An Analysis of the in vivo SEB response in a T Cell Receptor scid Transgenic mouse

Ruwaida Dhala

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of lmmunology

University of Toronto

O Copyright by Ruwaida Dhala (1 997)

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An Analysis of the in vivo SE5 response in a T Cell Receptor scidTransgenic mouse

Master of Science 1997

Ruwaida Dhala

Department of lmmunology

University of Toronto

Abstract

The superantigen SEI3 was used to analyze the immune response of a C.B- 17 scicl mouse

carrying a transgenic T cell receptor expressing V a 13 and Vp 8.2. There are no B cells

present in this mouse and the only T cells present express the T ceIl receptor transgene.

Therefore the response of a clonotypic population of T cells can be studied in the absence of

al1 other T or B cells. The splenic T cells from these mice mounted a response to SEB in vivo,

illustrating that a clonotypic population of T cells is able to mount an SEB induced immune

response in the absence of other T cells or B cells. IL-2 R a staining suggested that a majority

of the T cells had ken activated in response to SEB. In addition upon SEB injection a

population of CD4 TCR+ cells was detected in the spleens of SEB injected transgenic scid

mice. A similar population was seen in the thyms but not in the spleen ofcontrol mice.

Acknowledgments

1 am gratefùl to my supervisor Dr R.G. Miller for his advice and support. 1 would like to thank

the members of my advisory committee, Dr. P. Ohashi and Dr T. Watts for their helpful

suggestions. 1 would also l i e to thank Dr D. Spaner for his encouragement and advice.

1 would like to acknowledge members and fnends of the MiIler lab including Jacinth, John,

Juliet, Laszlo, Sam, Ruey, Raju and Audrey. A special thanks to John Shannon for his

expertise in tissue culture and Juliet Sheldon for her help with the flow cytometer as well as

with typing rnice. 1 would also like to recognize the members of the OC1 animal colony

including Christine and Robyn for their cooperation.

1 wish to thank my family including my mother Shamim, sister Sabiha and brother Hasnain for

their moral support. This thesis is dedicated to my mother, Shamim Dhala for her continued

encouragement and support.

Table of Contents

Page

Introduction

Materials and Methods

Results

Discussion

e References

List of Figures

Fi~ure 1 : OVA scid and BALB!c thymus and spleen

Figure 2: CD4 VB8 T cells respond to SEB

Figure 3: SEB response in OVA scid versus BALBtc mice

Figure 4: OVA scid T cells are detectable in C.B- 17 scid rnice upon

adoptive transfer

a Figure 5: CD4 and CD8 T cells from BALB/c reduce the number of

OVA scid T cells recovered

w Figure 6: Adoptive transfer of BALB!c splenocytes into OVA scid rnice

Figure 7: SEB response in OVA scid versus BALBtc mice

a Fime 8: IL-2 receptor a expression on OVA scid versus BALBIc

CD4 VP8 T cells

Figure 9: C D 4 TCR+ cells are seen upon SEB injection

Page

26

List of Tables

Table 1 : Adoptive transfer of BALBIc splenocytes into OVA scid mice

Table 2: OVA scid T cells are capable of mounting a

response to SEB upon adoptive transfer

Page

3 4

Abbreviations 82n1: beta 2 microglobi~lin

CM: cornplete media

EAE: experimental allergic encephalomyelitis

FITC: fluorescein isothiocyanate

GVHD: graft versus host disease

Ig: immunoglobulin

IL: interleukin

IL-2 R: interleukin 2 receptor

IFN: interferon

mAb: monoclonal antibody

MBP: myelin basic protein

MEM: minimum essential medium

MHC: major histocompatibility complex

MIP: macrophage inflarnatory peptide

MIS: minor lymphocyte stimulating mtigens

MMTV: mouse marnrnary tumor virus

OVA: DO- 1 1.1 O T ce11 receptor trmsgenic mouse

PLP: proteolipid protein

PBS: phosphate buffered saline

PE: phycoerythrin

RAG -1-: recombinase activating gene deficient

scid severe combined imrnunodeficiency

SE: Staphylococcal Enterotoxin

SDC: SEB dependent cytotoxicity

TCR: T ce11 receptor

TNF: turnor necrosis factor

Th 1 : T helper 1

Th2: T helper 2

7AAD: 7 amino actinomycin D

vii

Introduction

The immune response to foreign antigen involves the effector cells of the immune

system. Lymphocytes are the key effector cells of the immune system. These cells are

