Low IgG2 and polysaccharide response in a T cell receptor expression defect

¿3 JUL 200+

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

Pcdm Apsricio+.

receptor expression defect*

C¡dor Mslinez-A.+' Antonio Arnaiz-Villel|au

37:

tol.

2411

Low IgG2 and polysaccharide response in a T cell

P¡blo Morales ¡nd

ft.,

immunodcficiency

José

R. Regueiroo, Paloma Perez-Aciego.

ol,

function in Tc€ll receptor

Inmunología, Hospilal lj¿ de Octubre' DePsrtme¡tt of Pedialry Un¡versitY of Valladolido, I)eDrlmena of Medicine, University of Alcalá de Henaresu rnd Centro de Biologia Molecularr,

M¡drid

¡nd

,J.

B lymphocytcs require appropriate T lymphocyte cooperation to

synthcsize immunoglobulins (lg). Such interaction presumably takes place after engagement oftheTcell receptor (TcR) by antigen.The present work addresses B lymphocyte function (and phcnotype) in a novel type of ¡mmunodeficie ncy which is characterized by a TcR expression defect. In contrast to expcctat¡ons. the two affected siblings that were studied displayed normal irr vlvo antibody responses to both endogenous and exogenous protein antigens. However, they showed impaired responscs to ccrtain polysaccharidc antigens together with a selective IgG2 deficicncy.l'hese results suggest that some polysaccharide responses may bc mo¡e Tccll dcpendcnt than previously suspectcd, and support the notion that 'f cell dysfunctions (of this or other kind), rather than Ig gene deletions, may be the molecular basis of certain lgG2 deficicncies.To rule out a concomitant gross B cell dysfunction in these individuals, B lymphocyte phenotype and function were assayed in vitro, and found to be normal. A T cell linc dcrived from one of the siblings displayed an abnormal TcR on the cell surlace, but it showed sevcral normal TcR-mediatcd functions. This suggesls that the low number of peripheral

.IbR

lcvels in these lymphocytcs that have been found to express low immunodeficiencies may be operatiotral, and supplying sufficient "help" for thc obse¡vcci normal antibody rcsponscs to all testcd protein, but not polysaccharide.

T

y, P.

)80,

antlpens.

I

ln to freshly isolatcd peripheral blood T lympho-

polysaccharide antigens seems to be severely impaired.

Introduction

contrast

B lymphocytes are activated and synthesize lg in response to most antigenic challengcs only in the prescnce of T lymphocytes. This so-called Tcell "help" is excrted mainly through soluble lymphokines, secreted after the engagement of spccific TcR by antigen [1].

cytes, an lL2-dependent Tcell line (TcL) derived from onc of the affected individuals was found to bc functionally normal, supporting the notion that thc low TcR expression in this kind of TcRID may, under certain circumstanccs,

sufficc for protcin but not Polysaccharidc B cell responses.

A familial TcR immunodeficiency (TcRID) which consists of a selective expression defect of TcR structures on the surface of otherwise normal T lymphocytes has bcen previously descr¡bed by us [21. Functional analyses of these cells revealed an imoaired immunc rcsDonse to alloantigeos. tetanus toxoid and mitogens [3].'However. normal r¡actination schedules, including immunization with attcnuated measles. mumps, rubella and poliomyeliris virus. x¡ere followed without any incidenr l4l. This suggests that other mechanisms for immunitv mav sufficc in these cascs.

Í.e. nonspecific and Ig, and/ór oiher. This ohscrvarion Fompted us to pursuc a more dctailed study of B lymphocyte 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. alrhough tlre synthesis oi IgG2 ana of ipecific anribodics agai;r

lr

83431

|r¡ pan by FIS, CICYT and NATO grants. The R. R. and P P A. is equal and

I to this article by J.

ordet of authorshiD is árbitrarv.

¡deDc.: Jose R. Reguciro, Inmunología. Hospiral 12 dc E-28041

Madrid. Sóain

TcL: IL2-dep€ndent T ccll

Ve¡hgssesellschafr

linc

TCRIDT TcR

2 Materials

and methods

2.1 TcRID palients A 3-ycar-old hoy wilh severe TcRID (V) and h¡s 7-year-old brothe¡ with a mild form of TcRID (D) were studied. Their clinicaldata havc bccn prcviously described [2-4]. ln brief, sib V was a sevcrc combincd lD variant with normal number, but abnormal function, ofT lymphocytes. HisTcR exo¡cssion defect has been shown to correlate with an impai¡ed response to antigens (including skin tests with tuberculin) and mitogens [3]. A lack of CD3l (and probably CD3y; unpublished) may be involved in the development of the defective phcnotype fzl. The boy died in Junc 1986 (aged 32 months) aftcr a bronchopneumonia and a concurrent scvcre autoimmune hemolytic anemia. Sib D is essentially healthy (although rarc respiratory distress episodes have been recorded), and he is normal in other respects cxcept in hisTcell function and phenotyPe, which is similar to that of his brother. No Ig substitution thcrapy was uscd in cither of the sibs.

