NAVAL MEDICAL RESEARCH INSTITUTE.BETHESDA, MARYLAND

8 1 w99POLYCLONAL ACTIVATION OF MLRINELYMPHOCYTES BY ANTIBODY TO CELLSURFACE IGD

FD.FINKELMAN, J.JJIOND, ISCHERjS-KESSLER AND E,.SMETCALF

DTIC* Jo ~~Vorosmarti, CAPT, MC, USN EET

Commanding OfficerDE1

Noval Medical Research Institute BN~AVAL MED)ICAL RESEARC:H AND DEVELOPMENT COMMAND)

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I. REPORT NUMBER 8- AVGCCjSS y RECIPIENT'S CATALOG NUMBER

81-99,j,,,, ,,

4. TITLE (and Subtitle) S. TYPE OF REPORT & PERIOD COVERED

Medical Research ProgressPolyclonal activation of murine lymphocytesReportby antibody to cell surface IGD. R. PERFORt|NGORG.MEPORTNUMBER

6. ~ ~ ~ ~ ~ , PEFRMN OR. EOR UME

7. AUTHOR(&) a. CONTRACT OR GRANT NUMBER(#)

F.D.Finkelman, J.J.Mond, I.Scher, S.Kesslerand E.S.Metcalf

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Naval Medical Research Institute M0095.PNo001.1030"Bethesda, Maiyland 20814 Report No.32

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Naval Medical Research & Development Conmmand 1981Bethesda, Maryland 20814 13. NUMBER OF PAGES .

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'to, UPL MEN TARY NOTESP t, tuised in:

"Klinman, N., Mosier, D., Scher, I., and Vitetta, E., eds.B Lymphocytes in the Immune Response; Functional, Developmental, andInteractive Properties, New York, Elsevier, 1981. pp.201-210

IS. K~EY WORDS (Cont fngeon, everse side if n.ec*880dIdn7r pblhnib.Polyclonal B-Cell ActivationAnti-IgDMiceLymphocy tes

20. AsSTRACT (Continue an reverse aide It necessar and Identify by bWeek nimbe,)

see reverse side

DD ,AFM 1 1473 EDITION OF I NOV ,5 It O5OLETE

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The availability of hybridoma produced anti-S antibodies and murine

IgD secreting plasmacytomas made it possible to examine in vivo activation of

murine lymphocytes by anti-o antibodies using anti-6 concentrations similar

to those achieved in in vitro activation. Within 24 hrs after i.v. in-

jection of 800 ug of affinity purified goat anti-mouse 6 (GaM6) antibody

splenic B cells increased their quantities of cell surface (s) Ia antigen,

their size, and their rate of DNA synthesis. All three processes are T-

independent.

The data suggest that in vivo B cell activation by soluble anti-6"

proceeds through two phases. The first, includes increases in sla density,

cell size, and rate of DNA synthesis and appears to be T-independent. The

second phase, includes a further increase in the rate of DNA synthesis,

acquisition of sIgG, and Ig secretion, appears to be T-dependent and, to a

large extent, carrier dependent, which suggests that T cell help specific for

goat Ig may be polyclonally focused onto B cells by GaM6 antibody.

Accossion For

D"-"TI q ',",' e E

A~"L~ti Ity Codes;uAa. -••ia or

Kcorw) Dist :P-!

,MN 0102. LF-014"6601

SECURITY CLASSIFICATION OF THIS PAGE(MWl0 Dt0 ErINPrm)

201

"ý'zY-:DLcLC:; ACTI',A.i:; OF :'P.I:;- LY:..PHOfCYTES BY ANTIB(DY TO C=LL SURFACE" !-;D

+ + ÷+ 4-.•r•.•D. !ýI::KZUý.L'\ JA'U"'S J. ".O:AD 11ýWl'; SCMFR STE-'.'E ""., KESLR+++

+E,,partmvnt of :4'dicin.,, Uniformed Services University of the Health Sciences,

+:ý-AnartmUnt of Nelo.',, Naval :adical tesearch Institute, +++Departnent ofX.icrobiolog;y, Uniformt..d Services University of the Health Sciences, Bethesda,:.Id. 20014

Th: disov:ry that IgD is preecnt on most human B lVphocytes1-3 was rapidly

follow-1 by itr; ide!ntification as a major B l.-n7paiocyte surface isotype in4-7