responsible for recognizing, rnountin; a response to and clearing the antigen. In addition to the

response against antigen these cells miy also serve to regulate the immiine response. T cells

bearing the ap T ce11 receptor (TCR) are amongst the lymphocytes whjch are involved in

regiilating the immune response (1) (2) (3). Generally ap T ceils c m regulate an immune

response either by T ce11 mediated cytotoxicity or by the secretion of cytokines (4) (5). In

order for an ap T cell to exert its irnmunoregulatory activity it usually needs to be activated

(5).

ap T celis recognùe antigens in the context of cell surface proteins called major

histocompatibility complex (MHC) molecules (6) (7) . There are two seneml cIasses of ap T

cells, each of which have two major siibclasses. The two major subclasses of ap T cells

include helper T cells (CD41 and cyîotoxic T cells (CD8). CD4 T cells recognize antigen in the

context of MHC class II molecules, while CD8 T cells recognize antigen in the context of class

1 molecules. In addition to recognizins antigen plus MHC, T cells require costimulatory

molecules to be activated. One of the key costimulatory molecules involved in T cell activation

is CD28 (8). CD28 is a homodimeric molecule expressed on the surface of crp T cells. Its

interaction with n~embers of the B7 family of molecules expressed on antigen presenting cells

results in the activation of an US T cell.

T cells may regulate various phases of the immune response. For example, mice which

lack the TCR a chah and thus lack ap T cells had an increased number of B cells (1). These

B cells secreted increased amounts of autoreactive antibodies. The authors suggest that this

may point to a role for aB T cells in regulating B cells. The presence of a TCR (T cell

receptor) transgene specific for myelin basic protein (MBP) in a RAG 1 deficient (RAG -/- )

mouse resulted in spontaneous experimental allergic encephalomyelitis (EAE) in 100% of the

mice (9). Only 14% of TCR transgenic mice that were not RAG 1 deficient spontaneously

developed EAE. Since RAG I -i- mice are unable to initiate remangement of their antigen

receptors and thus are immunodeficient and EAE involves primarily T cells (10) ( 1 I), the

authors implicate the onset of spontaneous EAE in al1 MBP transgenic RAG -1- mice to be the

result of a lack of regulatory T cells that are absent in these mice but present in the RAG 1

proficient TCR transgenic mice. However the onset of spontaneous EAE may also be due to

other factors such as the EAE susceptible haplotype of the mice used.

T suppressor cells are proposed as a class of T cells which can actively downregulate

an immune response. These cells cm exert their suppressive activity through anti-idiotypic

suppression. The suppressor T cell cm recognize the mtigen receptor of the activated T cells

and thus suppress these cells. The suppressive activity of these cells could be adoptively

transferred to a secondary recipient ( 1 2). Both helper T cells (CD4) and cytoroxic T cells

(CD8) can influence the immune response. CD4 T cells exert tlieir immunoregulatory capacity

mainly by the secretion of cytokines (4) (5). CD8 T cells exert their immunoregulatoiy

capacity by their cytotoxic activity (13) and the secretion of cytokines (14) (15).

