Signaling and Ce11 Cycle Regulation

in B Lymphocytes

BY Iris S. Doyle

Department of Microbiology and Immunology

McGill University

Montréal, Quebec

July 19, 1999

A thesis submitted to the Faculty of Graduate Studies and Research in partial

fulfillment for the degree of Master of Science

Copyright O Iris S. Doyle

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Acknowledgements

1 would like to thank my supervisor, Dr. Trevor Owens, for his guidance,

encouragement and understanding throughout rny time in his lab. Thank you Trevor,

for giving me many opportunities to attend conferences, to present my work and to

learn fiom the best. 1 would aiso like to thank everyone in the lab, past and present,

Qioake Gong, Lyne Bourbonnière, Maria Caruso, Grace Chan, Gai1 Verge, Michael

Jensen, Mina Hassan-Zahraee, Elise Tran, and Esther Prince for showing me the

ropes and special thanks to Shelly Sud and Peter van den Elzen. I would like to

express my gratitude to Nick Crispe for inviting me to work in his laboratory at Yale

University in order to perform the knockout experhents.

My parents, Roger Doyle and Bérangere Vézina, have always been there to

love and support me, and for this 1 am forever grateful. Thank you for being great

parents and for teaching me what is important in life.

Most importantly, thank you Jean-Michel for aiways being there for me, for

understanding everything and for giving me balance.

Abstract

T-dependent activation of B ce11 proliferation is crucial to humoral immune

responses. Regulaîion of this activation is required to Iimit responses. The cyclin

dependent kinase (CDK) inhibitor (CKI) p2 1 cip-11~-1 is implicated in inhibition of

ce11 cycle progression. This thesis describes experiments that investigated the role of

p21"p'1-1 in regulating B ce11 proliferation. C57BV6 splenic B cells were

stimulated to enter ce11 cycle by crosslinking CD40. p21Pp1/"'-' expression was

upregulated by CD40 stimulation of B cells, and superinduced by CO-crosslinking

intercellular adhesion molecule 1 (ICAM- 1)/CD54, major histocompatibility

complex (MW) I or MHC II with CD40. Treatments that superinduced p2 1 cip-114-1

inhibited CWO-stimulated proliferation and induced apoptosis. Apoptosis was

abrogated in p21 cip-Ilwaf-1 -1- B cells, which otherwise responded equivalently to

controls. By contrast, B cells from p27'Up-1 -/- mice showed the same induction of

ceIl death as wild type mice. These results show that MHC and ICAM-1, Cipllwaf-l

specifically modulate CD40-mediated signals and this process involves p21

iii

Résumé

L'activation proliferative des cellules B par les cellules T est essentielle pour

la réponse immunitaire humorale, mais sa régulation est nécessaire pour limiter la

réponse immunitaire. L'inhibiteur de la kinase dépendante de cyclin, p2 1 cip-114-1 est

impliqué dans la régulation de la progression du cycle cellulaire. Cette thèse décrit

des expériences qui recherchent le role de p21 cip-rhivaf-1 dans la régulation de la

prolifération des cellules B. Des cellules B provenant de rates de souris C57BV6 ont

été stimulées pour entrer dans le cycle cellulaire par l'agglomération de CD40.

L'expression de p21 cip- l/waf- 1 a été augmentée par la stimulation de CD40, et a été

superinduite par l'agglomération de CDS4(ICAM- 1), MHC 1 ou MHC II avec CD40.

Les traitements qui ont supennduit p21 cip- 11wlf- 1 ont inhibé la prolifération induite par

CD40 et ont induit l'apoptose. L'apoptose a été abrogée dans les cellules B

provenant de souris p21 cip-~/waf-~_ / , qui autrement se comportaient comme les

contrôles. Par contre, les cellules B provenant de souris p27Ep-1-/- ont démontré la

même induction de mort cellulaire que celle des souris normales. Ces résultats

démontrent que MHC et ICAM-1 inhibent spécifiquement les signaux transmis par

CD40 et que ce processus implique p2 1 ap-i/~af,l

Table of Contents

. . Acknowledgernents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . - . . . u

. . . Ab stract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . - - - . . . - - - - . . . . . . - - - in

Résumé . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - . . . . . . . iv

Table of Contents. . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . v . . List of Figures. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . - - . . . . . . . . . v11

.. . Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . - . . . . . . . . . - - . . .wu . S . Contributions of Authors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - .wu

CHAPTER i

Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 1

A. T Dependent B Ce11 Activation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 1

B. Ce11 Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . - . . . -6

Marnmalian Cell Cycle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

C. Cyclin Dependent Kinase Inhibitors. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

PZ lnp-i'd-i ... .. . ... . .. .. . ... .. . ... . . . ,.. .. . . . . . . . .. . -. . .. . ...... ... . ..... .. . . . ,. . .a.... -9

p2 1 and PCNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 1 O

S ignaling Upstream of p2 1 .............................. ......... .......... 11

Signaling Downstream of p2 1 "P"*-' . -. . . . . . . . . . . . . .. .. . . . ... . . . . . . . . . . . . . . . . . . . -12

p2 1 and Apoptosis. . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

D. Rationale for Curent Project.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 14

E. Re ferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -1 5

CHAPTER 2 p21d~l- mediates apoptosis induced by ICAM-1 and MEIC in CD40-

stimulated B cells.

A. Title Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -3 6

B. Summary . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . -3 7

Cm Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . - . - . - -3 8

D. Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -40

E . Results .................................................................................. -43

F . Discussion .............................................................................. -47

............................................................................. G . References - 5 1

H . Figures ................................................................................... 59

CBAPTER 3

.................................................................. . A General Conclusions -65

............................................................................... . B References 70

List of Figures

.... Figure 1 : Regdation of p2 l"l/M-l expression in CD40-stimulated B ceiis.. -59

Figure 2: Co-crosslinking ICAM-1, MHC 1, or MHC II with CD40 inhibits CD40-

induced pro iiferation.. ......................................................... .60

Figure 3: IC AM- 1, MHC I, and MHC II CO-crosslinking with CD40 induces

apoptosis.. ................ .... ............................................... -6 1

Figure 4: p2 l"'l'*-L , but not p27~p1,is required for induction of apoptosis by

ICAM-1, MHC 1 and MHC iI. ................................................ .62

Figure 5: Fas L is not expressed on splenic B cells.. ................................... -63

Figure 6: High buoyant density B cells fiom p21np-1'M' -/- and p27'p1-/- rnice

........................... proliferate in response to anti-CD40 treatment.. ..64

vii

Preface

In accordance with the "Guidelines for Thesis Preparation" of the Faculty of

Graduate Studies and Research at McGill University, a paper that has been submitted

for publication is incorporated into this thesis. Instead of comecting texts between

sections, 1 will present an oveMew of thesis format. Chapter 1 is a review of

literature pertinent to the results in Chapter 2. It contains its own reference section

and a section entitled "Rationale for Current Research" which explicitly States the

rationale and objectives of the study. Chapter 2 is the long version of a paper that

was subrnitted for publication. Some parts of the original paper were kept in the

thesis, which were removed from the final version of the paper. It contains its own

Summary, Introduction, Materials and Methods, Results, Discussion, References and

Figures. Chapter 3 presents the general conclusions, an overall discussion of the

results in Chapter 2 and relates them to the literature. Future directions for the

project are also discussed in this section. Each section contains its own references

which are listed alphabetically and are not compiled anywhere in this thesis.

Contributions of Authors

1 am the f is t author of the included manuscript. I have done al1 of the

expenments and contributed to designing the project. 1. Nicholas Crispe is a

collaborator who provided me with the p21 cip-llwaf-1 and p27Lp1 knockout mice, and

invited me to perform experirnents in his laboratory at Yale University. Trevor

Owens is my supervisor.

viii

CKAPTER 1

Mammals have developed an immune system to protect themselves against

invading pathogens. This immune system consists of immune cells or leukocytes

falling into two categories: monocytes and lymphocytes . Lymphocytes include T

lymphocytes and B lymphocytes. B lymphocytes are ùnmune cells that produce

antibodies (Ab), the key players of the humoral immune system. Naïve B cells have

very shon half-lives. Contact wîth immunocompetent T cells is necessary for B ce11

proliferation and differentiation into Ab producing cells. Proliferation increases the

number of B ce11 clones specific for the pathogen. B cells progress fiom

proliferation and to differentiation and produce antibodies(Noel1e et al., 1983).

Some of the B cells become memory B cells that are capable of rapidly mounting a

specific response to this pathogen several years later. Once the pathogen bas been

removed, the immune reaction must be brought under control. This regulation of the

immune response involves inhibition of ce11 cycle progression and induction of

apoptosis. In this thesis, 1 will discuss how B cells become activated, how the

immune reaction is regulated through ce11 cycle regulation, and how the ce11 cycle

regulatory protein p21 is involved in the regulation of T dependent B ce11

proliferation.

A. T Dependent B Ce11 Activation

Naïve B cells leave the bone marrow and circulate in the penphery where

they encounter antigen. B cells that express surface immunoglobulin specific for

antigen, intemalize the antigen, process it and present it in their major

histocompatibility complex (MHC) class LT (Chesnut and Grey, 1981; Rock et al.,

1984; Chesnut and Grey. 1986; Gosselin et al., 1988; Lanzavecchia, 1985). This

signal primes the B cells to receive a second signal through CD40. The cuculating B

cells migrate in and out of secondary lymphoid organs such as lymph nodes, spleen,

1

tonsils and Peyer's patches. Then antigen presenting B cells migrate to the border

between the B ce11 follicle and the T ce11 nch paracortical region of the lymphoid

organ, where they interact with activated T cells (Jacob et al., 1991; Liu et a1.,1991;

Cyster and Goodnow, 1995; Garside et al., 1998).