;,,veral other mammalian specie-;, as well as by attemptj to ascertain its

function. Man., of these efforts have used antibodies prepared against IgD as

tunctio.al probos with the. thoug4ht that anti-' would reproduce polyclonally the

cL•:i:j.-; indaced in B cells when their surface IgD bound antijen. In vitro ex-

| .. ri:',nt,; hav- shown anti-I antibodies to block antigen specific responses to

a ".'cri. xtt d-1U and to induce I lymphocytes to proliferate. 1I-1-Such

cvIl;,, furth. r stLulated by macropha'e or T cell derived factors, dif-14

fkerntiaue into antibody secretirLi ceius. While the culture conditions and

)h'fical fors of anti-c antibodies reqtfired to achieve B cell activation have

varied, it is qenerally agreed that high anti-S concentrations (i.e., 50-100

iy/nl) arc requirod to maximize B cell proliferation.~ rl:' n .. .f~jr~d 1-17iBc. 0,197.: ;.' r ,_,f ., in ze:iky7';,15-17 nd mice have

getnerally indicated that anti-6 can increase the humoral immune response to

high do-es of shmultaneously injected antigen but does not induce polyclonal

H cell activation. The recent availability of hybridoma produced anti-S anti-

20,22bodies and rurine IgD secreting plasmacytomasu has made it possible for

v:; '• ,...1•n:• ••VI '., "t:.tý1 LAIt ifl :iiirin, '.! t ,,c;t, by.. anrtL-S ant-hkY)d~cs

'; Ln •.-- c:i•..n!.XI&. . inir ti to)-'e acaiev•d I:n Ln "vi.ro vitivat.lon

.*'L,.r i,.nk . We t :,mi rntt "w't,0.n n -" !,. attt tr t.. in0,ecti.,n Q.f •O 7 1g eL

i t ,n it.ty ! id ir ,t r! t L-mA,, " .. in -' ibod, io.nenli, !) -n z ; hav.o in-

crea:•t.d their quanttLtot, oat cr.ll surtaco tos Ia antiqgn, their size, and

their rat._ of UNA synthe-st.; All three pruct.s;.-es are T-independent. Six-seven

day:. after anti-, injection a :;econd phastie of activation is observed which is

T-dvpendt-t. Splenic T cells as well as B cells are found to be proliferatinq,

a 3-4 fold increase in the- number of spleen cells is seen, large numbers of B

celli with t;Igv; are found, and a polyclonal increase in the secretion of both

B I CopyBes

202

Ig4 and IgG is observed. This state of polyclonal immune activation persistsas long as anti-6 antibody remains in circulation; once injected anti-6 iscatabolized the Limmune system reverts to its initial level within one week.

GENERAL PROCEDURES

Fo]'owing j.v. injection of 800 pg of GaM6 or control antibody BALB/c micewere sacrificed after varying periods of time. Portions of spleen and lymphnodes were formalin-fixed, sectioned, stained with hematoxylin and eosin andexamined microscopically. Single cell suspensions prepared from these organswere analyzed for volume with a Coulter Channelizer and for DNA content bystaining with an ethanolic solution of Mithramycin and determining cellular

fluorescence intensity with a Becton-Dickinson FACS II fluorescence activated

cell sorter. Cells were analyzed for surface markers by staining with an FITC-labeled monoclonal antibody specific for Thy 1.2 (Clone 30H-12) or with FITC-

labeled affinity purified F(ab')2 fragments of rabbit antibodies specific for

mouse 6, ,!, or y chains or keyhole limpet hemocyanin (control) or with A.THanti-A.TL (anti-Ia) or normal mouse serum (control) followed by FITC-labeledrabbit anti-mouse y, after which the percentage of cells positive for each sur-face marker as well as the median fluorescence intensity of specificallystained cells was determined with a FACS II. Surface Ig was also studied by

radioautographic analysis of an SDS-PAGE electropherogram of an immunoprecipi-

tated NP-40 extract of 12,s surface labeled spleen and lymph node cells from

anti-6 injected and control mice. The percentages of cells with intracyto-plasmic Tg.M or IgG were determined by staining methanol fixed, cytocentrifuge

preparations of these cells-with the appropriate FITC-labeled antibodies and

examining the stained preparations by fluorescence microscopy. In some ex-periments the percentages of cells secreting IgM or IgG were determined by theProtein A reverse plaque assay and the percentages of cells secreting IgM anti-TrNP were determined by a modified Jerne plaque assay. In some experiments

mice were injected with '11-thymidine 18-24 hrs prior to sacrifice and theamount of JH incorporated per 2 x 106 cells was analyzed by scintillation

spectroocopy.