The role of CD4 T cells in the immune response

CD4 cells may be important in regulating the immune response. Mice deficient in CD4 cells

showed decreased antibody production illustrating that CD4 cells can help B cells make

antibodies (1 6). However since these mice c m still generate antibodies in the absence of CD4

cells, other cells may contribute to antibody production. In addition to helping B cells, CD4

cells can influence the immune response by the secretion of cytokines (1 7). Upon T ce11

activation, CD4 T cells c m differentiate into two distinct subsets characterized by their pattern

of cytokine secretion (1 7). These subsets, termed T helper 1 (Th]) m d T helper 2 (Th2) were

initially characterized in murine T helper cell clones that were divided into two distinct groups

based on their cytokine profile (18). Th1 and Th2 cells secreted distinct arrays of cytokines

which serve to regulate the other subset (4) as well as other immune responses such as

antibody responses and ceII mediated immune responses (17). Th1 cells produce IL-2, IFN-y,

TNF-fi, TNF-a ( 1 7) (1 8) (1 9) and are involved in ce11 mediated inflammation and in delayed

type hypersensitivity reactions (20). Th2 cells produce IL-4, IL-5, IL- 1 O, TNF-a (1 7) (1 8)

(19). Some of the Th2 cell produced cytokines encourage antibody production and are found

in association with allergic responses (17). In adddition the cytokine products of Th1 and Th2

cells serve to regulate each other (4). Thiis Th1 produced IFN-y selectively inhibits the

proliferation of Th2 cells. Th2 produced IL-10 selectively inhibits cytokine synthesis by Thl

cells. This cross regulation c m cause a bias in Th1 or Th2 responses in certain infections. In

addition other cytokines c m also serve to drive Th 1 or Th2 development. For example, IL1 2,

a cytokine secreted by n-ionocytes, c m drive the development of IFN-y producing Th1 cells

(21 ).

Development of the appropriate Th subset during infection can determine the outcorne

of infection. For example, a Th1 type response in C57BU6 mice correlated with the resolution

of the protozoan parasite infection L e i s h a n i n~crjoi. while a Th2 response in BALBIc mice

correlated with dissemination of disease and ultirnate death of the infected mice (5) . Th1 and

Th2 responses have k e n seen in autoimmune disease models. In EAE (experimental allergic

encephalomyelitis), a mouse mode1 of multiple sclerosis (22), there is evidence that Th1 cells

are important for the initiation of the disease (23) (24). EAE was first induced in mice by

Oliesky and Yager in 1949 (22). They treated albino mice of the Swiss strain with

intrarnuscular and subcutaneous injections of normal mouse brain mixed in Freunds adjuvant.

The mice developed syrnptoms such as wheezing and histological lesions similar to monkeys,

rabbits and guinea pigs with EAE. In most current models, myelin basic protein (MBP), a

dominant protein of the central nervous system myelin, c m in iused to intiate EAE (23) (24).

To determine the role of Th1 versus Th2 cells in EAE, two myelin basic protein peptide

specific ce11 lines were used (24). Adoptive transfers of these clones into mice showed that

only the Th1 clones were able to induce disease as measured by paralysis and

immunohistology. However the ability to induce EAE amongst the Th1 clones was dependent

on the presence of a4 integin (VLA-4). Furthemore lymphocytes that infiltrate the brain and

spinal cord of a mouse with EAE stained positively for IFN-y and IL-2. IL-4 and IL-5

infiltrathg lympliocytes were seen Iater on in the disease (23). Th2 ceils can protect against

EAE in sorne systems (25) but not in others (26). Mice oially administered myelin basic

protein (MBP) developed mucosally derived Th2 Iike cells (25). These cells produced TGF-B,

IL-4 and IL-IO. Cloned Th2 cells from these mice inhibited the development of EAE in mice

imniunized with MBP. However, TCF P, which is not classically a Th2 cytokine, inay play a

critical role in mediating protection in this model. Results from 'mother group that induced

EAE in SJL mice by proteolipid protein (PLP), a major component of the central nervous

system myelin membrane, showed that adoptive transfers of Th1 but not Th2 cells induced the

disease (26). However adoptive transfers of Th1 and Th2 cells still induced EAE . This

suggests that aIthough Th1 cells seem to be responsible for the initiation of EAE (23) (24) (26)

it is not clear whether Th2 cells protect against EAE (26).

