Indian Journal of Biochemistry & Biophysics Vol. 36, December 1999, pp. 405-4 14

The role ofLFA-I , Mac-I , ICAM-I and Ia in the induction ofTh- 2 type of immune response in spleen during murine syngeneic pregnancy

Umashashi C Hegde* and Rachel Nainan

Institute for Research in Reproducti on, Jehangir MerwanjiStreet, Pare!. Mu mbai 400 012. India

Received 16 J/(Iy 1999; accepled 1 Sell/ell/bel' 1999

Murine syngeneic pregnancy is charac teri zed by the transient splenomegaly at mid gesta ti on. Recent studies from our laboratory have ind icated the initiati on of T-ce ll dependent 8-cell response in the sp leen during ea rly pregnancy (Hegde and Nainan 1998). Present studies were carried out to understand the ro le of ce ll ad hes ion and MHC class II (Ja) molecules in the inducti on of Th-2 type of response in the spleen of pregnant Illouse. fmmunochemical localizati on of ICAM- I, LFA- I, Mac- I and fa in spleen have been carried out at differe nt stages of pregnancy and formati on of ce ll c lusters and natural cell adhes ion assay with splenocytes were ca rri ed out on day I (D I) pregnancy and compared with control. Upregulati on of ICAM- I, LFA- I. Mac- I and la was observed during earl y pregnancy. Thi s coi ncided with the formati on of germinal centers (GC) and T h2 type of interleuki ns in sp leen as reported earli er. Increased express ion of ce ll adhes ion and la molecules duri ng ear ly pregnancy provides add iti onal evide nce for the systemic shift to Th2 type of immune response in syngene ic murine pregnancy.

The mechani sms by whi ch the fetal a ll ograft is retained by the mother are not clearl y understood. lmmunomodul ati on during pregnancy appears to be a well orchestrated sequence of events with transient thymic atrophy and spl enomega ly indicating a signi ficant change in maternal immune systeml.5. In the mouse, the weight of the spleen is usuall y max imum on day lOCO I 0) pregnancy. Thereafter it regresses and reaches the we ight of non-pregnant control animals on day I post parium CPP)·'. The producti on and appearance of non cytotoxi c IgG I type of antibodi es during pregnancy is well documented6

.7

. These studi es suggest an acti ve involvement of the spleen in the changed immune status of the mother. Splenomegaly in the absence of active immunizati on or in fec ti on indicates acti vat ion of humoral immunity. For the inducti on of humora l immune response, acti vated T-Iymphocytes have to provide necessary signals to induce proli fe rati on of resting 8-cell s through direct T-8 cell interac ti on and the T cell receptor compl exx. In additi on, the CD4 molecule serves as an assoc iati ve rccogniti on structure by interac ting with the MHC class II molecule (la) on antigen presenting ce11 9

. The cognate

*Author to whom correspondence may be addressed. Telephone: 022-41321 I 1/2/617 ; Fax: 91-022-4 1394 12; E-mai l: li birr@icmrirr. bom.nic.in

interacti on between T and 8 lymp hocytes is cr it ical to I d 'b d d' III II 8 -cell growt 1 an anti 0 y pro lIct Ion . .

Another set of molecules capable of regul ating spec ifi c immunological adhes ions between T­lymphocytes and anti gen bearin g ta rge t ce ll s are intercellul ar adhes ion molec ul e I (lCAM- 1 or CD54) (CD II b/CD 18) and its li gand leukocyte function anti gell (LFA-I , CD II a/CD 18) and Mac-I (C D II b/CD 18) and medi ate the development of spec ific immunologica l responses in vo lving ce ll -ce ll inte racti o n s I 2 . 1 ~. Our earli er studi es In muri ne syngeneic pregnancy have demonstrated the in iti ation of T-ce ll dependent 8-ce ll respo nse in the mother on day I pregnanc/. A genera l increase in hemopoietic activity as we ll as the number of ig-producing ce lls can be observed during all ogene ic as ,veil as syngeneic murine pregnancyl :i.17 An increase in the JgG I type of asymmetric anti bod ies that block .. d d . IX I t) I I b antigen IS reporte uJ'Ing pregnancy . . t las een

suggested that Th-2 type of imlllune response is favourabl e for the continuati on of pregnancill

.21 but

not the Th- I type22. The ai m of the present study was

to assess the ex press ion of ICAM- I, LFA-I . Mac- I and Ia molecul es to see whether they support our earli er observati ons on the inducti on of hUIlloral immune response in the spleen during murine syngeneIc pregnancy .

