwere incubated with serial dilutions of antiJicL/B oranti-CD3 mAb (269"-27io. norrnal range SOio g5%).whereas other cell ma¡kers were normallv exDtess€d(HLA. CD2. CD4. CD8). ln conrrasr, an IL2-dcpendenlTcL derived from sib D PBMC was more efficientlv tvsed( > 50o/. ) by anti-CD3. but nor by anr i:Ticr/B larouná 3-0% ,normal range 95%-l00%r. The lysis of sib D TcL byanti-CD3 ¡ose to almost 100% at hieher concentrations ofOKT3 (t/10, l15, lll\, whereas ir remained consrant(around 307o) in the presence of anri{ic/F (1/1).

3.3 IbR function tui TCRID

In order to evaluate TcR function i¿ vivo in TcRID, Tceltactivation status tvas measured as CD25+ PBMC andserum-soluble CD25 and CD8 in sib D. This individualshowed higher CD25+ PBMC than conrrols (mean l47o,normaf range l9"-ll%1. and higher serum-soluble CD25(mean 1.187 U/ml. normal range 69-477).. bu( not CD8(mean 362 U/ml. normal range 138.533).

Prolife¡ation and cytokine synthesis upon stimulation ofTcRID PBMC or'IbL was next assessed in vitro, and theresults are summarized in Thble 5. Mitosen stimulation ofPBMC resulred in a vcry low synthesis of IL 2. This may bedue to an impaired memhrane signaling step bccause direcrsrimulation of PKC wirh phorbol ester PMA restorednormal levels of IL2 secreiion. Cell o¡olife¡ation assavsesseotially confirmed rhese findings (Thble 5). This isconsistent with the low surface TcR expression in TcRIDperipheral T cells (see footnote to lbble 5). In contrast, aTcL derived from sib D PBMC expressing higher levels of

Mean values of at least th¡ee determinations.Expressed as o/. of positive cells (lhose displayingfluorescence intensities above the upper limit of thenegative con(rol with rhe indicated anlibodies).

T¡ble 2. Serum Ig levels (mg/ml) in TCRID

a) Mcan values of thrce determinations (at 4,.5 and 6yea¡s ofagel.

b) Mcan valucs of rwo determinations (at 2 and 3 yea¡s of age).c) Ég/ml.

lbtal serum Ig lcvels are depicted in Table 2. The averagevalues show normal concentration of all isotypes exceptIgA, which is slightly raised in D, and IgG2, which isseverely diminishcd in both sibs.

The number and phenotype of peripheral blood B lympho-cytes and their proliferation to PWM were then assessed(Table 3). No¡mal numbers of pheootypically normal Bcells were observed in both sibs, and the response to pWMwas normal in th€ surviving sib D.The surface phenotype ofEBv-transformed B lymphoblasts was also analyzed in thissibling and found to be normal (Táble 3).

3.2 C-mediated cltoloxicity vs. FCM fo¡ the anslysis ofTcR expression

The fact thar the protein-specific funcrion ofB lymphocytesin these ID wa¡ nearly normal lpanicutarly ¡i¡ siU O)

I¡ble 3. B lymphocyte phenotype and functiona)

o,

g.&

fi|li a' I-t'rt'o"r'" phenotype by complement-mediatcd cy(oloxi-

Eur. J. lmmuno¡. 199{J. 20: 24Il.2416

a) Meao values of at least two determrnauons.

1{Ii

surface-IbRshowed normal proliferation and IL2 synthesisupon srimularion wirh plasric_bound anli_CD3 tTablc -5)although fL 2 produclion was low in threc other resrcdTcL.

In addition, the TcR-mediated cytotoxic effector functionof this TcRID TcL was rested, as indicated in fuú¡" O.Significant cytotoxicity of anri-CD3- and pHA_coated pgljtargets was observed. Direct specific cytotoxicity of feedercells ( GUS- l ) by TcRID TcL grown wirh feedei cells was

ili,?liil" normat levets. as compared roTrL grown onty

Thble 5. Prolife¡ation and lL2 syDthesis in ltRID")

a) Mean values of triplicates.

"ii;:,?;,X:i¿:fl.i:}".ily.t":;Ai8í;;lÍí;;.""í"""f llJ?l:resp€ctively, by FCM.

")3;l:¿l;.',::,"'!:i,',il:,#.ii'J8r:-I,fl l:_ii::RLñ1.

ln summary.Tccll phenotype and function are markedlyabnormat in TcRID pBMCis described t+1. ¡"i lánÁ.iá#culture in the presence of IL2 apparenily ."f".t"'fri!í",IbR-expressing T cells with mostly noimal functiónaiieatules.