Early studies showed that antigen alone or in combination with lymphokines,

soluble mediaton produced by lymphocytes, was not sufficient to induce resting B

ce11 proliferation and differentiation and that an antigen specific, contact dependent

collaboration between B cells and T cells was necessary (Hunig and Schimpl, 1979;

Marrack and Kappler, 1980, reviewed by Parker 1990). It was also found that the

contact dependent T-B interaction was MHC restricted (Jones and Janeway, 1981;

RatclSe and Julius 1982; Julius et al., 1982). That meant that some activated CD4'

T cells express a T ce11 receptor (TcR) that recognires the antigenic peptide

associated with MHC molecule on B cells. These activated T cells also migrate to

the edge of the follicle (Garside et al., 1998). C D ~ ' T cells recognize antigenic

peptides in MHC II molecules, while CD~' T celis recognize peptides in MHC 1

molecules.

For resting B cells to become activated to proliferate and differentiate, they

must interact with activated T cells (Whalen et al., 1988; Hirohata et al., 1988;

Owens, 1988). Direct evidence that T cells had to be activated pnor to B ce11

activation, was shown when plasma membranes from activated, but not resting T

cells, could induce B ce11 activation (Brian 1988; Hodgkin et al., 1990; Noelle et al.,

1991). Activated C D ~ ' T cells express CD154 (gp39 or CD40L), the ligand for

CD40, on their nirface (Annitage et al., 1992; Noelle et al., 1992; Lederman et al.,

1992; Lane et al., 1992). Crosslinking CD40 on B cells stimulates proliferation

(Clark and Ledbetter, 1 986; Clark and Lane, 199 1 ; Banchereau et al., 199 l), inhibits

apoptosis of germinal center (GC) B cells @lacLeman et al., 1992; Foy et al., 1994)

and promotes immunoglobulin class switching (Jabara et al., 1990; Lane et al.,

1992). When CD40/CD154 interactions were blocked in vitro with soluble CD40 or

mAbs, B ce11 proliferation and antibody production were abrogated (Armitage et al.,

1992; Hollenbaugh et al., 1992; Noelle et al., 1992; Spriggs et al., 1992). The Th3

contact, dong with cytokines produced by the T cells, induces the activated B cells

to migrate into B ce11 follicles (Gray 1988; Vonderheide and Hunt, 1990;

MacLennan et al., 1990), proliferate and form germinal centers (GC)(Garside et al.,

1998).

The initial step of GC formation is rapid B celi proliferation giving rise to the

dark zone of the GC (Hanna, 1964). The maturing B cells then migrate to the basal

light zone where they encounter T cells and follicular dendritic cells (Hanna, 1964).

The B cells then undergo somatic hypermutation in the variable region of the

immunoglobulin gene (Griffiths et al., 1984; Siekevitz et al., 1987; Jacob et al.,

199 1 ; Jacob and Kelsoe, 1992; reviewed by MacLennan, 1994). This produces B ce11

receptors (BcRs) that may have different affinities or even specificities from the

original BcR. B cells that coatain high afEnity receptors are positively selected

(MacLennan and Gray, 1986; Liu et al., 1989) and undergo isotype switchhg

(Siekevitz et al., 1987). These B cells can then mature into antibody producing

plasma cells (reviewed by MacLennan, 1994) or memory B cells (reviewed by Gray,

1993).

Although crosslinking mAbs to CD40 and soluble CD40L can induce B ce11

proliferation and survival without antigen in some expenmental systems, at

physiological CD154 concentrations, signal 1 through the BcR is also necessary.

Roy et ai. (1993) showed that activated CD4' T ceils from murine lymph nodes were

unable to activate resting B cells. These T cells were found to express 20 fold less

CD1 54 on their surface than the anti-TcRCD3-activated T ce11 clones used in most

experirnents (Roy et al., 1993). Therefure, CD40 engagement by CD154 is not

enough to fùlly activate resting B cells. Experiments f?om o u laboratory showed

that sIgM signaiing decreases the CD40 threshold necessary for B ce11 stimulation in

vin0 when C D ~ O L ' ' ~ T cells were used (Poudrier and Owens, 1994a). Both

signahg through CD40 on B cells or CD28 on T cells has been shown to be more

efficient afler signaiing through the BcR or TcK respectively (Clark and Lane, 199 1;

June et al., 1990). This ensures that interactions are specific.

T dependent B cell activation is a multimeric interaction. Many B ce11

ligands have counterligands on the T cell surface. The cellular membrane is fluid

and allows molecules to move on the ce11 surface. Along with the CD154KD40

interaction, another well-understood interaction takes place between CD28 on the T

cell and CD80 or CD86 (formerly known as B7.1 and B7.2) on the antigen

presenting cells (APC). Professional APCs that c m induce prirnary T ce11 responses

include activated B cells, bone marrow denved dendntic cells (Metlay et ai., 1989)

or activated macrophages (laneway, 1989; Anima et al., 1993; Freeman et al., 1993).

CD80 and CD86 are not expressed on resting B celis, so resting B cells cannot

function as professional APC (Jenkins et al., 1990). This signal through CD28

stimulates T ce11 proliferation and cytokine production (Lindsey et al., 1991). It also

induces CD154 expression on T cells (Klaus et al., 1994). Blocking this CD28-

CD80 interaction inhibits T ce11 proliferation and B ce11 maturation (Koulova et al.,

1991; Damle et al., 1991; Lindsey et al., 1992; Lindsey et al., 1991). CD80

expression is induced by crosslinking MHC II (Koulova et al., 1991, Poudrier and

Owens, 1994b) or CD40 on B cells (Ranheim and Kipps 1993). Therefore,

interactions that crosslink MHC II or CD40 on B cells, activate the B cells, so that

they can become efficient APCs.

Many ligand counter-ligand pairs are involved in B/T interactions. We have

discussed CD40KD154 and CD80KD28, but there are others. As an example, 1 will

discuss ICAM-ILFA-1 (CD54/ CD1 la,CDl8) and MHC IUCD4 that have been

involved in contact dependent activation and will be a focus of my experiments.

There is evidence suggesting that ICAM-ILFA-1 interactions play a role in

T dependent B ce11 activation. First, LFA-1 expression is rapidly induced on the T

ce11 surface after TcR engagement ous t in and Springer 1989) and its afinity

increases with T ce11 activation (reviewed by Hogg et al., 1993). Second, during

antigen specific T ce11 activation, an LFA-l/ICAM-1-dependent signal is transmitted

to the B ce11 (Lane et al., 1991). Third, CD40 crosslinking on the B ceIl causes

allogeneic T ce11 proliferation through ICAM- 1 /LF A- l -de pendent signals (B arrett et

al., 199 1). Fourth, anti-LFA-1 antibodies block antigen recognition and T ce11

activation in cognate or bystander interactions (Whalen et al., 1988). In non-cognate

interactions, anti-ICAM-1 or LFA-1 antibodies have no effect on B ce11 responses to

activated T cell membranes (NoelIe et al., 1992; Gascan et al., 1992). Moreover, in

CD3 activated T ce11 interactions, the ICAM-l/LFA-I interaction seems to enhance

T ce11 help (Tohma and Lipsky, 1991; Lohoff et al., 1992; Owens 1991; Tohma et

al., 1991). Furthermore, in a T helper 1 (Th1)-dependent helper system, ICAM-

ILFA-1 interactions were shown to induce expression of MHC II and CD80 on B

cells and contributed to immunoglobulin secretion (Poudrier and Owens, 1994b).

ICAM-1 was also shown to induce IL-2Ra expression when CO-crosslinked with

MHC II (Poudrier and Owens, 1994b). Finally, CO-ligation of ICAM-1 and sIgM

inhibits IgM-mediated calcium mobilization (van Horssen et al., 1995). ICAM-1

signaling in B lymphomas involves pS3/561yn, Raf-l and MAPWERK2 (Holland

and Owens, 1997)

MHC II also signals in B cells. Anti-MHC II mAbs have been shown to have

stimulatory and inhibitory effects (St-Pierre et al., 1989; Cambier and Lehman, 1989;

Lane et al., 1990; St-Pierre and Watts, 1991; Bishop, 1991; Carnbier et al., 1991).

Anti-MHC II induces proliferation when used in combination with suboptimal anti-

Ig or anti-Ig and IL4 (Cambier et al., 1991; Baluyut and Subbarao 1988). In

contrast, MHC II crosslinking on resting B cells induces apoptosis through a cyclic

AMP dependent pathway (Newell et al., 1993). This may reflect signaling through a

TcR independent pathway. CD4 can bind MHC II irrespective of the antigenic

peptide within the groove (Doyle and Strorninger 1987). In addition, LAG-3, a

molecule closely related to CD4, can also bind MHC II (Baixeras et al., 1992). Mice

deficient for both LA and 1-E molecules have been used to show that MHC Iï is not

necessary for immunoglobulin secretion and isotype switching in response to Th1 or

Th2 cells activated with anti-CD3 (Markowitz et al., 1993) and that the rapid calcium

and inositol phospholipid response to activated T ce11 clones is not afEcted (Lane et

al., 1991).

Therefore, ail of these signals are involved in the induction of proliferation by

the B ce11 and differentiation into Ab producing cells or into memory B cell.

However, when the pathogen is removed, the immune reaction is limited. This

entails rapid deletion of unwanted cells and inhibition of proliferation.

B. CeU Cycle

Every living ce11 divides at some point (for review see Aiberts et ai.1994).

There are five phases to the ce11 division cycle: Gi, S , G2, M and GO. Both Gl and GZ

are growth phases where cells double in mass before dividing again. In Gl or gap 1

cells have diploid (ZN) DNA Gi follows mitosis and precedes the DNA synthesis

phase. DNA repair also occurs in this phase to prevent propagation of mutations

dhng DNA replication. EDNA is too damaged to be replicated the ce11 undergoes

apoptosis or programmed ce11 death. When conditions are appropriate, cells enter

the DNA synthesis phase or S phase where the DNA is replicated. This phase is

followed by another gap phase or G2, see above. Gi, S and G2 make up what is

known as the interphase. In G2, the cells also verify that the DNA was correctly

replicated before entering mitosis or M phase. Mitosis is the process in which one

ce11 divides into two identical daughter cells. Cells in Gl can enter a specialized

resting state called Go, where they remain for days to years before resuming

proliferation. This is the phase where most B cells are found.