406 INDIAN J BIOCHEM . BIOPHYS., VOL. 36, DECEMBER t999

Materials and Methods

Animals Pathogen free young syngeneic male and female

Swi ss albino mice, 10 to 12 weeks old, bred (inbred for 75 generations) in the Animal Facility of the Institute were kept for mating. Virgin mice on the day of diestrus were used as contro l animals. The day the vaginal plug (V.P) was observed was considered as day I of pregnancy. Pregnant mice were sacrificed on days I, 3, 5, 10, 15 (D I-D 15) and day I post partum (PP). Each group consisted of a minimum of three animals on each day of gestation.

Aggregation of splenocytes by cell cluster assay alld cell adherence assay

Single cell suspension of splenocytes was prepared by teasing the spleen gent ly from 0 I pregnant and non pregnant mice. As most of the changes in the spleen were found to occur on D I pregnancy, these assays were carried out only with splenocytes of D I pregnant mice. Cells were gently passed th rough pasture pipette to make a uniform cell suspension. The large clusters of ce ll s were allowed to settle down for 10 min at 20°C. The ce ll suspension was adjusted to I x 106 ce lls/ml and transferred to a 25 ml culture flasks in DMEM containing 10% FCS. The formation of cell clusters were observed after incl.:;ation at 37°C for I hI' in a CO 2 incubator and photographed. For the cell adherence assay, the cell suspension was incubated for 24 hr. The unattached cells were washed out as described earl ier2:1 and observed under Zeiss in verted microscope and photographed.

Preparation of splenic section Groups of control virgin mice on the day of

diestrus and different groups of pregnant mice were killed, and the ir sp leens were removed and embedded in Tissue Tek OCT compound (Mi les) frozen at -70°C. Fi ve micrometer secti ons were cut on cryostat (Leit z, Germany) and mounted on gelat in coated slides, sec ti ons were allowed to air dry for 10 min fixed in ice co ld acetone (BO H) for 10 min air dried and used for immunohi stochemical stainin g.

Monoclonal antibodies (I11Ahs ) Rat mAbs directed to mouse LFA-I and ICAM- I

were obtained from Sekagaku Corporati on, Japan . Mabs direc ted to mouse Mac- I and la were obta ined fro m Boehringe r, Mann heim (Germany) .

Immunohistochemical staining alsplenic sections

Spleen sections were rehydrated and endogenous peroxidase activity was blocked by a 10 min incubation with 0.3% H20 2. The sec tions were then washed, blocked with PBS containing I % normal rabbit serum (NRS). The sections were incubated overn ight at 4°C with rat monoclonal antibodies directed to mouse LFA-I, ICAM-I , Mac- l or la (I: 100). Rabbit anti rat i mmunoglobu lin conj liga ted to horse raddi sh peroxidase (HRP, OAKO) wa:, added (I: 100) to the sections and incubated for one hour. After washing the sections , co lour was deve loped with DAB (S igma) and H20 !. Normal rat serum (I: 100) was used as the negative co ntrol. Microphotographs were taken ( IOOx) with a Zeiss microscope, Germany .

Results

Cell cluster and cell adhcu' llce !l/Oleelll e.1 ill splenocytes

Splenocytes of 0 I pregnant mi ce (Fig . I b) but not those of comro l mice (Fi g. I a) formed large ce ll clusters on incubation . The interacti on of lymphocytes with other lymphoid cells is show n in Fig. Ie. This cluster format ion and ce ll adherence arc known to be due to the upregulati on or acti va tion or LFA-I molecu le l). The attachment of acti vated ce ll s to fibroblast s are shown in Fig. I d ancl Ie indicating the upregulation of lCAM-1 and its li ga nd LfA- 1 in these cells.