A differential roIe for Th 1 and Th2 cells has also ken implicated in grdl versus host

disease (GVHD) (27) (28). Chronic GVHD and acute GVHD are two forms of GVHD that

have ken weIl characterized in mouse models. While acute GVHD is induced by the transfer

of C57BU6 splenocytes into unirradiated FI recipients (C57BL6 X DBAtS), chronic GVHD

is induced by the transfer of DBN2 splenocytes into these FI recipients (27). Acute GVHD is

characteriized by the involvement of cytokines such as IL- 1, IFN-y and TNF cl while chronic

GVHD is characterized by the involvement of cytokines such as IL-4 and IL-10. (28). AlIen et

al. found that in the chronic model of GVHD (DBN2 into C57BU6 X DBN2), splenic

mRNA showed more IL-4 signal than in control mice or mice with acute GVHD (C57BU6

into C57BL6 X DBN2) (27). In contrast, mice with acute GVHD showed more IL-IO, IFN y

and MIP-la (a chemokine) signal and less TNF-p (lymphotoxin) signal. Since the mics with

acute GVHD had increased IL-IO and decseased TNF-P, the ThltTh2 distinction in acute

versus chronic GVHD may not be clearcut. Recently, Krenger et al. showed that polxized

type 2 CD4 and CD8 T cells failed to incluce acute GVHD as measiired by reduced IFN-y and

TNF-a production as well as lethality though LPS challenge (28). LPS is used in models of

GVHD to induce Tm-a production and the extent of TNF a production is related to the

severity of GVHD. They generated polarized alloreactive CD4 or CD8 T cells in vitro by

adding rIL-2 plus or minus rIL-4 to a mixed lymphocyte reaction directed against class 1

differences (bml into C57BL16) or class II (bm12 into C57BU6) differences. Type 1 cells

were generated in the cultures that lacked IL-4, while type 2 celIs were generated in the

cukures that had IL-4. Transplantion of polarized type 2 CD4 or CD8 plus donor bone

marrow cells failed to induce GVHD as measured by reduced IFN-y production, LPS induced

lethality and reduced TNF-a production. Polarized type I CD4 and CD8 cells induced

increased IFN-y in response to Con A after transplantation and vinuaily no IL-4. Polarized

type 2 CD4 cells produced IL-4 and reduced IFN-'y, while type 2 CD8 cells produced very low

levels of IFN-y. Mixing experirnents of polarized Th2 CD4 cells with naive CD4 cells and

donor bone rnarrow showed that these polarized type 2 cells could inhibit acute GVHD as

measured by IFN-y production in a secondary mixed lymphocyte reaction. Thus CD4 cells

may play a role in regulating the immune response.

The role of CD8 T cells in the immune resoonse

CD8 cells cm play a role in the regulation of an immune response. These cells c m mediate

suppression of an immune response by directly killing activated T cells (13), by secreting

cytokines (15) (28), or by controlling the activity of CD4 cells (2) (3). CD8 T cells can also

suppress the immune response by a functional elimination of the reactive T ce11 . For example,

veto cells are specialized antigen presenting cells that delete activated T cells that recognize

them (29). Veto cells generally carry the molecuk CD8 on them and inhibit class 1 mediated

immune responses.

Although classically CD8 cells have not been irnplicated in cytokine production, more

recently alloreactive CD8 T cells clones have k e n shown to secrete a Th 1 pattern of cytokines

(1 4). CD8 T ce11 clones were stimulated with a panel of antigens and the supernatants were

assayed for the production of cytokines by ELISAs and bioassays. These clones produced

IFN-y, TNF-B, and TNF-ci and low arnounts of IL-2. CD8 T cells cm also be polarized in

vitro to become type 1 and type 2 CD8 cells depending on their cytokine profiles (15). CD8

cells fiom H-Y transgenic mice (30) stimulated with B6 mtigen presenting ceiis and cultured in

the presence of IL-2 and IL-1 2 made high amounts of IFN-y and IL-2 and no IL-4 or IL-5

upon restimulation with anti-CD3 antibody. CD8 celIs generated with IL-2 and IL-4 made IL-

4 and IL-5 but very little IL-2 and IFN-y. The role that type I and type 2 CD8 cells may play

in intluencing the immune reponse in vivo was discussed earlier in a murine mode1 of acute

GVHD (28). These findings have important implications for type 1 and type 2 CD8 T cells in

the regulation of the immune response.