The celi cycle is controlled by a braking system that stops the ce11 at specific

checkpoints. These prevent tnggering the next downstream process before the

previous one has finished. Cyclins and cyclin dependent kinases (CDK) regulate

passage through these ce11 cycle checkpoints. Cyclins are proteins whose expression

cycles according to the phases of the ce11 cycle. There are two main classes of

cyclins: mitotic cyclins and Gl cyclins. Both of these bind CDKs, activate the CDKs

phosphorylat ing capacity, and regulate their specificity . Activated cyclin/CDK

complexes phosphorylate downstream proteins on serine or threonine residues.

The fission yeast, Schizomcc~omyces plombe, was the 6 r s t organism in

which the ce11 division cycle was studied. In it, a 34 kDa kinase called cdc2 was

discovered (Nurse et al., 1976) that was responsible for crossing the Gi checkpoint

(start) and the Gt/M checkpoint (initiation of mitosis) by biading to different cyclins

(Nurse and Thuriaux, 1980; Nurse and Bissett, 1981). In G2, it associated with the

mitotic cyclin to fom the maturation promoting factor W F ) , while in the Gi it

associated with the Gi cyclins, CLNl and CLN2 (reviewed by Morgan, 1995). The

specificity of cdc2 is therefore dependent on the cyclin. Homologous proteins were

also discovered in Saccharomyces cerevisioe (cdc28) (Nasmyth and Reed, 1980), in

the vertebrate Xenopus oocytes @34"c2)(~a~tier et al., 1988), and in mammals

(CDKl)(Lee and Nurse, 1987). These can be used interchangeably. For instance,

mutant yeast lacking their own hnctional cdc28 gene can be rescued by transfection

of a mammalian one (Lew et al, 199 1; Elledge and Spottswood, 199 1).

Mammalian Ce11 Cycle

In the mammalian system, multiple CDKs are necessary to enable a ce11 to

progress through ce11 cycle: CDK1, CDK.2, CDK3, CDK4, CDKS, CDK6 and

CDK7. Similarly, so far six cyclin families have been discovered named cyclin A,

B, C, Dl-3, E and F. As in yeast, in proliferating marnmalian cells, the concentration

of CDKs remains constant throughout the ce11 cycle and it is the concentrations of

cyclins that vary.

Mammalian cyclins C, D, and E are normally expressed in GI (Sherr and

Roberts 1995). Cyclin C levels are generally constant throughout the ceIl cycle and

only slightly increase in early Gi. Cyclin E, however, peaks at the Gi/S transition

(Lew et al., 199 1; Koff et ai., 199 1). D type cyclins are quickly degraded when

growth factors are withdrawn and appear first d e r readdition of growth factors, but

generally do not oscillate much during the cycle (Matsushime et al., 1991). They

seern to act as growth factor sensors. in rnid-Gi, cyclin D-CDK4 and cyclin D-

CDK6 complexes phosphorylate the retinoblastoma protein @RB) and related family

members which release members of the E2F family of transcription factors thereby

allowing transcription of genes required for S phase entq and progression (Jacks and

Weinberg, 1998; Bates et al., 1994). CDK2 is essential for GIIS transition in

rnammalian cells and becomes active by binding cyclin E dunng late GI early S

phase (Koff et al., 1992; Dulic et al, 1992;).

Ln rnammalian cells, the p34*" also known as CDKl is present throughout

the ce11 cycle but its catalytic activity is confined to the M phase (reviewed by Sherr,

1993.). Its activity is regulated by phosphorylation and binding to cyclin B. Cyclin

B is synthesized during the S phase and as it accumulates, binds to phosphorylated

CDK 1 (S herr, 1993). This phosp horylation on threonine- 16 1, perforrned by CDK-

activating kinase (CAK) is necessary for the kinase activity and may stabilize

CDKl's interaction with cyclin B @ucomrnun et al., 1991; Desai et al., 1995). As

the cells approach the G&l transition, the complex is maintained inactive by

phosphorylation on threonine- 14 and tyrosine- 1 5 by the kinase Weel (Parker and

Piwnica-Worms, 1992, McGowan and Russell, 1993), within the ATP-binding site.

These two phosphates are then removed by CDC25, and the ce11 enters mitosis

(reviewed by Morgan, 1995). Cyclin B is abruptly degraded during the anaphase of

mitosis through a ubiquitin-mediated process, thereby releasing the inactive CDKl

monomer (Draetta et al., 1989; reviewed by Sherr, 1993). Cyclin A is synthesized

near the Gi/S transition but is not essential for the initiation of S phase (Fang and

Newport, 1991) however, it is necessary for chromosomal DNA replication (Girard

et al., 199 1 ; Pagano et al., 1992; Zindy et al., 1992).

Ln multicelIular organisms, differentiated cetls are not growing and dividing

but instead are in a resting state called Go. To grow and divide, cells must receive

proliferative signals from their environment, such as growth factors, that ovemde the

intracellular negative signals. When cells are in Go, their concentrations of CDK and

all of the Gi cyclins are depleted. Therefore, quiescent cells do not simply stop their

proliferating machinery; rhey also disrnantle their cell-cycle control system. This

ailows for very tight regulation of entry into ceIl cycle.

C. Cyclin Dependent Kinase Inhibitors

Cyclin dependent kinase inhibitors (CKls) inhibit ce11 cycle progression or

induce cells to exit the active proliferative cycle and enter Go (Jacks and Weinberg, INK 4b 1998). There are two families of mammalian CKIs: the Mc4 family (pl5 ,

1 6mx4a, p 18" 1 9"K4d) and the Kip 1 family (p2 1°p-"dl, P271rip-1, and p57 l " ~ ~ )

(Sherr and Roberts, 1995). The Mc4 family of CKIs have a restricted CDK

specificity and preferentially associate with CDK4- and CDK6-cyclin complexes

(reviewed by Morgan, 1995 ; Gartel et al., 1996). They finction by competing with

cyclins for binding to CDKs (Ragione et al., 1996). In vivo experiments show that

p16'"L4 recognizes CDK4 monomer and blocks cyclin binding (Serrano et al., 1993).

On the other hand, the Kipl CKIs have a broad specificity but interact mostly with

the Gr-associated CDK2- and CDK4-cyclin complexes (reviewed by Morgan, 1995;

Gartel and Tyner, 1998). They inhibit ceIl cycle progression by binding to Thr

160/161-phosphorylated cyclinlCDK complexes and inhibiting their kinase activity

(Morgan 1995). Kipl family mernbers are phosphorylated by CDKs suggesting

attachent near the substrate binding site (Zhang et ai., 1994).

p21 was the first CKI to be identified. It was cloned by five different groups

and is variously referred to as CIP-1 (CDK interacting protein)(Harper et al., 1993;

Xiong et al., 1993), WAF-1 (Wild-type p53 activated factor) (El-Deiry et al., 1993),

SDI 1 (senescent ceil-derived inhibitor)(Noda et al., 1994), and MDA-6 (melanoma

differentiation associated)(Jiang et al., 1994).

9

p21 plays multiple roles. It functions as both a cell cycle activator and as a

CDK inhibitor. Basal levels of p21 are always present in actively proliferating

human (Zhang et al., 1994) and mouse fibroblasts (Gu et al., 1993), suggesting that it

might play a role in proliferation. In 1994, p21*-1m1 was found to fùnction as an

assembly factor for binding of D-type cyclins to CDK4 early in the ceii cycle (Zhang

et al., 1994; Kato et al., 1994; Matsushime et al., 1994; LaBaer et al., 1997; Hiyama

et al., 1997). In vitro experiments show that, in the absence of p21, assembly of

cyclin D/CDK4 is so ineficient that most complexes remain dissociated (LaBaer et

al., 1997). This cyclin DKDM interaction is necessary for progression nom Gi to S

phase (Jacks and Weinberg, 1998) and so p21 acts as a ce11 cycle potentiator.

Moreover, p21 cip- l/waf- 1 contains a nuclear localization sequence on its C terminus,

which allows the cyclin D/CDK4 complex to be imported into the nucleus where

CDK4 is phosphorylated by CAK and becomes maximally activated (LaBaer et al.,

1997; Diehl and Sherr 1997). In addition, p21 can displace preassociated p27 fiom

CDK2 complexes, thereby activating the complex (Mantel et al., 1996). There is

evidence that at least two p21 molecules must bind one cyclidCDK2 cornplex to

inactivate it (Zhang et al. 1994; Harper et al., 1995). At higher stoichiometries,

~2 l"P-l/d-L inhibits CDK activity either by binding to substrate recognition motifs on

CDKs (Adams et al., 1996), by rendering CDKs inaccessible to phosphorylation by

CAK (Aprelikova et al., 1995) or by preventing the dephosphorylation of CDKs by

cdc25 (Saha et al., 1997). p21"'/d1 effectively inhibits CDK2, CDK.3, CDK4 and

CDK6, those that have a direct role in Gl/S transition, but does not inhibit CDKI,

CDKS and CDK7 (Harper et al., 1995). The conformational changes that occur

when cyclins bind CDKs enhance p21 cip-l/wif-1 , s ability to bind to the complex

(Harper et al., 1995). Therefore, at low stoichiometry p21 is necessary for CDK

activation, while at high concentrations it is an inhibitor.

p21 and PCNA

In no& human fibroblasts, p21 is part of a quaternary complex that

contains a cyclin, a CDK and a proliferating ce11 nuclear anîigen (PCNA) (Zhang et

al., 1993). We have seen that p21 inhibits GiIS transition through interactions with

CDKs (Harper et al., 1995), but it can also inhibit proliferation by binding to and

inhibiting PCNA PCNA is a processivity factor for DNA polymerase S @olS). It

assembles into a trïmenc ring around the DNA and allows the DNA polymerase 6 to

remain associated to the DNA throughout replication (reviewed by Kelman 1997). It

plays a crucial rote in nucleic acid metabolism, DNA replication and DNA excision

repair. When p21 is associated with the complex in excess stoichiometry, it directly

cornpetes with the pol6 core for binding to PCNA (Podust et ai., 1995), prevents

interactions between po16 and PCNA (Gulbis et ai., 1996) and interferes with DNA

replication (Flores-Rozas et al., 1994; Li et al., 1994; Waga et al., 1994). p21 does

not inhibit synthesis of the short DNA fragments necessary for repair, but it does

prevent the transcription of long DNA templates (Flores-Rozas et al., 1994; Li et al.,

1994; Waga et al., 1994; Podust et al., 1995), as the DNA polymerase falls off of the

template due to lack of association with PCNA. p21 was found to inhibit repair of

DNA damaged by UV irradiation, alkylating agents and mismatched base pairs

through its PCNA binding (Pan et al., 1995; Umar et al., 1996).