II17I17Ul7oliistocli emicalloc({/i-:.(/tioll oj"lCt1M- 1 ill spleen

These studies show an upregulati on of ICAM-I on D I pregnancy (Fig. 2) . In the spleen of control mouse, the staining of rCAM- 1 was mainl y in the germilial follicl es (GF) and al ong the capillari es. However on day I, intense staining of the network of dendritic ce ll s (DCs) in the germi nal ce nt ers (GC )

was observed indicatin g the initi ati on of humoral immune response in the sp leen. The germin al center formation could be vis uali zed i1 [) I. These developed into mature folli cles on [)~ . The intense staining for ICAM- I was observed in the T-ce ll area where substantial numbers of DCs are known to be ICAM- I +. Subsequentl y, the ex press ion was considerably reduced on ]).') and few stained celh cou ld be observed on D I () and the cap i Ilari es \Ve re clearly seen. The spl een of" 01 .'1 mouse ex hibited vc ry

HEGDE & NAINAN: IMM UNE RESPONSE IN SPLEEN DURING MURI NE SYNGENEIC PR EGNANCY

-, • '-

.It ~~~ ~---.-.----'--------.

Fig. I- (A) : Splenocytes of control virgin 1l111USe on the day o f diestrus in DMEM with 10% FCS Incuhated ~ 11

37°C in CO2 incubatur fur 30 min and ohserved ami photographed under Zeiss in verted microsc(l pe. \' ·:1 y few clu sters are observed (x 40) (B): Splenocytes of day I pregnant mOUSe in DrYl EM wi th 10% FCS incubated for I hr at 37°(, in CO, incuh at(lr . Large number of cell agg lut inates arc ohserved indica tin g the upregulation of ce ll adhes ion IIlO\cCU\cS (x oW )

(C): Sp lenocytes from day I pregnant mouse forll1 lng agglut inates wi th an tigen present ing cells aud lymphocytes observed ancr 24 hrs indi cat ing the acti vation of cell adhesion mo lecules. (0 & E): Splenocytes fro m <J ay I pregnant tllo use Adherence of lymphoblast to fibrob lasts. I As ahllVL' incubated for 24 hr and photographed ullLkr Zcis'-. inverted microsco pe]

407

408 INDIAN J BIOCHEM. BIOPHYS .. VOL. 36, DECEMBER 1999

little staining in the T-cell area and the PP an imal showed some staining around the germinal follic les, mainly in the red pulp (RP).

Immullohistochemicallocalizatioll of LFA- / LFA-I is most ly present on T-cell s and is shown to

be important for the interacti on of T and B­lymphocytes. In the non pregnant mouse sp leen some staining was observed in the T cell area of germinal fo llicle (Fig. 3). In the spleen of 0 I pregnant mouse the staining was significantly increased both in the germinal center and also in the red pulp. By 03 and 05 mostly the T-cell areas of mature fo llicles and cells in the red pulp were stained. The spleen of 0 I 0 pregnant mouse had LFA + cell s in the T cell area, in the red pulp and along the capillaries. Thereafter, on DIS pregnancy and PP the staining was observed in the red pulp .

Imll1unohistochemicallocalizarioll of Mac- /+ cells In addi ti on to LFA-I , Mac- I is also known to be a

li gand for ICAM-I . It is an antigen present mainly in macrophages. Our studies show Mac-I + cell s in mature germinal fo llicles of the sp leen of virgin mouse (Fig. 4). The formation of three germinal centers wit h MAC- I + ce ll s were observed on 0 I pregnancy . These MAC-I + ce ll s were in terspersed in the small fo llicles and red pulp on 03 gestat ion. Later, on day 5 the margin al zone macrophages appeared to be interrupted by other cell s. Thereafter, MAC-I + macrophages were observed onl y in the marginal zones (Table I).

1lIll11l1nohistochemicalloc({lizatioll oj' /o+ cells

In the sp leen of virgin mouse, sO~le staining of la was observed around the germinal foll icles and red pulp (Fig. 5, Tab le I). On day I gestation , the newly formed GCs and some cells in the RP exhibi ted intense staining. The T-cell area of the GF of 0 3 and

D5 spleen were strongly la+. Thereafter some staining was observed only in the red pulp. Detai ls of staining of different cell membrane markers are presen ted in Table I. Discussion

Germinal center is a speciali zed microenvironment whi ch deve lops in the B cell folli cles of secondary lymphoid ti ssllle during T ce ll dependent anti body response. The B-cell s that give ri se to GCs init iall y have to be acti vated outside germinal folli cles in the T cell rich zones in association with interdi gitating cell s or follicu ~ ar dendrit ic ce ll s and T ce ll help soon after the admini stration or entry of antigen. Activated B cell s undergo affin ity maturation and immunoglobulin (Ig) isotype swiiching foll owed by the formation of memory B cells and antibody forming plasma ce ll s2~.