RESULTS

I. Early Events. Twenty-four hrs after injection of GaMS the ratio ofwhite pulp: red pulp in spleen is greatly increased and the small B lymphocytesof the mantle layers of spleen and lymph node follicles appeared to have dif-ferentiated into cells with increased amounts of pale staining cytoplasm

203

a::;--"r, pale, .ucl,-.i wit". rron2nent nucluoli. Surface IgD was aLmost totally

I''.-j * frf-'; B which retained most of their s-;:I. No

S:.,fi_--'t ýr.anr;.s 'ert :-en in percentages of gig. or 31a cells, although

::- :rj•en, intensity of sta staining was markedly increased (Table 1).

:~' (-S F Ga:.. -; SPLZEEN CELL Sr':?PACE MAFYERPS

ay At- : Perci-nt positive (I-odian ý'luorescence Intensity)In 0:ted g g'-1 IgG Ia Thy I

NI 55.3 (100) 55.8 3.8 51.1 (71) N.D.

47.1 5.9 47.5 (125) N.D.

'.c Iis; ,. (7-1) 52.5 3.6 49.tU (83) 33.63•.",. (12) 64.3 2.@ 63.5 (156) 21.8

"(l i J 5 . 1 7) 55.5 5.6 S9. 8 (61)) 34.3

/a' 9.o *(311) 43.4 38.1 t . 27.8

N 1; "yj 54.7 '•6) 55.3 7.4 N(.D. 30.6

14 Ga: 37.7 (73) 40.8 10.3 N.D. 45.7

;l G I 47.5 51.5 5.8 N.D, 32.8

A~~~ 1. 1 .A '~30.3

Day:s aftvr injetction of 800 ý;q of not-nal goat !qG or Ga4..PooL; (f ;ple,n ct.IL frcn 3 mice were stain,.d directly for TgD, Ig:.M, IgG,

or T.hyi 1, or ind2.•ectl-. lot Ia (A'11 .anti-ATL + FITC-Ra~y) and analyzed witha fluore:Žco.rce activatod cell ::jrttr. Media.n fluoret'cence izitensity is a

A ~. .: ~ *~' . '''~*'I wi' i .a.'~ Trr.A r.;~ .)'r .ALA" en. . . . . .. . . • '.•.. : .: .F, :• .. r .lw rt -•••.: O .,••l r % ,G ,r •,I

'.'A, ' ch.insg.'-: w,.r.' az.;- ia.t.d wLth marked inrea.-ot-. in splh',n ct, 1 di.Ž

(Tale 2) an, L'A :,'itt..e;i:. (Tabl.e 3). Twenty-four hours after GaM. injection

the. -r|,:,,nta,;,, of Lplvvn co-l-n with ,;r,-ater than 2C ,::A was more than 2 fold

jnc.ea.sd a: cor~.utred to control valu.s (Table? 3). Tir.e lays after Ga.MS

int~ ction .;pletun cell,; from ant.i-) tr,:at,:d mice show,.d 4 time's ,re in vivo3I-thp'nidine irncorporation on a p,,r cPel bai•,i than spleen celln from control

204

TABLE 2

EFFECT OF Ga.45 ON SPLEEN CELL SIZE

Day Antibody Percent of Spleen Cells with a Volume of

Injected 113-248 p 3 253-423 p3 429-626 p3

N1 G IgG 74 ± 1 21 ± 1 6 t 31 oa.M 51 ± 1 34 1 1 15 ± 1

NI G IgG 82 ± 2 15 ± 1 3 ± 0.2GaM6 37 ± 3 44 ± 1 18 2 2

N1 G IgG 85 ± 2 13 t 1 2 ± 0.5Ga.m6 29 ± 2 46 ± 1 24 ± 2

N1 G IgG 84 ± 1 14 ± 0.9 2 ± 0.314 Ca:.i5 79 t 4 18 ± 3 3 ± 0.6

N1 G IgG 76 ± 7 20 ± 5 5 ± 213 Ga-S 35 ± 3 43 ± 1 22 ± 4

,*Days after injection of 800 ug of normal goat IgG or GaM6*Mice were injected with 800 1g of normal goat IgG or GaM6 on days,0,1,5,7,10,11 and 12.