Signaling Upstream of p21

Several cellular receptors can induce p2 1 expression: NGF, TNFa, TGF-P,

IFNa, IFNP and IFNy as well as growth factors such as PDGF, FGF and EGF

(reviewed by Gartel and Tyner, 1998). Ligand binding to ce11 surface receptors often

activates the JAK-STAT pathway (reviewed by Ihle 1996). There are three STAT

transcription factor binding sites in the p21 promoter (Chin et al., 1996; Matsumura

et al., 1997). When these STAT dimers bind to the p21 promoter, p21 expression is

induced and replication is inhibited (Chin et al., 1996; Matsumura et al., 1997). The

p21 promoter also contains a functional vitamin D3-responsive element (Liu et al.,

1996b), and a fùnctional retinoic acid-responsive element (Liu et ai., 1996a).

PZ laP""""l can be transcriptionally regulated in a p53-dependent and - independent marner. Its transcription is induced by p53 in response to DNA damage

(El-Deiry et al., 1994; Dulic et al., 1994), ionking radiation (Macleod et al., 1995)

and senescence (Noda et ai., 1994). p21 induction during development in most

tissues, except the spleen, is p53-independent (Parker et al., 1995; Macleod et al.,

1996).

Signaling Downstream of p2l

p2 1 has been associated with MAPK pathway. p2 1 can be activated by Ras

through Raf and p53 leading to growth arrest. Nene Growth Factor (NGF) inhibits

growth of NIH-3T3 cells through upregulation of p21 (Decker et ai., 1995). When

these cells are treated with the MAPK kinase (MEK)/MAPK pathway inhibitor

PD98059, growth inhibition is reversed (Pumiglia and Decker 1997). These data

suggest that CD& are negatively regulated by the MEWMAPK pathway through

PZ l"L'w.'-L mediated cell cycle arrest (Pumiglia and Decker 1997). Furthemore,

p21 binds to and inhibits c-jun amino terminal kinases ( M s ) aiso calied stress-

activated protein kinases ( S m ) , in vivo and in vi~o (Shim et al., 1996). MK is the

enzyme that phosphorylates c-jun and this leads to the activation of many immediate

early gene promoters that contain AP-1 sites.

p2l and Apoptosis

Apoptosis and mitosis resemble each other in that they have similar

cytoskeletal changes, nuclear envelope breakdown and chromatin condensation

(Heald and McKeon 1990). It has been suggested that apoptosis results fkom

"catastrophic mitosisy' or aberrant ce11 cycling (Shi et al., 1994; Evans et al., 1995;

Hakem et al., 1999). Catastrophic mitosis refers to irreparable mistakes occuning at

some stage of the ce11 cycle inducing the ce11 to "commit suicide". For instance, if

CDKl is activated at an inappropriate time during the celi cycle, apoptosis will be

induced (Shi et ai., 1994).

There is some evidence that p21 cip i / 4 - 1 is involved in apoptosis induction.

p2 1 is important for BcR mediated apoptosis of WEHI 23 1 B lymphoma cells (Wu et

al., 1998). Ectopic p21 expression in MCF-7 breast carcinoma cells induced

apoptosis and levels of p21 cip~twaf-l increased during apoptosis of RT4 human

bladder tumor ce11 lines (Sheikh et al., 1995; Sheikh et al., 1996; Chresta et al.,

1996). Okadaic acid (OA) induced apoptosis of MCF-7 cells was associated with

overexpression of p21 (Sheikh et al., 1996) while nitrïc oxide (NO) induced

apoptosis of h u m cancer cells increased both p21 and p53 (Ho et al., 1996).

Similarly, pRb 4- mouse embryos undergo extensive apoptosis of the central nervous

system, and this apoptosis was accompanied by an increase in p21 and p53

expression (Macleod et al., 1996). mV-1-transfomed lymphocytes induced to

apoptose using the genotoxic h g adriarnycin, increased p21 expression in a p53-

dependent and -independent manner (Gartenbaus et al., 1996). p53-independent

expression of p21 in osteosarcorna cells led to expression of the Wilms tumor

suppressor gene WT1 and apoptosis (Englert et al., 1997). In serum-deprived 3T3

fibroblasts, apoptosis is accompanied by increased p21 expression and introduction

of antisense p21 RNA delays apoptosis (Duttaroy et ai., 1997).

There is also some evidence that p21 protects cells from apoptosis. p21

protects Bac3 hematopoietic cells fiom apoptosis caused by radiation in the absence

of IL-3 by inducing G1 arrest (Canman et al., 1995). Transient overexpression of

p21 prevented apoptosis of C2C12 myoblasts during differentiation of wild-type

(Wang and Walsh, 1996) but not pRb-/- myoblasts (Wang et al., 1997). Low levels

of p21 expression were associated with apoptosis while higher levels induced Gi

arrest in response to cyclopentenone prostaglandin A2 (PGAz), phenyacetate or p53

overexpression, in RKO human colorectal carcinoma cells (Gorospe et al., l996b), in

NIH-3T3 cells m t o m i et al., l996), in MCF-7 cells (Gorospe et al., 1996a) or in

colorectal cancer ce11 lines (Polyak et al., 1996). Therefore, it seems as though the

role of p21 in apoptosis depends on many factors including ce11 type, growth

conditions and expression of other genes.

D. Rationale for the Current Project

T dependent B ce11 activation induces B cells to enter and progress through

the cell cycle. B cells suMve and proliferate in response to CD40 ligation. The Th3

cellular interactions involve signals fiom multiple ligand pairs. Some of these CO-

signals are stimulatory while others are inhibitory. Negative signais are of interest

because, afier elimination of pathogens, B ce11 proliferation must stop and the lack of

such control can lead to autoimrnunity or cancer. The signaling cascades involved in

limiting immune responses are not fully understood. However, regulation of

proliferation must involve ce11 cycle inhibition. p2 1 a p - l ~ ~ a f - 1 is a weil established ce11

cycle inhibitor as well as a potentiator and inducer of apoptosis. To better

understand the regulation of T dependent signaling in p n m q B cells, 1 chose to

study the role of p2 1 "P-"*-' in modulation of CD40 induced proliferative activation

of % cells.

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CHNTER 2

A. Titie Page

~ 2 l ~ ~ ~ ~ * ~ mediates apoptosis induced by ICAM-1 and MHC in CD409 stimulated B cells

Iris S. ~oyle ' , 1. Nicholas crispe2, Trevor Owensl.

1 Department of Microbiology and Immunology, McGill University, Montreal, Qc; 2 School of Medicine, Immunobiology Section, Yale University, New Haven, CT

AaWess for corre~pondence :

Trevor Owens

Neuroimrnunology Unit

Montreai Neurological Institute

3 80 1 University Street

Montreai, Quebec

H3A 2B4.

Fax: (5 14)398-7371

E-mail: trevor@med. mcgill. ca

The cyclin dependent kinase (CDK) inhibitor (CKI) p21 ap-116- i is implicated

in control of proliferation and ce11 cycle arrest. Here, we show that p21 cip-l/waf-1

expression was upregulated in CD40 stimulation of murine splenic B cells, and

supennduced by CO-crosslinking intercellular adhesion molecule 1 (ICAM-

1)/CD54, MHC 1 or MHC II with CD40. Supennduction of p21*44-'

correlated with inhibition of CD40-induced ceIl cycle progression and

induction of apoptosis. Proliferation of Lipopolysaccharide (US)- or B ce11

receptor @CR)-induced B cells was not inhibited by these treatments, and

mAb-induced association of CD40 with other B ce11 surface molecules did not

have these effects. Apoptosis was abrogated in p21 ap-114-1 ,/, B cells, which

otherwise responded to anti-CD40 equivalent 1 y to controls. B y contrast, B

cells lacking expression of the CKI p27Ep-1 showed the same induction of ce11

death in response to CD40 crosslinking with ICAM-1, MHC 1 or MHC 11, as

wild type mice. These results show that MHC I and II and the adhesion

molecule ICAM-1 specifically modulate CWO-mediated proliferation by

induction of apoptosis, which is controlled by the CKI p21 cip- L/waf- 1 . This

demonstrates the importance of Gl/S checkpoint mechanisms in integration of

signals for B lymphocyte sumival and proliferation.

C. Introduction

Induction of B lymphocyte entry to ce11 cycle is fiuidamental to the humoral

immune response. Clonal expansion and isotype switching both require proliferation

of antigen-specific B cells. The major proliferative stimulus for B ce11 activation is

CD40, Ligated by CD40LKD154 on activated CD4' helper T lymphocytes,

themselves directed to engage with antigen-presenting B lymphocytes via the

TCR/CD4/MHCII + peptide multirnolecular interaction (Parker, 1993; Noelle 1996;

Kehry 1996). Other molecular interactions occur during this interceilular contact,

and although some of these may be purely adhesive, it is likely that many of them

signal (Owens, 1991; Clark and Ledbetter, 1994; Holland and Owens, 1997). Co-

stimuIatory or counter-stimulatory interactions are likely to contribute to the eventual

outcorne. Negative signals are necessary for limiting immune reactions and

maintenance of self4olerance. It is important to understand those signals for fil1

understanding of the B ce11 response.