Cel l adhesion molecu les are th ought to have severa l important roles in immune response in vo lving cell -cell interacti ons. Tram:ient ad hes ion of T.B lymphocytes and/or ant igen presenting ce ll s are crucial for the initi at ion and induction of humoral immune response. T-cell activa ti on through the TCRIClass II molecule interaction faci li tates subsequent B-cel l acti vat ion requiring the LFA­I/JCAM- I pair of adhes ion receptors . This fi nall y leads to B-cell proliferat ion and ant ibody production. It was observed that when the sp lenocytes of virgin mice and day I pregnant mi ce were suspended in tissue cu lture media and incubated . the lat ter forilled large cell clusters. Under hi gher Illagni ficat ion these appeared to be clusters of dend ritic ce ll ~; with charac teri sti c processes interdi git ing between lymphoid cells . Follicular dendritic cell s (FDC) and T cell s, which are present in the GC, playa crucial ro le in the regulati on of immune res ponse25 This indicated the upregulation of cell adhesion molecules resu lting in cluster formation"!'. The attachment of

Table I- Immunobcali zation of ICAM- I, Mac-I and la in spleen during murine syngcnei c pregnancy

Markers Cont rol DI D3 D5 DID DI5 Pust diestrus Prcgnancy Pregnancy Pregnancy Pregnancy Pregnancy P ;lrllllll

ICAM-I GF, RP GC GF GF Gf .C GF RP + +++ ++ + + + +

LFA-I GF GC GF GF Rfr',C RP RP + +++ ++ + + + +

Mac-I GC GC GF GF GF GF RP ++ +++ ++ ++ + + +

I a-I GF,RP GC GF GF RiP RP RP ++ +++ ++ ++ + + +

GF, germinal folli cle; GC, germinal centre; RP, red pulp; C, capillaries

A

o

G

(A) NORMAL RAT SERUM (8) NORMAL MOUSE (C) DAY.1 PREGNANCY (D) DAY3 .. (E) DAY 5 .. (F) DAY 10 .. (G) DAY 15

(H) DAY 1 POST PARTUM

Fig. 2-l llllllunoh islOchclll ica l IllCa li /<lti lln o f ICA M - I , l a in spl ccn during pregnancy. Ori ginal Illagni ficatiun x 100. (A ): Stain ing o f ,cc tilli1\ o f spiecnnorll1a l rat ,crUIll: ( 8 ): Staining on the day pl'diestrus (Nonprcgn3nt l'ont rn l ): (e): Staining o f day I prcgnanLY D-H are as

ex plained in th e Fi gure

:r: m Cl o m R<> z ~ z ;.. z 3:: 3:: c: z m ;:0 m VJ "0 o Z VJ m z VJ "0 r m :n z o c: ;:0

z Cl 3:: c: ;:0

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(G) DAY 15 .. G (H) DAY 1 POST PARTUM

Fig . .1 - -l llllllullohi slOchcll1i cal l\lCali zJlioll of LFA- I (X 100)

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NORMAL RAT SERUM NORMAL MOUSE DAY 1 PREGNANCY DAY3 .. DAY 5 II

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DAY 1 POST PARTUM

:r: m Cl I:) m ~ z :> z :> z 3: 3: c: z m ~ m en "1:1 o Z en m z en "1:1

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NOR MAL RAT SERUM NORMAL MOUSE DAY 1 PREGNANCY DAY3

" DAY 5 DAY 10 ., DAY 15 " DAY 1 POST PARTUM

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HEGDE & NA INAN: IMM UNE RESPONSE IN SPLEEN DURING MURINE SYNGENEI C PREG NANCY 4 13

Iymphoblasts to fibrocytes present in sp leen cell cu lture also demonstrated the activat ion of these molecules in the sp lenocytes of early pregnant mice23.

Upregulation of ICNvl-1 and its natural li gands LFA-I and Mac-I on day I pregnancy, when the germinal centers were also visua li zed suggest their role during the early phase of immune response. lCAM - I is expressed on diverse types of cell s and tissues in response to inflammatory mediators or the

. . ?7' , I h f T d d . actlvatlon - '-' . n t e case 0 - epen ent antigen, dendritic ce ll s, macrophages or B ce ll s take up protein anti gens, proteo lyt ica ll y degrade them and present peptide fragments to Ag-specific T cell s28

.