tArithmetic mean ± standard deviation of spleen cells from 3 mice.

mice. Sorting of these cells into sIa and sta fractions prior to Coulter

analysis and scintillation spectroscopy demonstrated that only the sa + cells

frm G&aMS treated mice showed increased size and 3H-thymidine incorporation.

These events appeared to be T-independent, since congenitally athymic (nu/nu)

mice and mice tolerized to goat IgG by i.v. injection of 2 mg of ultracentri-

fuced goat IgG 2 weeksz prior to GaMS injoction b.haved'similarly to normal non-

toterLzed Ga&M6 treated mice during the first 3 days after anti-6 injection.

Anti-S induced Lncrease! in sla density, B c-ll size, dnd B cell DNA synthesis,

are, to '.ome ,!xtent, independent ev-ent:i in B cell activation. Injection of

eOU ug ot CaMd induced a substantial increase in sla density but not B cell

size. B colls from mice with the BA/N X-linked immune defect23 increased in

size in response to GaM6 both in vivo and in vitro (A. DeFranco and W.E. Paul,

personal communication) but did not show increased DNA synthesis.

II. Late Lvents. Six - seven days after GaMS injection the immune system

demonstrated further evidence of polyclonal activation. The frequency of 91gG+

205

2 TABLE 3

EFFECTS OF GaMd6 ON SPLEEN CELL NUMBER AND DNA SYNTHESIS

N t.u7 Antibody Cell Number 3H-Thy CPM/2 x 10 Cells Percent >2C DNANN Injected x 10-6

NI G IgG 101 (1.13) 787 (1.10) 4.7 (1.60)GaM6 86 (1.10) 714 (1.30) 10.4 (1.26)

NI G IgG 78.6 (1.06) 548 (1.17) 5.5 (1.23)3 GaM5 119 (1.10) 2,257 (1.18) 12.2 (1.08)

N1 G IgG 75.9 (1.17) 551 (1.17) 5.5 (1.11)

GaM,1 352 (1.22) 1,866 (1.85) 12.1 (1.34)

NI G IgG 1i1 (1.08) N.D. 3.4 (1.08)14

GaM6 115 (1.15) N.D. 4.6 (1.17)

NI G IgG 169 (1.53) N.D. 6.4 (1.70)13,tGaM6 263 (1.63) N.D. 22.6 (1.21)

,CDays after injection of 800 ug of normal goat IgG or GaM6.***Determined by fluorescence intensity after staining with mithramycin.

Geometric mean and standard deviation of spleen cells from 3 mice.tCPM of 3H-Thymidine/2 x 106 Nucleated Spleen Cells 18 hrs after i.v.injection of 100 lCi of 311-Thymidine.

t-•tice were injected with 800 jig of normal goat IgG or GaMS on days 0,1,5,7,10,11, and 12.U.D. = Not Done.

spleen cells increased to 4-8 times the normal level (Table 1). Some of this

increase may have been due to formation of GaMS - mouse anti-goat Ig immune

complexes that bound to Fc receptors. However, the appearance of a large per-

.sntaae of Thy I -IqM cells at this time favorti the possibility that in-cr(,-3ed numbers uf B cells with intrinsic siqG were appearinq. In addition,

D;-PAiE analysis of reducud "51-labeled sIq from these cells indicated the

prennnce of a major band with mobility sliqhtly faster than that of 6 chain and

slower than that of the heavy chain of serum IgGa. This major band, which is2a'

found only in trace amounts on lymphoid cells from control mice, has a mobility

similar to that of previously described surface y2a from murine tumor cell

lines.2 4 , 2 5

Spleen cells from GaM46 treated mice 7 days after injection continued to

show increased size and DNA synthesis. However, analysis of FACS purified

206

TABLE 4

EFFECT OF Ga:..S ON GE-1.ERATION OF SPLEEN CELLS THAT EXPPZSS CYTOPLASMUC IG

Day Antibody Percent of Cells Expressing Cytoplas-icInjected IgM IgG

NI G IgG 0.9 (1.45) <0.2GaMS 2.2 (1.53) 0.4 (2.03)