Entry and progression through the ce11 cycle is regulated by cyclin/CDK

complexes and molecules that govern their activity. CDKs associated with cyclins

can phosphorylate retinoblastoma protein @RB), or related moleniles, that then

release transcription factors essential for progression into S phase (reviewed by Jacks

and Weinberg, 1998). This association is itself regulated by CDK inhibitors (CKI)

(reviewed by Morgan, 1995). CKIs bind to cyclin/CDK complexes and directly

inhibit their kinase activity (Morgan, 1995).

The CKI p2 1"~'~'~' has multiple functions. The association of one p21~p- 114-1 molecule with a cyclin/CDK/PCNA (proliferating ce11 nuclear antigen)

cornplex activates ce11 cycle progression. It facilitates CDK4 association with D-

type cyclins (Zhang et al., 1994; Kato et al., 1994; Matsushime et al., 1994; LaBaer

et al., 1997), a necessary step for transition fiom Gi to S phase. It also allows the

complex to translocate into the nucleus where the CDK is activated through

phosphorylation by CDK activating kinase (CAK) (LaBaer et al., 1997; Diehl and

S herr, 1 997). At higher stoichiometry, p2 1 ap- 11wrf-I inhibits Gr progression. It

associates with and inhibits cyclin E-cdkî complexes which are essential for the Gi

to S transition @ulic et al., 1992; Koff et al., 1992; Lees et al., 1992; El-Deiry et ai.,

1 994). 1*"**' also contains a binding domain for the PCNA of DNA

polymerase (Waga et al., 1994). This suggests that it might be able to act directiy as

a DNA synthesis inhibitor.

The negative association of CK?s with ce11 cycle progression fiequently

extends to induction of apoptosis. It has been suggested that apoptosis is the result

of aberrant ce11 cycling called "catastrophic mitosis" (Shi et al., 1994; Evans et al.,

1995; Hakem et al., 1999). Induction of p53 by DNA damage or other ce11 stress

induces p21 cip-llwaf-1 (El-Deiry et al., 1993; Harper et al., 1993; Dulic et al., 1994),

whose binding to and inhibition of cyclidCDK complexes arrests DNA replication

until DNA damage has been repaired (Kuerbitz et ai., 1992; Dulic et al., 1993; Deng

et al., 1995). If DNA damage is too extensive, the ce11 then undergoes apoptosis

(reviewed by Evans et al., 1995). In this way, p21 cip-IIW-1 plays a critical role in cell

cycle arrest and induction of apoptosis. Apoptosis also occurs during normal

development and helps to maintain homeostasis in complex organisms.

The majonty of studies of CKIs and ce11 cycle regulation/apoptosis in

mammalian cells have been carried out in transformed cells. In one of the few CKI

studies in non-transformed lymphocyte activation, Solvason et al. (1996) showed

that p27kp1 expression decreased when low buoyant density B cells were ligated

with anti-CD40. Nourse et al. (1994) implicated p27fipL in growth arrest of human

peripheral blood T lymphocytes, whi le p2 1 cip- 1 1 4 - 1 was associated with IL-2 driven

ce11 cycling. Moreover, MuUins et al. (1998) showed that crosslinking CD40 on

small resting B cells induced an increase in p21 cip-Ilwaf-1 expression, while cycling B

cells needed both BcR and CD4O-mediated signals to maintain increased p21

expression. Both Tetsu et al. (1998) and Solvason et al. (1996) showed that ligation

of BcR decreased p27 expression and upregulated p21 expression - the latter peaked

at 24 hours and then decreased. To further address the role of p21 in B ceU

activation, and to examine how its induction and activity relate to signaiing derived

from contact with activated T helper ceils, we screened mAbs against B ce11 surface

ligands for their effects on p21 levels in murine splenic B cells. Our results show

that MHC I and II, as well as the adhesion ligand ICAM-1 superinduce p21 cip 114-1

when CO-ligated with CD40, and that this signals induction of apoptosis. These

findings speak to the regulation of T-dependent B cell proliferation, in which conte-

they are discussed.

D. Materials and Methods

Monoclonal Antibodies

Hybridomas secreting anti-murine CD40 (FGK-45 rat IgG=) (Rolink et al., 1996),

and CD2 (12.15A rat IgGl) were kindly provided by Dr. P. Hugo (McGill

University) and Dr. Bruno Kyewski GMBL), respectively. Other rat anti-murine

mAbs used were YNU1.7.4 (Ig% anti-ICAM-1), 2362 (IgG2. anti-CD45RB), 53-

7.3 (IgG2, anti-CDS), E4.2 (IgGi anti-p), FD44 1.8 (IgG2b anti-LFA-1), IM78 1 (IgG2b

anti-Pgp- l/CD44), Ml142 (IgG2, anti-MHC 1), P7/7 (IgGZb anti-MHC II), PC61

(IgGl anti-IL-2Ra). MaR18.5 (mouse IgG2, anti-rat K) was used as a cross-linker.

These mAbs were obtained fiom ATCC (Rockville. U.S.A). R52- 120 (rat IgGl)

anti-IL-SR was generated by Devos et al. (1990). mAbs were affinity-purified fiom

culture supernatants using Protein-G-Sepharose columns (Pharmacia, Baie d'Urfe,

Canada).

B Cell Preparation

Spleens from C57BV6 female mice (CharIes River Laboratories, St-Constant,

Canada) were disnipted using a wire mesh. Red blood cells were lysed using 0.83 %

ammonium chloride for 10 minutes at room temperature. T cells were depleted

using 172.4 (rat IgM anti-murine CD4), F7D5 (mouse IgM anti-Thy-1.2) and 53-

6.72 (rat IgGza anti-murine CD8) hybridoma culture supernatants for 20 minutes on

ice and Low-Tox-M Rabbit Cornplement (Cedadane, Homby, Canada) for 40

minutes at 37OC. The remaining ceils were fiactionated by centrifugation on

discontinuous Percoll gradients. High buoyant density B cells (behveen 1.090 and

1.075 gh l ) were cultured at 5 x 105 cells/ml in RPMI 1640 (Flow/ICN Biomedicals,

Mississauga, Canada) supplemented with 10 % FCS (Flow), 2mM L-glutamine

(Gibco, Burlington, Canada), 5 x 1 0-5 M O-mercaptoethanol (Sigma, Oakville,

Canada) and penicillin (100U/ml)-streptomycin (100pg/d) (GibcolBRL,

Burlington, Canada) plus 10 pgh l mAbs or 20 p g h l LPS (Sigma) for 48 hours.

Proll~eration and Cell Cycle Anabsis

Proliferation was evaluated at 48 hours in culture, &er a 6-hr pulse with 2.5 pCi/ml

3~-thymidine (ICN Biornedicals, Mississauga, Canada). Cells were harvested ont0

g l a s tilters and incorporated radioactivity was measured by scintillation counting.

Ce11 cycle analysis was performed by propidium iodide (PI) staining for DNA

content. M e r culture, the cells were washed twice with Dulbecco's Phosphate

Buffered Saline and permeabilized for 2 minutes with ice cold 70Y0 ethanol. The

cells were then washed again and incubated with 3 p g m l PI (Sigma), 50 @ml

RNAse A (Boehringer Mannheim/ Roche Molecular Biochemicals, Laval, Canada)

and 0.625 % FCS (Gibco/BRL) for 5 minutes at 37°C. Staining was assessed using a

FACScan (Becton-Dickinson, Oakville, Canada) and analyzed using Ce11 Quest

Software (Becton-Dickinson).

Annexin V Staining

1 x 106 cells were washed twice with Dulbecco's Phosphate Buffered Saline and

resuspended in 100~1 of calcium binding buffer (1mM Hepes +2.5 mM CaC12). The

cells were then incubated for 15 minutes in the dark with 2 @ of 10 pg/d Annexin

V-FITC (PharMingen, Mississauga, Canada). Staining was assessed using a

FACScan (Becton-Dickinson) and analyzed using Ce11 Quest Software (Becton-

Dickinson).

Surface Staining

5 x 1 O' cells were washed 3 times with Dulbecco's Phosphate BufFered Saiine and

the incubated with 10 pg/d of biotinylated anti-FasL mAb (PhMingen) for 20

minutes on ice. The cells were washed in FACS buffer @-PBS + 2% FCS + 0.1%

azide) and incubated with 1 &ml of PE-avidine (Southern Biotechnology

Associates, Inc., Birmingham, USA) for 20 minutes on ice. The ceils were washed

again and resuspended in FACS buffer. Staining was assessed using a FACScan

(Becton-Dickinson) and analyzed using Ce11 Quest Software (Becton-Dickinson).

Lysis for Western B lots

Cultures were set up at IO' celldml in 5 mi wells (Falcon/Becton Dickinson,

Oakville, Canada) and stimulated with 10 pghl anti-CD40 crosslinked with 10

@ml MaR with or without 10 &ml ad-ICAM-1 or controls for the indicated

times. Cells were then washed 3 times with Hanks Buffered Saline Solution (Gibco)

and 4 times with Dulbecco's Phosphate Buffered Saline + ImM Sodium

Onhovanadate (NaVO4)(ICN Biomedicals, Mississauga, Canada). The cells were

then lysed by incubation in Lysis Buffer (1% NP40 (ICN), 250 mM NaCl, 1 mM

HEPES, pH 7.5 (Gibco/BRL), and 1 mM dithiothreitol (Gibco), with PharMingen

Protease Inhibitors Cocktail added at a 1 X concentration (16 pgfrnl benzarnidine

HCI, 1 Opg/ml phenanthroline, 10pg/ml aprotinin, 1 Opg/mi leupeptin, 1 Opglml

pepstatin A, 1pg/ml PMSF)) for 45 minutes on ice. Cell debris was removed by

centrifugation (15 min at 13000 x g) at 4OC. Protein concentrations were calculated

using Protein Assay Kit (Pierce, Rockford, USA) according to manufacturer's

procedures and lysates were stored at -20 O C .

Western Biotting

85 pg of lysates were separated by 12% SDS-PAGE at 120 volts for 1.5 hours. The

proteins were transferred onto Irnmobiloa polyvinyildene difluoride (IPVDF)

membranes (MiHipore, Bedford, USA) at 100 volts for 1 hour at 4OC using a Mini-

Protean II Transfer Apparatus (Bio-RAD Laboratones, Mississauga, Canada).