CD8 T cells may also influence the fate of CD4 cells (2) (3) (3 1). LCMV virus

infected b2m -/- mice (lack class 1 MHC) had exaggerated CD4 T cell proliferation itz vivo

(3 1). These CD4 cells also developed cytotoxic function. Although the authors impIicate a

role for CD8 cells in controlling CD4 T ceIl proliferation and cytotoxicity, a direct role wczs not

demonstrated. Since CD8 T cells lyse virally infected cells (32), the exaggerated CD4 T cell

responses could lx due to an increased viral load. A role for CD8 cells has also been suggested

in EAE (2) (3). Although, EAE is primarily rnediated by CD4 cells (10) (1 l), in mice with

EAE a lack of CD8 cells prevented recovery fiom the disease (2). Mice lacking CD8 cells

were bred with EAE susceptible PIlJ H-2" mice for four generations. Although the disease

onset and susceptibility in CD8 -1- mice was the same as in wild type mice, the mutant mice had

milder acute EAE (less deaths) and suffered from a chronic (relapsing) form of EAE. This

sugested that CD8 cells may play a role in the pathogenesis or regulate recovery from EAE.

The role that CD8 cells have in EAE was also shown by Jian,o et a1 (3). They depleted

B 1O.PL (H-2") mice of CD8 cells and showed that mice depleted of CD8 cells were no longer

resistant to a secondary challenge with MBP. The authors suggest that CD8 cells may

participate in the resistance to a secondary induction of EAE.

Superantiqens Superantigens stimulate particular populations of T cells (33) (34). In contrast to peptide

antigens which activate T cells carrying the specificity for the peptide MHC cornplex,

superantigens act ivate T cells that bear particular VP chains (33) (35). Since superantigens

involve the activation of T cells with particular VP chains they are usefùl in following antigen

specific responses in vivo.

The T ce11 superantigen family consists of bacterial superantigens produced by

Staphylococcus aureus and Sireptococcus pyogenes as well as virally encoded superantigens

such as the minor lymphocyte stimulating (Mls) antigens (36). Mls antigens are encoded by

open reading fiames in the 3' long terminal repeat of mouse mammary tumor viruses (MMTV)

(37). Staphylococcus ciureus produces eight similar enterotoxins; SEA, SEB, SEC 1, SECZ,

SEC3, SED, SEE, and TSST (toxic shock syndrome toxin) which are supermtigens (38). The

superantigens SEA-E are involved in food poisoning while TSST is involved in toxic shock

syndrome. Strcptococcus pyogeries produces three superantigens SPA, SPI3 and SPC which

have been associated with scarlet fever and toxic shock. Both the staphylococcal and

streptococcal toxins are 27-28 kDA polypeptides. TSST is a 22 kDA poIypeptide. The

staphylococcal and streptococcaI toxins share similarities in their primary amino acid sequence

suggesting common origins. The crystal structure of SEB shows that it consists of a main

chain with two domains (39). The first domain consists of residues 1 - 120 and has five p-

strands and three a-helices. The second domain consists of residues 127-239 and has seven P-

strands and two a-helices. A shallow cavity formed by these two domains is postulated as the

TCR binding site. The MHC class II rnolecule binds to an adjacent site.