This cognate interacti on between T and B lymphocytes is crit ical to B ce ll growth and antibody production29

. T cell dependent signaling of the B ce ll line was strongly inhi bited by either mAb to CO 18 (b chai n of LFA-I ) on the T cell or mAb to rCAM-1 on the B cell demonstrating the importance of thi s pair of adhesion molecules in early T-B ce ll interaction30

.

The inhibi to ry effect of the mAb to LFA- I or ICAM­I in B-cell activation was largely caused by blocking direct contact between ac ti va ted T cells and B cell s and was secondary to dimini shed cytokine production . These results clearly indicate th at LFA-I , rCAM-1 interaction plays an important role in T-B cell collaboration' i and al so that mo lecules oth er than MHC class II (la) mo lecules and the TCR and/or the C04 accessory molecule are required earl y in T cell dependent B ce ll activati on and suggest a ro le for the LFA-I /ICAM-I pair of ad hesion molecules in T ce ll dependent B ce ll signaling.

After mating, the constituents of semen may be recogn ized by the immune cell s in the female genital tracr12

• The sensitized immune cell s from the uterus appear to move to the secondary lymph oid organs, such as spleen whi ch has the microenvironment conducive for the development of germinal center for the activati on and proliferation of B ce ll s and production of antibod/,l The increased ex press ion of LFA-I/ICAM- I observed during earl y pregnancy appears to be important for the interact ion of ant igen present ing cell s, T cell s and B ce ll s and al so in T cell dependent B ce ll signaling which is cruci al for the initiation of immune response. Our earli er studies have shown the fo rmati on of GCs and presence of Th2 type of cytokines on D I gestation followed by the appearance of Ig+ cell s on 03 ind icating the induction of T cell dependent immune response culminating in the producti on of an ti body forming

plasma cell s in the spl een4. The Signi ficant upregulation of ICAM- I, LFA- I, Mac- I and la molecules with the formati on of multipl e germinal centers on 0 I ges tation is a clear indication of the crucial role of these molecul es in the ini tiation of T cell dependent B ce ll response. The humoral immune response to ant igens (Ag) is initiated by Ag binding to specific receptor membrane immunoglobulin s on B lymphocytes"". Antigen li ga ti on of these receptors prepares the B cell s for a subsequent physical interaction with T helper ce ll s by initi ating the processes of Ag internali za ti on. proteolys is, and reex pression in assoc iation with class II molecul es encoded by the MHC (la). Subsequent T-B interaction involve ab TCR binding to antigeni c peptides in assoc iat ion with la mo lecul es on the B cell surface.14. It has been show n that direct contact between T he lper cell s and B ce ll s deli vers an act ivati on signal to B ce ll s throu gh surface la . Therefore the presence of MHC class II molecu les (I a) in newly forming GCs is indicative of the direct contact of T and B cell s in the initiation of Th-2 type of immune response in the sp leen. · Loca li zati on of Mac- I in GCs corre lates with our observation on the ac ti vation of T and B cell s. Anti gen presenting ce ll s are necessary to initi ate the humoral Immune response. The CD4 glycoprote in on T cell s can interact direc tl y with La molecul es on B ce ll s, perhaps transmitting an activation signal. Taken togethe r the upregul ati on of ICAM- I, LFA- I, Mac- I and la molecules during earl y pregnancy sup port our earl ier observations th at the T cell dependent B ce ll respo nse occurs in the spleen of mouse duri ng earl y pregnancy. These antibodies may have an im munoregul at ory role' ). The identifi cation of thi s anti gen will hclp LI S

to unders tand the immunoll1odulatory c h~ln ges of the pregnant mother.

Acknowledgem ent We wish to thank our Direc tor. Dr. H S .l unei a ro r

hi s encouragement , Ms. Doris D'Souza for typi ng the manusc ript and Mr. G K Murihy for hi s assistance in the animals house.

References I Maroni E S & DeS uu za M A 13 ( I <) XO) Clill 1"' 11 111 1111111101 :I I .