Lysates of A20 B lymphoma or irradiated L-ce11 fibroblasts were used as positive

controls. Membranes were dipped in methanoi, le& to dry ovemight and blocked for

3 hours in blocking buffer (5% powdered Milk (Carnation)+ 1% bovine serum

albumin fiaction V (Gibco/BRC) in wash buffer (10 mM Tris-HCI, pH 7.5

(Boehringer Mannheim), 50 mM NaCI, 2.5 mM EDTA, pH 8 .O and 0.1% Tween-20

(B io-RAD)) . Membranes were incubated wit h ant i-p2 1 cip-~vvlf-I (NeoMarkers, Union

City, U.S.A), at 1:100 in wash buffer for 2 hours, washed 4 x 5 minutes and

incubated for 1 hour with goat anti-rabbit Ig conjugated to horseradish peroxidase

(Cedarlane) at 1 : 5000 in blocking buffer. Membranes were washed 4 x 5 minutes

and proteins were detected using ECL (Amersharn Corporation, Oakville, Canada) or

Supersignal West Femto Maximum Sensitivity Substrate (Pierce) according to

manufacturer's protocols. Equivaiency of protein loading was confirmed by

cornparison of background bands and by staining membranes with Ponceau Red.

E. Results

p21c1P*'wuF' expression increases with ICAM-1, Mc 1 and II co-cmss~inùing

with CD40.

Given that lymphocyte ce11 cycle entry is initiated by receptor signaling, one

rnight expect ceil surface receptors to be implicated in regulating p21 activity. An

increase in p21 cip- l/waf- 1 expression was detected after 30 minutes of treatment with

anti-CD40 and continued to increase for 42 hours (Figure la). These results are in

agreement with recently published results fiom Mullins et al. (1998). We were

interested in how B ce11 surface molecules that are implicated in T ce11 help would

fiec. p2 1 +-l/W-l expression in CD40 triggered cells. Figure lb shows that CO-

crosslinking ICAM-I, MHC I or MHC II with CD40 superinduced expression of

~ 2 1 " - " ~ - ~ after 42 hours Figure 1 b). In contrast, cm-crosslinking LFA- 1 or CD44

with CD40 did not increase p2 1 expression (Figure 1 b). Furthermore, crosslinking

ICAM-1, MHC 1 or MHC II alone did not affect p21 cip-l/wrf-1 levels (Figure lc).

Therefore, p2 1 cip- 1 / 4 - 1 is upregulated by anti-CD40 treatment and is superinduced by

CO-crosslinking CD40 with the adhesion molecule ICAM-1, or with MHC 1 or MHC

II. The uicrease in p21 expression is consistent with previously published data that

p21 acts as a celi cycle potentiator at low stoichiometry and an inhibitor at high

stoichiometry .

ICAM-1, MHC I ondA4HC I l spec@caUy inhibit anti-CD40 zndixedprolijerarion.

Superinduction of p21 cip-l/waf-l is suggestive of a shift in CKIKDK

stoichiometry towards ceIl cycle inhibition. We tested the etfed of p21"p-1'M1-

inducing mAbs on CWO-triggered B ceIl proliferation. Whereas some mAbs, from

a panel of 9 tested, enhanced proiiferation (not shown), o d y those that superinduced

~2 lapl'VL''l i.e. ICAM- 1, MHC 1 and MHC II, were inhibitory (Figure 2a). We also

tested whether modulation was specific to CD40 induced proliferation, by testing

effects on B cells stimulated with LPS or Sepharose-coupled anti-p. Neither

stimulus was inhibited by crosslinking ICAM- 1, MHC 1 or MHC 11 (Figures 2b and

Zc). Therefore, ICAM-1, MHC 1 and W C II specificaily inhibit anti-CD40-induced

proliferation.

ICAM-1. MHC I and MHC II inrfuce cpoptosis of CD40-stzmulated B celis.

Untreated mouse splenic B cells spontaneously undergo apoptosis in culture,

while stimulation through CD40 reverses this and induces B ceil s u ~ v a l (Figure

3a). When ICAM-1, MHC 1 or MHC II were CO-crosslinked with CD40, B cell

apoptosis increased to between 32.58% (Figure 3a). This effect was again specific

to p21-inducing mAbs, as anti-LFA-1 (not shown) and anti-CD44, did not induce

this increase in apoptosis. These results were confkmed by PI staining for DNA

content (Figure 3b), which also show the lack of effect of LFA-1, in a compilation of

data from 11 experiments. Although there was inter-experiment variability in

absolute values, the direction of effect was highiy consistent for ICA.-1, MHC II

and the control rnAbs. In our hands, MHC 1 did not induce PI-detectable apoptosis

as reliably as ICAM-1 or MHC II but Wallen-Ohman et al. (1997) have shown it to

specifically induce apoptosis in CD40-stimulated B cells. Representative

experiments are shown in Figure 4a and 4c, where 49-62% of untreated B cells

apoptosed and ody 5% were in S+G2+M d e r 48 hours in culture. CD40

engagement reduced apoptosis to 10- 15 %, and CO-crosslinking CD40 with ICAM- I

reversed this (3 3 -45% apoptosis).

Apoptosis is not mediated by F a

Resting B cells do not express Fas, but signaling through CD40 makes them

susceptible to Fas-killing. Signaling through BcR renders CD40 stimulated B ceils

resistant to Fas mediated apoptosis (Rothstein et al., 1995; Schneider et al., 1999).

However, in our experiments, CO-signaiing through the BcR using anti-p mAbs did

not prevent inhibition of CD40-stimulated ce11 growth by ICAM-1, MHC 1 or MHC

KI (Figure 2d), suggesting that this is not Fas-mediated. Moreover, we could not

detect FasL staining on CD40-stimulated B ceiis, whether or not their growth was

rnodulated by ICAM-1, MHC 1 or MHC II (Figure 5).

p2 1 " ~ " ~ ' ~ ' is required for upoptosis of CD40 stimuluted B cells.

To ask what role p21 cip-llwaf-1 plays in induction of apoptosis in CD40

stimulated B cells, we repeated these expenments using B cells fiom p21 cip-114-1-

deficient mice. Wild-type C575V6 mice were used as controls. We also tested B

cells £kom ~ 2 7 ~ ~ " -/- mice. The CKI p27Ep', has been implicated in apoptosis

induction in T lymphocytes (Nourse et al., 1994) and signaling through CD40 has

been shown to decrease expression of p27 (Solvason et al., 1996). Thus, p27~p1

represented an additional candidate for regulation of CD40 proliferation. p21 cipl/waf-

l-deficient mice are defective in Gl checkpoint so that embryooic fibroblasts nom

these mice are unable to arrea in Gi in response to DNA damage and nucleotide pool

perturbations (Deng et al., 1995). p27'Up1 knockout rnice have increased T ce11 and

hematopoietic progenitor ceil proliferation and female stenlity (Fero et ai., 1996).

These mice were larger than controls, as reported. Nevertheless, gene-targeted mice

were of normal appearance and had spleens of grossly normal size, fiom which were

obtained equivaient numbers of high buoyant density B cells to those fiom C57BV6

mice. Both p21"-1'd-'-deficient and p27fip1-deficient B cells proliferated in

response to anti-CD40 (Figure 6). The high stimulation index (SI) for p27Ep1-/- B

cells is consistent with other aspects of thei phenotype (Fero et al., 1996), but did

not ovemde the pro-apoptotic effects of MHC 1 & II or ICAM-1 (see below).

In ~ 2 l " ~ ~ ' ~ - ~ -deficient B cells, rates of spontaneous apoptosis were in the

same range as wild-type controls (23% to 49%), although at the low end of the range

(Figures 3b and 4b) which may reflect lack of this CKI. Anti-CD40 reduced this

(5% apoptosis) similar to its effect on control B cells (Figure 4b). However,

apoptosis induced by ICAM-1, MHC 1 or MHC II CO-crosslinking with CD40 was

significantly reduced (7- 1 1 %) in p2 1 cip-l/w;if- 1 -deficient B cells compared to wild type

(2 1-45%) (Figure 4b and 4a, respectively). Therefore, p2 1 cip- l/waf-1 is required for

induction of apoptosis by ICAM-1, MHC 1 and MHC II in CD40 sthulated B cells.

It was noteworthy that whereas the percentage of cells with less than diploid (2N)

DNA decreased compared to wild type controls, the percentage of cells with 4N

DNA was not affected. Consistently, the predominant effect of p21-deficiency was

on the percentage of cells in Gi and in apoptosis, suggestive of an effect on Gl-S

transition. Ce11 cycle inhibition-related apoptosis has been associated with Gl-S

block (Dao et al., 1997) although Boehme et al. (1993) showed that propriocidal

apoptosis of T cells occurs in S phase.

The same effects were not seen in p27kP-1-deficient B ceils (Figure 4d).

Unstimulated cells had a rnid-range level of apoptosis (58%) which matched the

percentage of B cells apoptosing in unstimulated wild-type cultures (62%).

Treatrnent with anti-CD40 mAbs reduced the apoptosis of both knockout (21%) and

wild-type (15%) 8 cells, as predicted by proliferation data in Figure 6. However,

unlike in the p21 cip-vwaf-1 -knockout B ceIls, CO-crosslinking ICAM-1, MHC 1 or

MHC II with CD40 did not abrogate the induction of apoptosis, and ICAM-1, MHC

1 or MHC II treated B cells showed exactly the same increase as did wild-type B

cells. This argues against a role for ~ 2 7 ~ ~ ' in regulation of T dependent B ce11

activation through ICAM-1, MHC 1 or MHC II.

F. Discussion

By showing that p21*1'd-1 is upregulated by CD40 stimulation and

supennduced by ICAM-1 and MHC 1 & JI, we provide noveI insight into the bioIogy

of both the CKI and into the proliferative activation of the primary B lymphocytes.