2.2 B lymphocyfe fünctional

asssys

Total scrum IgG, lgA and IgM levels were determined by rate nephelometry (Beckman, Brea, CA). lgc subclasses | | 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 specific mAb as described previously [5].

Vaccination with bacterial polysaccharides and poliomyelitis antigens was performed following the manufacturer's

instructions. Briefly, an s.c. dose of either pneumococcal (Pneumovax 23; Merck,West Point, PA) or Haemophilus 6

(Prohibit, Connaught Labs., Swiftwater, PA) or Salk's polio (Inst. Berna, Madrid) vaccine was injected to both patients and age-matched controls. Serum was obtain€d before and 15 days (Salk's), I month (Prohibit) or 4 monrhs (Pneumovax) afler vaccinat¡on.

Eur. J. Immunol.

2.3 lsolation of lymphoid cells, generation of B and T cell lines ¡nd FCM analysis Téchniques for the isolation and FCM analysis of PBMC have been previously described [4]. B lymphocytes were identified by immunofluorescence on PBMC with FITCconjugated F(ab')2 goat anti-human Ig (Kallestad, Ausrin, TX) and also with va¡ious FlTC-conjugated mAb. EBVtransformed B cell lines [6] and IL2-dependent Tcell lines

[4] were obtained f¡om fresh PBMC, and their

surface

phenotype was analyzcd by FCM and/or cytotoxicity (see below). assays

f

lymphocyte functional ¡ssays

for measuring proliferation and synthesis of IL2 by PBMC or lL2-dependent Tcell lines (TcL) have been described [4]. An anti-CD25 rnAb (Mar 108, kindlv

Techniques

provided by M. Lopez-Botet) was added to the cultures tó avoid autologous use of secreted IL2. Serum soluble CD25 and CD8levels were measured by ELISA (T-Cetl Sciences,

Cambridge, MA). In addition. the specific cytoroxicity of a TcR-defective TcL against a mouse plasmacytoma (P815) or against allogeneic

EBVtransformed 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 with irradiated GUS-l cells and allogencic PBMC were mixed at

different

cef l

ratios

(Eft : l{Yl,2lI)

with P8l5 or cUS-1 in

the presence or absence of anti-CD3 mAb (T3bsv65, or of PHA as cross-linking reagenr.

3 2.4 C-mediated cttoloxicity

. 20:24111416

(Raritan, NJ); Leu-16 (anti,CD20) from Becton Dickinso¡ (Erembodegem, Belgium); ts pan (anti-CD19) fror¡ Eurodiagnostic. Apeldoorn, 'Ihe Nethe¡lands); Ib-seriis mAh from lmmunolech (Marseille. France); TSt/2 (anti_ HLA-DR) was obtained from F Sanchez-Madrid (Hospirat de 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 (Netherland Cancer Institute. Amsterdam,The Netherlands).

2.6 Specific serum lg titers against dilferenr virus were derermined by C fixation or by neutralization. Specific IgG titers fo¡ rubella were tested by commercial fluoro-immunoassays (Whittaker, Walke¡sville, MD). Specific Ig titers against polysaccharide antigens we¡e performed by standa¡d ELISA using the vaccines as a source of antigen.

19

1/10)

Resuhs

3.1 B lymphocyte function and phenoaype

The surface expression of various T (and B) lymphocyte antigens was also evaluated by a standard two-step micro-

The highest specific antibody titers againsr different exo-

2,5 mAb

contrasl. serum iso-hemagglutinins wcre absent in sibV and sib D showed very low levels of specific antibodies against bacterial polysaccharides (from pneumococcus or I{aemophilus) both before and after vacclnatlon.

lymphocytotoxicity technique using monomorphic mAb diluted to their end-point [7].

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

l.

genous and endogenous antigens io both TCRID sibs are shown in Table 1. All the virus-sDecific antibodv titers analyzed were normal in both siblings. and seroconversion was observed upon infection (sib V) or vaccination (D). In

Srrccific scrum antibodv titersa)

i:

,: sibv , :' p¡a .Pa6!

,

Normal rarge ' posl

F¿

v16 = > vl6 (= *Uló E4 x pre VI6 ¿4 x p{e = >11 2

a)

due to recognized infection (Pa-

>4xpft

vaccinalion (Polio, Pne¡rmococcus, llacmophilus). titers before (prc) and after (post)

rainfluenza, Adenovirus) or

>1/ 2 >4xpre ¿1/ 2 ¿4xpre

>6

conv€¡sion are shown. Highest values are indicated in allother cases.