107-124 2 Chambers S P & Cl arke A G ( 1979) .I i?l'llmri h 'rl il 55. 3()l) -

3 15 3 Gu pta R & Hegde U C ( I <) 9 I) in : lI iollll'lII iJ rullf".l ill HI'lilih

alld Diseases (A M Kid w:li . R K Upreti and P K Rav Ed, ; Vo l I pp 43-52. Today and T0 1110 ITOW, Pr ill ie,', ,mel Publi shers. ew Delhi

414 INDIAN J BIOCHEM . BIOPHYS., VOL. 36, DECEMBER 1999

4 Hegde U C & Nainan R (1998) AIIl J Reprod IIIlIllUIIO! 40, 424-430

5 Clarke A G & Kendall M D (1994) 11Il1I1I/1l01 Toda)' 15, 545-551

6 Bell S & Billington W D ( 1980) Nalllre, LOlldoll 288, 387-388

7 Dresser D W, Popham A M, Herrera & Carter J ( 1989) J Reprod Immullol 16, 55-70

8 Tedder J F, Schmidt R E, Rudd C E Kornacki M, Ric J & Schlossman S F ( 1986) E liI' J 111111l1l1l01 16, 1539- 1543

9 Lanzavecchi a A ( 1990) AliI/II Rev IlIllllllllol 8, 773-793 10 Abbas A K (1988) Imlllllllol Todav 9, 89-94 II Noelle R J & Snow E C ( 1990) IlIllllllllol Toda)' I 1,36 1-368 12 Dustin M L, Rot hlein R. Bhan A K, Dinarell o C A & Springer

T A ( 1986) J 11Il1ll1ll101 137,245-253 13 Dougherty G J, Murdoch S & Hogg N (1988) E liI' J /illl nllll ol

18,35-39 14 Hamann A, Jablonski-Westrich D & Heinz Gunteher T ( 1986)

EliI' J 111111111110116, 847 -850 15 Fowler J H & Nash D J ( 1968) Dev Bioi 18, 33 1-353 16 Carter J & Dresser D W (1983) IlIllllllllol 49. 481-490 17 Mattson R & Mattson A ( 1984) Dev COIllP 1IIllll lll lO i 8, 92 1-

929 18 Margni R & Binaghi R (1988) A 1111 II Rev 1111111111101 6, 535 .. 554 19 Gentile T & Margni RA ( 1995) .I ReJirod 1111 III 11/1 01 28, 1-3 20 Wegmann T G, Lin H, Gui lbert L & Mosmann T R (1993)

IlIImlll/ol Today 14, 353-356

21 Lin H, Mossmann T R. Guilbcrt L. Tun!ip(lpi!a! S & Wegmann T G ( 1993) J /illlllllll ol 15 1. 4562-457:1

22 Raghupath y R ( 1997) 1111111111101 Tod((.\' I ~. 4n-4X2 23 Dustin M L, Rothi ein R, Bhan A K. Dinarcllo C A & Sp rin~c r

T A (1986) J fll1l1lllllol 13, 245-254 24 MacLennan I e M ( 1994) A 1111 II ReI' 111111111/101 12 . I 17· I:1lJ 25 Butcher E C. Rouse R V. Coll man R L. N()! t enilel '~ C N.

Hardy R R & Weissman I L ( llJR2) Jill /lillI/wi 12lJ .. 26lJX-2707

26 Temponi M. Ronano G, D'U rso C M. Wan~ Z. Kckshish U & Ferrone S ( 1988) Selllill ar Oil ()lIcologr 15. 5lJ5-607

27 Springer T A ( 1990) Nalllre, Loudoll :146. 425-4:14

28 Lanzavecchi a A (1985) Nalllre. LOlldo/l 31 '+. 5:17-5:1lJ

29 MacLennan I C M ( 19lJ4) AI/I/II Rev 1111111111101 12. 11 7- I.W

30 Lane P J L. McConnell F M. Clarke E A & Mel li ns E ( l lJlJl) J Immullol1 47 , 4103-4 108

3 1 Loughman M S & Nossal G J (llJXlJ) N(J(/Iu·. LOlldoll 340. 76-79

32 Robertson S A (1998) AIIl .I Rel'md 111111111/101 Ails! No 26. 40 127

33 Campbell K S. Tustement L B & Camilil'r J C ( l lJ!JO) (J C Cambier Ed) ASM Publication Washington DC)

34 Pierce S K, Morri s J F, Grusby M 1. KaulTlaya P. Van Buskirk A, Srinivasan M, Crulllp B & Smolcnski LA ( IlJXX) lilli/ III/wi Rev 106, 149- 180

35 Hegde U C (1991) Medica l HI'Jlothe.l' i.l' :15. 15lJ- 1 M