4 Discussion

{.1 B lymphoryre furction and phenot}pe

Two main conclusions emerge from the data on B cellluncrton and phenotype obrainetl in the two TCRID ana_lyzed: (a) hoth individuals sharc an impaired anrihodvJeslolse.to polysaccharides, and this is rÁflected by theiifgc.:. .de

fi91e ncV, and_(h) all orher resled phenotypic andI uncr ¡onal leatures of B lymphocytes are nórmat, includingotner-. tg_ tsorype levels. thc response to pWM. and théspecrnc lg responses Io thc cxogenous and endogenousprotern antrgens ana¡yzed-

IgG subclass deticiencies arc vcry seklom caused by H chaingene deletions l8l.Therefore. thc clefecr probahly iesides arthe level o[ the regulation of lg gene expression by Blymphocytes. This may be due in sóme cases lo an abnormalI cell conlrol o[ B cell function. since lgC subclasdeficiencies are often found in disorders with añ impaircd Tlymphocyte. function. including AIDS l9l and Bü trans-pla.ntation [101. Our observarion t_rf a selecrive IgG2deficiency in ID with a selectiveTbR expression defect lJndsypporl to the notion lhat IgG2 synthesis by B cells isdependent on normal surface TcR expressioí by T cells.However, the possibility that aTcell anú a B cell de'fect haveindependently met in these iodividuals is, alrhough veryunlikely, not completely ruled out.WhereasIgGl añ<l IgG3generally provide the specific response ro pr;rein antitenso[ bacteria. viruses. vaciines and fboJs. IsG2 antibodies arepredominantly direcled against carbóhydrate antigens(such as hlood group B llllt. anr.l are impórtant in protec-

tlvfmrefnal

Bur. J. l͡munol. 1990 20:2411-2416

tion against polysaccharide-encapsulated organisms (such'o<

S. ineumoniue, H. influenzae an<J N neningiriJts ll2l\ 'lU ú sho*e¿ low serum lgG2 lcvels anr.l indced lacked

isoaggfutinins and protect¡on against H influenzaa (grown

in-hír"t"tpituroty secretion\ and pulmonary biopsics [4])i'tris may-hc due to the quanlitatively more profound dcfcct

áUt.*.á ¡n sib v. as compared to sib D. in TcR exprcssioni¡-¿-fot¿) an¡l function (2 2O-fold) [4] and. probably as a

tnser¡uence. also in tgC2 levels (2-3-fold.Table 2). Sib D

.hows-notmal isoagglutinin ti{ers and is healthy probably

because of a quantitatively higher overall Protection His

hinh.r r"tut lgA level\ (Table 2) may. in addition. confer a

beiter surlacc protection. Vaccination of this individu¿lrrith Pneumococcus and Haemophilas polysaccharides

induced some seroconve¡sion' but revealcd very low titersof sDecific antibodies both before and after vaccination

l.fable l). Also, his partial response to the latter may havc

lieen due to the minor protein component (diphthcriatoxoid) prcsent in the Yaccine.

Thken togethe¡, these results suggest that TcR expression

may be iritical for the synthesis of specific antibodies(máinty tgCZ) against polysaccha des, but not cerlainorotein aot igcns. This is a striking finding becausc polysac-

charides arc considered thymus-independenl antigcnsHowevcr. the so-called thymus-independent antigens have

long been found to be heterogeneous, some of them

ltyfc t ¡ Uelng higltty Tcelldcpendent l13l and in some cases

beñaving like thymus-dePenden{ ones Il4l lt is thercforepossible that a simila¡ situation is taking place in TcRID.

The no¡mal response to PWM in sib D PBMC contrastswith the low response of TcRID lymphocytes to othermitogens like PHA [41. However, normal resPonses 10

PWM have been reported previously in th€ absence of a

normal rcsDonse to PHA and Con A in severe combined lDvariants with normal T cell numbers but abnormal T ccllfunction that might be presumed to be TCRID [15]. Ourdata suppo¡t the view that sufficient T:"B ccll interactionsfor a normal response to PWM (and Protein antigens, see

above) may take place in the presence of T lymphocyteswith abnormal TcR expression.

4.2 TcR expression ¿nd function

TcR is exoressed in verv low levels inTcRID T lymphocytes!n vivo, ás judged by cytoto*icity (present results), FCM,

lhuc 6. Cylotoxic function in TcRlD-derived Tcell lincso)