T cell superantigens stimulate mature T cells bearing particular T cell receptor V(3

chains (33) (35). Exposure of mature T cells to Mlsbtigens stimulates a strong T ce11

proliferative response in vivo (4.0) and in virro (36). Staphylococcal enterotoxin B (SEB)

potently stimulates murine T cells that bear the following T cell receptor B chains: Vp 3, 7,8.1,

8,2,8.3, 1 1, 17 (33) (34) (38) (41). In general superantigens bind outside the peptide binding

groove of the MHC molecule and to the TCR Vb thus crosslinking the two molecules (42).

Heterogeneity in the interaction of particular SEB reactive VP chains with superantigens

suggests other contributing elements to superantigen recognition. For exarnple the CY chain has

also been implicated in influencing the response to SEB (43) (44) as well as MIS (45) (46) (47).

Another factor that influences T ce11 recognition of SEB (34) or MIS is the MHC molecule

(48). Fui-thermore, in human T cells, mutations in the peptide binding groove of class II MHC

cm effect the binding of toxic shock syndrome toxin- 1 and SEB with the class II MHC

molecule (49).

Su~erantiqen response in vivo The in vivo response of mature T cells exposed to superantisen is chaacterized by an initial

phase of proliferation followed by a deletion (after day 4) of CD4 and CD8 cells bearing the

appropriate V(3 chah (50) (51) (52). Results fiom Kawabe et al indicate that the deletion seen

is partially due to ce11 death via apoptosis (53). Since deletion is defined as the loss of V(38.l T

cells in the spleen, lyrnph node or blood, after day 4 of SEB injection (50) (51) (52), it is

possible that this loss may be due to other factors such as migration of the T cells to other

anatomical sites or downregulation of the both the TCR and the CD4 molecule. For the

purposes of this thesis deletion is debed as the loss of CD4 Vp8 T ceHs from the spleen,

lymph node or blood of mice afier day 4 of SEB injection. This loss could be due to migration,

apoptosis or a combination of both. The superantigen SEB can activate both CD4 and CD8

cells (54), however CD4 cells undergo more deletion than CD8 cells (50) (5 1) (53).

Additionally only CD4 cells are impaired in theù ability to respond to SEB upon in vitro

restimulation and are termed anergic (50). These anergic T cells exhibit impaired tyrosine

phosphorylation in response to TCR mediated stimulation (55). It is important to note that the

anergy exhibited by these T cells is transient (56). Furthemore, competent antigen presenting

cells and high concentrations of SEI3 can overcome anergy itt virro (57) (58). A second

exposure to SE6 irt vivo results in the production ofsimilar arnount of IL-2 and tumor necrosis

factor by CD4 VB8 cells as is present in mice given ri single challenge of SEB. However this

secondaiy challenge only results in expansion of CD8 V(38 cells (59). Therefore the CD4 V(38

cells are still unable to proliferate in vivo upon subsequent challenge with SEB and rnay be

anergic or may lack the ability to respond to SEB. Clonal anergy to superantigens has also

k e n shown to exist with human lymphocytes (60).

An effector function of CD8 cells upon SEB injection is SEB dependent cytotoxicity

(SDC) (54). Upon SEB injection CD8 cells are able to directly lyse class II bearing target cells

incubated with SEB ex vivo . This effector function of CD8 ceIl has been documented in rirro

and is termed SEB dependent cytotoxicity (SDC). SDC peaks after 2-3 days of SE3

injection. The role of SDC in vivo has not been addressed.

Factors which influence the su~erantiaen - response in vivo The response to superantigens in vivo is detemined by a number of factors. These factors

include age of the host (33) (61), dosage of the superantigen (62) (63), haplotype of the host

and the particular VB studied (48). In neonatal rnice, encounter with superantigen leads to a

deletion of the reactive T ceii without the induction of a proliferative response, while in mature

mice encounter with superantigen leads to proliferation of the mature reactive T cells followed

by a deletion of some of the superantigen reactive cells (33) (35) (64). In aged mice, encounter

with superantigen leads to less expansion and less deletion (61). If the superantigen is injected

in a very high dose in adult mice along with the hepatotoxin D-galactosamine, then there is an

expansion of T cells and the mice die of toxic shock (63). If the superantigen is repeatedly

injected into adult mice, there is a profound deletion of the SEB reactive T cells (62) (65).