The mAbs that superinduce p21 cip- 114-1 also inhibit proliferation induced by CD40

stimulation and induce apoptosis. Furthemore, in B cells deficient for p2 1 cipl/waf-L 1

ICAM- 1, MHC 1 and MHC II do not induce apoptosis of CD40-stimulated B ceus.

Therefore, p2 1 cip- I/Waf- 1 is Ïnvolved in the induction of apoptosis by ICAM-1, MHC 1

and MHC II on CD40-stirnulated B cells.

~ 2 l " ~ - ' / ~ - ' fiinctions as both a ce11 cycle activator and a CDK inhibitor. This

explains the basal level of p21 cip- llwaf- L found in constitLtively active cells (Gu et al.,

1993; Zhang et al., 1994; and see data for A20 lymphoma in Figure 1). It also

accounts for the upregulation of p2 1 by CD40 signaling in resting B cells (Mullins et

al., 1998; Figure la). At higher stoichiometries, p21 cip-vwaf-1 inhibits ce11 cycle

progression by either binding to substrate recognition motifs on CDKs (Adams et al.,

1996), by rendering CDKs inaccessible to phosphorylation by CAK (Aprelikova et

a.., 1995) or by preventing the dephosphorylation of CDKs by cdc25 (Saha et al.,

1997). Thus, superinduction of p21 by ICAM-1, MHC 1 and MHC II CO-

crosslinking to CD40 is associated with inhibition of proliferation. Therefore, our

results show p 2 1 cip- l/wa£-1 acting both as a ce11 cycle potentiator and as a CDK

inhibitor in primary B cells in response to T-dependent signals. We have also shown

that p2 1 is necessary for induction of apoptosis.

The upstream signaling that induces p2 1 remains poorly characterized. Our

data suggest that ICAM-1, MHC 1 and MKC II may play a role. Biochemical

signaling has been descnbed for al1 three of these molecules. ICAM-1 CO-ligation

with surfâce IgM inhibits surface BcR signaling (van Horssen et al., 1995) and LFA-

IACAM-1 interactions are necessary for Fas-mediated killing of B celis (Wang and

Lenardo, 1997). Crosslinking ICAM-1 induces p53/561yn phosphorylation, as well

as activation of Raf-1 and MAPWERK-2 (Holland and Owens, 1997). Activation of

lyn in B cells is associated with negative signahg (Hoiland and Owens, 1997,

Pedersen et al., 1998) in generai consistency with our findings. In T ceils ICAM-1

engagement induces phosphorylation of CDKl ( ~ 3 4 ' ~ ~ ) thereby inhibithg its kinase

activity (Chirathaworn et al., 1999, another instance where negative signaling is

predicted. MHC II and ICAM-1 crosslinking on B cells upregulates B7 and CD25

(Nabavi et al., 1992; Watts et al., 1993; Poudrier and Owens, 1994) and promotes

CD40 responses in a B lymphoma (Bishop 1995). MHC II signaling induces IL4

beta expression (Al-Daccak et al., 1994), phosphatidyl inositol turnover, protein

tyrosine phosphorylation and proliferation (Lane et al., 1990) and also induces

apoptosis of primary B cells. Newell et al. (1993) showed that the latter involves an

increase in intracellular cyclic AMP. Leveille et al. (1999) showed that MHC II,

CD20 and CD40 are physically associated to one another and that this may

contribute to modulatory effects. MHC 1 has been shown to induce apoptosis in T

lymphocytes (Sambhara and Miller, 1991; Skov et al., 199%). Signaling for

apoptosis through MHC 1 is complex and involves Jak3, PUY ZAP70, p561ck, and

STAT3 depending on the T ce11 type (Skov et al., 1997a; Skov et al., 1997b; Skov et

al., 1998). p56J53lyn and p72syk have also been shown to mediate MHC 1 signal

transduction in B lymphomas (Pedersen et ai., 1998) although this is not

demonstrable in al1 laboratones (Holland and Owens, 1997). This may relate to the

use of human versus murine B lymphomas.

The physiological significance of CD40-associated growth arrest and

apoptosis probably relates to its role in T-dependent help for B cells. Effector

ligands for these molecules are expressed on T cells. ICAM-1 binds LFA-1 on T

cells. Our data open the possibility that this interaction and its associated negative

signal may prevent B ceii proliferation. It is not clear when or under which

circumstances it would be physiologically appropriate for ICAM-1iLFA-1

interactions to inhibit B ceil proliferation. One possibility is that the higher affinity

interactions with LFA-1 on activated T ceils (Dusth and Spnnger, 1989) prornotes

distinct signaling and that inhibition of proliferation may be associated with

ditférentiative events involved in isotype switching. It remains to be detemined

whether this is the case, and whether the mAbs used in our experirnents rnimic high

or low &nity interactions. ICAM-1 can also bind to LFA-1 on other cells including

other B cells or epithelial cells. Homotypic interactions between B cells are

implicated in promotion of B ce11 growth (Mourad et al., 1990). The lack of effect of

anti-LFA-1 mAbs in our experiments argues against blocking homotypic LFA-

l/ICAM-1 interactions as a mechanism for inhibition and suggests that ICAM-I

itself signals growth arrest.

Ligation of MHC IT, with or without CD40 activation, induces B ce11

apoptosis. The apoptosis induced by M X class II Ligation alone might correspond

to suppression of B ce11 response to binding of MHC II to CD4 on resting T celis.

These B cells would be deleted in order to avoid inappropriate activation. The

enhanced apoptotic effect of CD40 and MHC II CO-ligation, that we have shown,

might correspond to a stronger negative influence, that suppresses bystander, non-

cognate, activation of B cells by activated, CD~OL' T ceils during an immune

response against an irrelevant antigen. In the absence of antigenic peptide, CD4

becomes a major ligand for MHC II. Whether the apoptosis that we describe reflects

CD40-potentiated MHC II killing as descnbed for lymphomas by Leveille et al.

(1999), or whether it reflects more wmplex CO-signaling by MHC II and CD40, our

resubs demonstrate its dependence on p21 cip- I {waf- 1 for effect. Since CD40 ligation,

but not MHC II ligation, upregulates p21 tip-l/waf-1 , MHC II signals alone cannot

prornote p21 tip- uwaf- I -dependent de& The same p21 cip- llwaf- 1 dependence was seen

for MHC 1- and ICAM-1-induced apoptosis, also specific to CD40-stimulated B

cells- We do not find either of these to be directly apoptotic for B cells.

MHC I CO-crosslinking to CD40 on B cells can be induced by a variety of

counter-ligands. The principal ligand for MHC 1 is CD8 but, KIRS on T cells and

Natural KiIler (NEC) cells, and the BYSS ligand expressed on human NK ceils, a

subset of circulating CD8' T lymphocytes and al1 intestinal intraepithelial T cells

(Agrawal et al., 1999) can also bind MHC 1. The mechanism by which CD8'

suppressor T celis inhibit B ce11 activation is not yet understood. My results show

that MHC 1 crosslinking by CD8 inhibits CD40 sthulated B ce11 proliferation and

induces apoptosis. Thus, this could mode1 suppressor T cells mechanisms to prevent

B ceIl activation.

The CKI p21"'1**' is a weil known central player in the Gi/S cell cycle

checkpoint in many ce11 types, and is intimately Linked to apoptosis in cells that "fail"

the checkpoint test. Our present data showing that biologically important ce11

surface receptors on normal B lymphocytes modulate cell cycle arrest and apoptosis

through a p2 I * - ' ~ - ' dependent process suggest that the signals are acting through

Gi/S checkpoint mechanisms. The GlIS checkpoint is believed to have evolved to

prevent the propagation of cells with defects in DNA that impair normal replication.

Our data suggest that this ancient mechanism has been CO-opted in lymphocytes, and

now acts as an integration site for lymphocyte surface receptor signals.

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H. Figures

Figure 1: Regdation of p2 1 cap- llwaf-1 expression in C MO-stimulated B cells. High

buoyant density splenic B cells fiom CS7BV6 mice were treated by crosslinking a rat

anti-CD40 mAb with a mouse anti-rat Ig mAb, with or without rat mAbs against the

indicated molecules. Lysates were resolved by PAGE and immunoblotted for p21" 1 iwaf- 1 using a rabbit antibody and HRP-goat anti-rabbit Ig. a) Kinetics of p21

expression after CD40 engagement. b) EfTect of CO-crosslinking B ce11 surface

molecules with CD40 on expression of p2l after 42 hours. c) Effects of single B ce11

surface molecule engagement on expression of p21 d e r 42 hours. A20 B

lymphoma lysate was used as positive control.

Figure 1

a)

CD40

A20 - ICAM-1 MHC I MHC II LFA-1 CD44

CD40 ICAM-1 MHCI MHCII LFA-1 CD44

Figure 2: Co-crosslinking ICAM-1, MHC 1, or MHC II with CD40 inhibits CD40-

induced proliferation. B cells were cultured for 48 hrs wiîh a) anti-CD40 and MaR

mAb b) LPS c) Sepharose coupled anti-p or d) CD40 plus anti-p F(ab')2 fragments

and MaR mAb, in the presence or absence of mAbs against the indicated ce11 surface

molecules. Proliferation was assessed by '~-th~midine incorporation. Data are

shown as means of triplicate cultures, bars represent standard deviation. Data shown

in a) are representative of 1 1 experiments.

Figure 3: ICAM-1, MHC 1, and MHC II co-crosslinking with CD40 induce

apoptosis. a) Annexin V staining was used to assess induction of apoptosis in cells

treated for 42 hrs with anti-CD40 in the presence or absence of the indicated rnAbs.

Data shown are representative of 3 experiments. b) Summary graph of 1 1

experiments in which PI staining was used to determine the percentage of cells with

subdiploid DNA following mAb treatment.

Percentage cells Apoptosing

Figure 4: p21 ap-l/waf'-l , but not p27kpL, is required for induction of apoptosis by

ICAM-1, MHC I and MHC II. a) and c) C57BV6 B cells, b) p21 cipl/\nf-1 -1- B cells

and d) p271"p1-/- B cells were treated for 42 hrs with the indicated mAbs.