B cell function in Tcell rcceptor immunodeficiency 2415

and functional and biochemical studies ([2-4] and Presentresults). However, upon culture of PBMC (60-90 days) onIL 2-containing media, it is possible to select stable CD4+TCRID TcL that apparently exPress detectable TcR struc-tures by cytotoxicity (although with 3-fold lower membranedensity than control cells as ascertained by using FCM. no-t

shown).The higher cytotoxi€ity ofTcRID'IbL by anti-CD3as compared to antijfio./p probably reflects that some TcR

epitopés (OKT3) are more conservcd than other(ilMA03l) in the abno¡mal TcR variant expresscd on these

cells.'I'hese in vitro sglected TCRID TcL display normal invit¡o Tcell functions through their mutant TcR as assessed

bv at least three criteria: (a) antibody-dependent cellcytotoxicity (Thbles 4 and 6), (b) specific cytotoxicity offeeder cells (Table 6), and (c) proliferation and in some

cases IL2 synthesis upon stimulation with immobilizedanti-CD3 mAb (Table 5). Therefore, it is Possible thatsufficient T l¡imphocyte helP is available l¿ viv¡'¡ fo¡ normalB lymphocyte synthesis of antibodies to protein antigens int"Étd u""uuti, small fractio¡ (around l0% by FCM) ofthcir T lymphocytes may bear functional TcR structures as

shown í¡ riiro. Indeed,Tcell activation is apparently taking

Dlace ir¡ vivo in sib D. asjudged by his higher CD25' PBMCand serum soluble CD25 levels. High local conccntration oIantigen in lymphoid organs or other compensating mecha-nisrns laccessory molecules ?) may allow for thc partiallyunimpaired ü¡ r,ivo B cell function reported here. In fact, a

high ixpression of HLA-DR is obsered ¡n TcRID PBMC(dble j) aod also in the lymph nodes of sib V (unpub- 'lished).

'[he authots reish to thank S. Fotrtdn, A ]'uertes. F de Ory' G. de ta

Fuute. A. Jimenez. M. Perez-Blas and M. C. Medina for their help'Hoffmunn-La Rothe an.l Sando. Ltd. ure Eratelully acknowledged

foí <torriruous supply of rlL2 an¿ cyclosporin' rcsPectivell

Rcccived February 26, 1990; in final revised form July 5' 1990

5 References

1 Mefchcrs. F. and Andersson. J.,,4rr¡¿¡. Reu lmnunol. 1986 4:

13.

2 Alarcon,8., Reguei¡o. J. R.. Arnaiz-Ville¡a. A. and Tcrhorst'C.. N. Engl. J. Med.l9a8.3l9: 1203.

3 Rcguciro,l. R., Arnaiz-Villena. A.. Or(iz dc Landázuri. M etal.. La cet 1986. i: 1271.

;ellna-)dyrei¡rndingthe)u$

ainsat'It

.ass

dT

s|l:lls,

a)

b)

Presence ofNK cells is unlikely (TcLwcre ) a0% CD2'CD4+ and <3% CD56+ CD57'\.TcL srown only with IL2.TcL grown with IL2 + irradiated fceder cel¡s

(GUS-l).

' 4 Regueiro, J- R., Lopez-Botet, M., Oniz de Laridázuri, M.,Alcami, J., Corell, A., Martin-Villa, J. M.,Vica¡io, J. L. andArnaiz-Villena. A.. Scand. J. Iññunol. 19a7.2ó:699.

5 Jimenez, A., Lopez{rascasa, M. and Fonlan,G.,lnmunología1988. 7: 52.

6 Miller, G., Shope, T., Lisco, H., Stitt, D. and Lipman, M.,Proc. Natl. Acad. Sci. USA 7912. ó9:383.

7 Arnaiz-Villena, A., Rodriguez-Cordoba, S.,Vela, F., Pascual,J. C., Cervero, J. and Bootello, A., Hum. Genet. 7981. 58:

8 Smith, C. l. E. and Hamñarstróm, L., in Hanson, L. A.,Sóderstrdm, T. a¡d Oxelius, V A. (Eds.), ImmunoglobulinSubclass Defíciencies, Karger, Basel, 1986, p. 18.

9 Aumuturier, P, Bremard-Oury, C. and Clauvel, J. P, inHanson, L. A., Siiderstróm, T. and Oxelius, V A. (Eds.),Immuho glo b ulin Subc lass Deficiencies, Karger, Basel 1986,D.62.

10 Morgao, G., Tume¡, M. w. and Seymou!, N. D., in Vossen. J.

and Griscelli, C. (Eds.), Progrcss in Immunodeficíency

Research and Theropy, vol.ll, Excerpta Medica. Amsterü¡l19 , p.229.

1l Oxelius,V A., Am. J. Mecl. 1984.76:7.12 Morgan, G. and Levinsky, R. J., Arch. Dis. Child 1988.8:

171.13 Pike, B. L. and Nossal, G. J.,l. Immunol. 1984. /32. 1687.14 Amfot, P L-, Grcnnan, D. and Humphrey, J. lf., Eur L

Immunol. 1985. 15: 508.15 Gehr¿, R. C., McAuliffe. J. J., Linner, K. M. and Kersey, J. H.,

Clin. Exp. Immunol. 1980. J9:344.

Note added in proof: Thc low antibody responses of sib D afte¡vaccination with P/¡¿umococcus a¡d Haefttop,,tl¿¿.r did not contaiospecific IgG2 or IgG4, but ooly some IgG3 and IgGl, respec-tively.

Rcceived September 10. 1990.