N1 G IgG 0.4 (1.09) 0.5 (2.14)GaM,5 2.5 (1.67) 15.7 (1.28)

Ni G .gG 0.2 (1.46) 0.3 (1.88)14 Ga:*i 0.7 (1.29) 1.4 (1.60)

Ni G IgG 1.5 (1.14) 3.1 (1.54)13 GaM3 6.9 (1.39) 33.0 (1.26)

.,Days after injection of 800 tug of normal goat IgG or CaMS.Mice were injected with 800 ug of nornal goat IgG or GaMS on days0,1,5,7,]0,11, a-d 12.

tGeometric mean and standard deviation of spleen cells from 3 mice.

sIa sia-, sThy 1 and smhy 1 spleen cell populations from these mice showed

that bcth B and T cells had now increased in size and rate of DNA synthesis.

Thuse findinqs are 'onsistent with the- observation that absolute numbers of

both B and T cells in spleen were now substantially increased (calculated from

Tables I and 3).

Increases in synthesis of IgG and IgM by spleen cells were also noted 7 days

after GaMS injection. This was evident from the percentages of cells that con-

tained intracytopla•nmic rqM Aind 1,4( (Table 4) .2s well axs from approximately 10

foLd increases in treqw,%ncles ot Iq secretin4 coll:- (do.terminet; by the protein

A rev,'rse plaque tiachnique) total Ig gecretion (determined by in vitro incorpo-

ratL,:i of '!1-lhucino into cjM4 and [rg), and cells secretinq i.4 anti-'.iP anti-

hodi-_c. 71ii.; last finding is of particular import.wtc-- as it indicates that theincreased Ig synthesis stimulated by GaM. represents a polyclonal antibody re-sponse rather than simply an enhanced anti-goat Ig response. In congenitallyathymic mice no increase in the frequency of slgG÷ lymphocytes, rate of lymph-

ocyte UNA s-nthesis, spleen cell number, or Ig synthesis were observed 7 days

aftoer GatS injection. Similarly, mice "tolerized" with 2 mg of ultracentri-

207

fued goat IgG showed much less evidence of polyclonal a:tivation I da-ys after

*a:!5 i•nection than did noMtoler~zed mice.

Ill. Peversal of Activation. Ten days after GWAS injection circulating

anti-S was no longer detectable and considerable numbers of s~gD cells were

seen. Decreases in the size of splenic lymphocytes and a reduction in the

+nw:.ber and frequency of sIqG cells were also observed. By 14 days after

anti-3 injection these parameters (Table 1-3), as well as the frequency of

cell3 with intracytoplasr ic Ig (Table 4) and the histologic appearance of the

;pleen had almost completely normalized. One abnormality was present, howeverl

the percentages of B lymphocy-tus in spleen and lymph nodes were considerably

below the unstiraulated values.

If multiple injections of GaM: were given to maintain circulating levels

of this antibody the activated immune state persisted. As many as 40% of

spleen cells contained intracytoplasmic Ig 13 days after the initial anti-S

injection and hi.toloqic sections of spleen demonstrated many lymphoid cells

with relatively large amounts of darkly staining cytoplasm in both the red and

white pulp.

DISCUSSION

Our data suggest that in vivo B cell activation ty soluble anti-i proceeds

through 2 phases. The first, which includes increases in sla density, cell

size, and rate of DNA syntrhesis appears to be T-independent. The second phase,

which includes a further increase in rate of DNA synthesis, 2cquisition of

n[g,;, and [4 3ecretirn, drpars to to T-decniJent and, to a large extent, car-

rier dependent, which suggests that T cell help specific for goat Ig may be

polyclonally focused onto B cells by Ga&M antibody.