Intracellular DNA content was assessed by PI staining, and the percentages of

apoptotic cells (subdiploid DNA content) are show as numbers above each profile.

Figure 5: FasL is not expressed on splenic B cells. C57BY6 small resting B cells

were cultured for 48 hours with the indicated treatments and stained for FasL

expression. A20 B lymphoma was used as a positive control.

Figure 6: High buoyant density B cells corn p21*1'M1 -/- and ~ 2 7 ~ ~ - ' - / - rnice

proliferate in response to anti-CD40 treatment. Unstimulated (black) and CD40-

stimulated (checkered) B cells fiom C57BU6, p21 4- and p27~~-1-/. . mice

were measured at 48 hours of culture by 3~-thymidine incorporation. Data are

show as means of triplicate cultures 9 SD. Stimulation indices are indicated above

each set of bars.

Thymidine Incorporation (x 1 Os cpm)

CHAPTER 3

A. General Conclusion

Ce11 cycle regulation is necessary to maintain homeostasis and to prevent

uncontrolled proliferation. The modulation of CD40-induced ce11 cycle progression

during T-dependent B cell activation that 1 have described is a case in point. 1 was

interested in studying the role of the CKI p21 cip-t~waf-1 in modulation of T-dependent

B ce11 activation.

To determine if the CKI p21 cip-Xlwaf-1 was involved in T dependent B ce11

activation, we stirnulated B cells with mAbs against CD40, CO-crosslinked potential

CO-receptors to CD40 and looked for a change in p21 cip-~waf-1 expression. CD40

stimulation upregulated p21, induced proliferation and increased B ce11 survival. Co-

crosslinking ICAM-l, MHC 1 or MHC II with CD40 superinduced p21 expression

and induced apoptosis. Using gene-targeted mice, we found that p21 was required

for induction of apoptosis by ICAM-1, MHC 1 or MHC II: CO-crosslinking with

CD40.

ICAM-1 and MHC II have long been thought to act as w-stimulators of B

ce11 proliferation and dwerentiation. However, under the experimental conditions

described here, they were shown to modulate CD40 induced proliferation and to

induce apoptosis. This may be due to digerent expenmental conditions. Perhaps the

rnAbs that were used in my experiments had much higher binding affhities than

natural ligands, and this may have ovemdden the proliferative signals. It was

surpnsing to find that BcR signaling did not rescue B cells from ICAM-1, MHC I

and MHC II modulation of CD40-induced proliferation (Figure 2d) because antigen

recognition dong with CD40 signaling are the main requirements of cognate

interactions. To test whether this was due to the use of anti-p mAbs rather than

soluble antigen, BcR transgenic B cells could be stimulated using antigen and anti-

CD40 and the effects of the inhibitory mAbs could be observed. The use of soluble

ligands and non-cognate culture systems is discussed below.

Engagement of FcyRlIB, the only Fc receptor @CR) expressed on B cells

(Ravetch et al., 1986), inhibits BcR induced B celi proliferation (reviewed by

Ravetch, 1994) and induces apoptosis (Ashman et al., 1996; Ono et al., 1997; Pearse

et al., 1999). To control for the possibility that the inhibitory effects of ICAM-1,

MHC 1 and MHC II were due to FcR signaling, isotype control mAbs were used.

These mAbs did not inhibit CMO-induced proliferation. A better way to control for

FcR signaling would be to use F(ab')z fragments of anti-ICAM-1, anti-MHC 1 or

anti-MHC II mAbs. At the time when these experiments were performed, these

F(ab')2 fiagments were not comrnerciaily available. 1 therefore started to make anti-

ICAM-1 mAbs into F(abY) 2 fiagments but have not wmpleted the process. The use

of these fragments would definitely rule out the possibility of FcR signaling.

Al1 of the experiments done for this thesis were performed using mAbs to

crosslink B ce11 surface molecules. These experiments should also be done under

more physiological conditions. To do this, soluble ligands could be used to stimulate

B ce11 surface molecules. A soluble CD40L-CD8 molecule is available that can be

crosslinked by using anti-CD8 antibodies. This interaction cannot be rnirnicked

exactly since the natural CD40L is a trimeric molecule, but perhaps soluble CD40L

would tnmerize nahrraily. To crosslink ICAM-1, soluble LFA-1 shouid be used.

LFA- 1 is a heterodimer consisting of CD I l dCD18; therefore, it would be necessary

to conjugate these subunits with molecules that naturally associate to one another.

This could perhaps be done by conjugating both of them to one molecule. MHC II

would have to be crosslinked with a soluble CD4 conjugate. Soluble TcR could not

be used because it recognizes MHC II with its associated antigenic peptide. MHC 1

could be crosslinked with a soluble CD8 conjugate. Another way to make these

experiments more physiologically relevant would be to establish CO-culture systems

for T-dependent help for B cells using B cells £?om MHC 1 or LFA-1 deficient mice

or activated T cells fiom mice deficient for ICAM-1 or MHC II. These could be

used to observe the effects on B celi proliferation and apoptosis in the absence of

these molecules. The following step would be to take these effects in vivo and see

physically where p2 1 is superinduced.

The modulation of CD40-induced proliferation by ICAM-1, MHC 1 and

MHC II may play a role in B ce11 differentiation or selection in the germinal center.

The expenments described in this thesis show that in splenic B cells, p21 c i p - l / d - l is

nipennduced when CD40-stimulated proliferation is inhibited by ICAM-1, MHC 1

and MHC II. It would be interesting to see which subpopulation of B cells

superinduces p21 and whether this relates to GC development. This could be done

by staining adjacent sections of splenic and lymph node tissue nom control and

antigen primed mice with d b s to p21 cip- 1 /waS 1 , B220 (a B ce11 marker) and peanut

agglutinin (PNA) (a germinal center rnarker) to see which B cells express very high

levels of p21 ap-uwaf-i . This would identie whether there is a relationship between

germinal center B cells and p2 1 c i p - l / d - 1

ICAM-1, MHC 1 and MHC II reverse the B ce11 çurvival induced by CD40.

This mechanism seems to take place both at the Gl/S checkpoint and at the Gz/M

checkpoint. In the absence of p21 cip-uunf-~ , the induction of apoptosis from Go/Gi

seems to be abrogateâ, but not that nom G2M. Phosphorylation of CDKl has been

shown to inhibit transition into mitosis. ICAM-1 signaling, in T cells, has been

shown to involve phosphorylation and inhibition of p 3 4 C d c 2 / ~ ~ ~ 1 (Chirathaworn et

al., 1995). It would be interesting to see whether the ceil cycle inhibition of CD4O-

stimulated B celIs induced by ICAM-1 also involves inhibition of CDKl by protein

tyrosine phosphorylation. Results from Our lab have demonstrated that ICAM-1

signaling in A20 lymphoma induces the phosphorylation of proteins with

approximate molecular weights of 150, 100, 55-60 and 3 5 kDa (Holland and Owens,

1997). Perhaps this last band may be CDK1. To see whether this is the case, CDKl

could be immunoprecipitated fkom B ce11 lysates treated with mAbs to CD40 and

ICAM-1 and western blotted with anti-phosphotyrosine antibodies. This would

provide a second mechanism for ce11 cycle inhibition in this system. Thus, the

decrease in cells fiom GdGr and f?om G2/M during the increased apoptosis induced

by ICAM-1, MHC 1 and MHC II may be mediated by two different mechanisms: the

first being p21 cip- ~ w a f - L binding to cyclidCDK complexes and the second being

phosphorylation of CDK1. To see if this was the case, chernical inhibitors of ce11

cycle could be used to observe the effects on induction of apoptosis f?om certain ce11

cycle checkpoints.

Recognizing under which circumstances ICAM-1, MHC 1 and MHC II can

inhibit CD40-stimulated growth and induce apoptosis is important for understanding

regdation of T dependent B ce11 activation. ICAM-1, MHC 1 and MHC II signaling

might be involved in B ce11 differentiation. The signals involved in differentiation

into plasma cells are not completely understood but there is a general association

with reduced proliferation. It is hypothesized that the B cells shift fiom proliferation

to differentiation into antibody producing plasma cells. Consistent with this

hypothesis, my results show that several B ce11 surface moIecules, i-e. ICAM-1,

MHC 1 and II, specifically modulate CD40-induced proliferation and Our lab has

shown that blocking IC AM- 1 /LF A- 1 or MHC WTcR interactions inhibited T-

dependent induction of Ig secretion (Poudrier and Owens, 1994b). Together, these

point to a role for ICAM-1 and MHC 11 in B ce11 differentiation. However, the

induction of apoptosis of CD40-stimulated B cells suggests that these signals could

be also involved in deletion of autoreactive B celIs or prevention of inappropriate B

ce11 activation. After somatic hypermutation, CD40-stimulated B cells must be

rescued by recognizing antigen through their new BcR. If antigen is not recognized

before encountenng an activated T cell, the B ce11 must be destroyed. These signals

may be transmitted by ICAM-1, MHC 1 or MKC II. It is more efficient for non-

specific B cells encountering activated T cells, to be deleted before activation than to

eliminate activated autoimmune B cells. Still, these inhibitory interactions can

always take place. One must consider how then, inhibition can be modulated to

allow immune responses to occur. In this thesis, 1 have focused on ïnhibitory

interactions, but there were also some stimulatory interactions. CD45RB and CD44

repeatedly increased B ce11 proliferation when CO-crosslinked to CD40. Perhaps the

varying intensity of each interaction determines whether proliferation, inhibition or

apoptosis occurs, so that the outcome is detennined by the net sum of the interactions

between a B ceU and a T ceil.

The regulation of immune responses is necessary to prevent uncontroled

proliferation and tissue injury. The downstream signaling that leads to ce11 cycle

inhibition remains to be explored. A lot of questions are lefi unanswered, but

showing that p2 1 npl~~af-I is involved in the induction of apoptosis by ICAM-1, MHC

1 and MHC II of CWO-stimulated B cells brings us one step closer to understanding

the regulation of T dependent B cell activation.

B. References

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