>11 g ¿ 3000

When scroconve¡sion occurred

lznt

x pre

=4 x prE >4

b)

The blood group of siblings was Al.

c) Titer

expresscd as 7o

both

of a con-

¡rol positive normal serum.

j

Eur J. fmmunol. lgql.20: 24ll-2416

B cell function inTcell receptor

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

immunodeficicncy

2413

Prompted us to look more closely into the surface exp¡ess¡on of different TcR epitopes on T c€lls using mAb and C-mediated cytotoxicity (Thble 4).Very low levels of cytotoxicity (comparable to the low cell surface expression observed by FCM [4]) were apparent when sib D PBMC

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

were incubated with serial dilutions of antiJicL/B or anti-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-dcpendenl TcL 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 by anti-CD3 ¡ose to almost 100% at hieher concentrations of OKT3 (t/10, l15, lll\, whereas ir remained consrant (around 307o) in the presence of anri{ic/F (1/1).

agel.

b) Mcan valucs of rwo determinations (at 2 and

c)

3 yea¡s

of age).

Ég/ml.

3.3 IbR function

tui

TCRID

lbtal serum Ig lcvels are depicted in Table 2. The average values show normal concentration of all isotypes except IgA, which is slightly raised in D, and IgG2, which is severely diminishcd in both sibs.

In order to evaluate TcR function i¿ vivo in TcRID, Tcelt activation status tvas measured as CD25+ PBMC and serum-soluble CD25 and CD8 in sib D. This individual showed higher CD25+ PBMC than conrrols (mean l47o,

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

Prolife¡ation and cytokine synthesis upon stimulation of TcRID PBMC or'IbL was next assessed in vitro, and the results are summarized in Thble 5. Mitosen stimulation of PBMC resulred in a vcry low synthesis of IL 2. This may be

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).

due to an impaired memhrane signaling step bccause direcr

3.2 C-mediated cltoloxicity vs. FCM fo¡ the anslysis of TcR expression The fact thar the protein-specific funcrion ofB lymphocytes

in these ID wa¡ nearly normal lpanicutarly ¡i¡

siU

O)

srimulation of PKC wirh phorbol ester PMA restored normal levels of IL2 secreiion. Cell o¡olife¡ation assavs esseotially confirmed rhese findings (Thble 5). This is consistent with the low surface TcR expression in TcRID

peripheral T cells (see footnote to lbble 5). In contrast, a TcL derived from sib D PBMC expressing higher levels of

I¡ble 3. B lymphocyte phenotype and functiona)

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

g.&

Eur. J. lmmuno¡.

fi|li

a' I-t'rt'o"r'" phenotype by

199{J.

20: 24Il.2416

complement-mediatcd cy(oloxi-

1

{ I i

a) Meao surface-IbRshowed normal proliferation and IL2 synthesis

upon srimularion wirh plasric_bound anli_CD3 tTablc -5) although fL 2 produclion was low in threc other resrcd

TcL.

Significant cytotoxicity of anri-CD3- and pHA_coated pglj targets was observed. Direct specific cytotoxicity of feeder cells ( GUS- l ) by TcRID TcL grown wirh feedei cells was normat levets. as compared roTrL grown onty

ili,?liil" Thble

5. Prolife¡ation

and

lL2

syDthesis in

of at least two determrnauons.

ln summary.Tccll phenotype and function are markedly abnormat in TcRID pBMCis described t+1. ¡"i lánÁ.iá# culture in the presence of IL2 apparenily ."f".t"'fri!í",

IbR-expressing

In addition, the TcR-mediated cytotoxic effector function of this TcRID TcL was rested, as indicated in fuú¡" O.

values

ieatules.

4

T cells with mostly noimal

functiónai

Discussion

{.1 B lymphoryre furction

and phenot}pe

Two main conclusions emerge from the data on B cell luncrton and phenotype obrainetl in the two TCRID ana_ lyzed: (a) hoth individuals sharc an impaired anrihodv Jeslolse.to polysaccharides, and this is rÁflected by theii fi91e ncV, and_(h) all orher resled phenotypic and fgc.:. .de

ltRID")

I uncr ¡onal leatures of B lymphocytes are nórmat, including otner-. tg_ tsorype levels. thc response to pWM. and thé specrnc lg responses Io thc cxogenous and endogenous protern antrgens ana¡yzed-

IgG subclass deticiencies arc vcry seklom caused by H chain gene deletions l8l.Therefore. thc clefecr probahly iesides ar the level o[ the regulation of lg gene expression by B lymphocytes. This may be due in sóme cases lo an abnormal

I cell conlrol o[ B cell function.

since

lgC

subclas

deficiencies are often found in disorders with añ impaircd T lymphocyte. function. including AIDS and Bü trans-

l9l

pla.ntation [101. Our observarion t_rf a selecrive IgG2 deficiency in ID with a selectiveTbR expression defect lJnds ypporl to the notion lhat IgG2 synthesis by B cells is dependent on normal surface TcR expressioí by T cells. However, the possibility that aTcell anú

a) Mean values of triplicates.

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

aB

cell de'fect have

independently met in these iodividuals is, alrhough very

llJ?l:

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

unlikely, not completely ruled out.WhereasIgGl añ