Our results have a number of implications. First, they establish the valid-

ity of a two staqe model of a cell activation26,27 under physiolouic3l condi-

tion;. Second, rh..y support the. view th4t B calls acquire L-gcG in the process

of differentiatinq into IqG secreting cells. Is Third, they indicato that Ln

tht! presencx,• of endoq'nc•unly generated T cell ielp, the interaction of a

ligqan- with sIgD can activate B lyI:phocytes to secrete antikody even when that

ligand dons not interact with sigM. As other papers in this volume suggest

that the interaction of ligand with sIqD is not required for B cell differen-

tiation into antibody secreting c-lls 29i30t seems likely that the interaction

of ligand with either slgD or sIgM 7an lead to B cell terminal differentiation

in the presence of appropriate helper factors. This interpretation suggests

208

•> • f i.. v xtra imu-.- resan~ies Ly anti-_ or anti-6 antibodies is

-A :11 ' -1yu.-*•J ~ :.e th t'e bindinq of these 11 cands to

)r .Ip rjth.•r t -. e t thý.!ir bloQki:k.! of anti4en binding by sIg.M or slgD.

in• =:re;out; :aper we have pri,- =ntd our belief that an important functional

diffý.re:.c. betweý.n s:gD and 59g:4 is the relative strernth of the souppressive

ett.-t 4enerated by lganJ - slg interaction. with the binding of ligand to

s514. proddcing the greater supprecsive effect.19

It is of in.ttrest that the anti-) concentrations required to achieve poly-

cl,_:al activati'n in vivo and in vitro are higher than the concentration of

sý,-. antigt-ns tht.at are regquired to induce !e.,cific antibody secretion under

si:mil:r coidJitivn5. The lad epitc~e density ana helper factor stimulating

abilities of soluble anti-Ig antibodies relative to some antigens may contri-

buti to this difference. For example. Parker has found that binding anti-Ig

antibodies to an inzoluble matrix and adding helper factors to an in vitro

cultrre systt-n .•ubstant..ally reduce the quantities of anti-Ig antibody required14

for b cell activation.

, •&,. by wh-cn T lr.'..•iocytet are stimulated to proliferate in the

t-,cun:d phit-, of anti-5 activation are not, clvar. Possibilities include pro-

du:. t;on of factors by activated B cells that induce T cell proliferation,

induition of T cell proliferation by goat Ig-mouse anti-goat Ig complexes,

ant z ;t•mulat ion uf an autologous MLR by new antigenic determinants on GaM4

V.- redAoonu for the collapse of the.activated immune system once anti-6 is

-a .L., is.- tl .) nb xur-. Hit,;t.iLoic dAta siuyjest that cells die in ;ittu

in t,.e spleen, rather than migrate to other organs. Specific or non-specific

suppressor macrophaqes or T lymphocytes, killer cells specific for activated

lym.jhocytes, and an immunologic network in which polyclonal activation results

Ln socrption of 1'1 molecules that induce "c.molimentanrv" suppression by bind-

, ac:. . t...tr, Ld •dt)t' iete.mir.ant ,i ay tll contribute to the col'apz•.-

"hL.-' t Z• ,2 MVC.1At,L..nZ. h3%'ver, leAdn inevit.ably to :upprosnion of the acti-

.," 1. ,S Is,•.J 7 ;.I'.| ,, , . d' t ita-! ,t|p:. k ., to hov" tr a.Lnt .d as long a-s rir-

In many wayu polyclonal activatiura by (a'. antibody seems to closely par-

a11kd activation of antigen binding clones by a T-dependent antigen. The re-

lat-v., ease with which the in vivo (aM3 system provides large populations of

activated a and T lmphocytes should therefore be of great value in the study

of antigtn acti%.atud cells and the molecular b•ises of B lymphocyte activation

anJ regulation.

209

W_. tý.af.k M•. Sha:.r.on Wilburn and Xr. L-'eraldo Dacc for their expert techni-

,:i azsistance and :".s. Janet -,Tomson for typing this manuscript. This work was

;.: :rted by US%.HS protocol nu•mbers P08307, R08308, C08410, and the Naval

"*eic.± P•?.earch and Develonment Command Work Unit M0095.PN.00l.1030. The

.znicn and assertions contained herein are the private ones of the writers

3nd are not to be constraed as official or as reflecting the views of the Navy

neFartment or the naval service at large. The experLments reported herein were

conducted according to the principles set forth in the Guide for the Care andu*e of Li.Wratory knL-als, Institute of Laboratory Resovrces, National

'..f:'earch Council, Department of Health, Education and Welfare Publication (NIH)

78-23.

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