The proliferative response to superantigens in vivo is dependent on the presence of

many ce11 surface molecules (66) (67) (68) (69). CD28 is a homodimeric molecule expressed

on T cells (8). Its binding to B7- I or B7-2 molecules, which are expressed on the sutface of

antigen presenting cells, facilitates the activation of a T cell. While CD28 allows for the

activation of T cells, its homolog CTLA-4, which also binds members of the B7 family, inhibits

activation (70). In contrast to CD28, which is constitutively expressed on T ceiis. CTLA-4 is

transiently expressed on activated T ceus. Antibodies that block the interaction of CD28 and

B7 are able to block SEB induced proliferation in vivo (66) and in vitro (7 1 ) . Evidence from

CD28 deficient mice showed that though the initial proliferation is dependent on CD28,

deletion is not (67). In contrast to anti CD28 antibodies, Fab fiasments of anti CTLA-4

antibodies in vitro and in vivo enhance T ce11 responses to SEB (68). This shows that CTLA-4

and CD28 have opposing effects in T ceIl activation in response to SEB. Additionally the

adhesion molecule, ICAM- 1 , also seems to play a role SEB induced T ceIl proliferation in vivo

(69). Mice that lack ICAM-1 have impaired T cell proliferative responses to SEB but are able

to undergo SEB induced deletion.

Factors which influence superantiaen induced deletion in vivo There are many factors which c m intluence superantigen induced deletion in vivo. These

factors include the ce11 surface molecule Fas (72), other T or B cells (1 3) (73) (74), the initial

proliferative response to the superantigen (73, as well as the cytokine IL-2 (65) (76). Fas

plays a partial role in the apoptosis of T cell activated in response to SEB (72). Fas is an

integral membrane protein which is a member of the tumor necrosis factor receptor family. T

cells from both gld mice, which Iack fimctiona1 Fas ligand, and young MRUlpr mice, which

lack functional Fas, are able ta proliferate in response to SEB (72). However only a fraction of

the T cells undergo superantigen induced deletion. Thus Fas may play a partial sole in the

deletion of SEI3 activated T ceBs.

While the role that T and B lymphocytes play in the in iiin response to Mls responses

is defined, the role that T and B lymphocytes play in the in vivo response to SEB response is

less clearly defined. For the MIS response, B cells seern to be the most efficient presenters

although other lymphocytes can present MIS (73). Furthemore both B cells and CD8 cells but

not CD4 cells c m induce peripheral deletion of Mls reactive T cells (73). In neonates CD8

cells seem to be more efficient than B cells in inducing deletion (52). The role of B cells in the

SEB response has k e n recently addressed in B cell knockout mice (58) (77). B cells do not

seem to be required in the initial proliferative response to SEB in vivo (77). However T cells

from B cell knockout mice are not anergic in vitro (58). The role of B cells in irz vivo anergy

or deletion was not addressed. The role of other CD8 or y6 cells in the in vivo response to

SEB response is not clearly defined. There are two reports that discuss the role of CD8 cells in

the SEB induced deletion of CD4 VP8 T cells (13) (74). One report using CD8 depleted mice

suggests that the role for CD8 cells is minimal if any, since CD8 depleted mice (depleted by

anti-CD8 monoclonal antibody treatment) have CD4 VP8 T cells deleted (74). This study

found no qualitative difference in the responses of untreated and CD8 T ceIl depleted mice

(depleted by anti-CD8 monoclonal antibody treatment) to SEB. However they did find a small

quantitative difference in deletion. Another report using h t h CD8 depleted and 02m

knockout mice (which lack class 1 MHC) suggested that CD8 cells are required in SEB

induced deletion of CD4 Vp8 T cells ( 1 3). Both CD8 depleted and B2m knockout mice had a

partial deletion of CD4 VB8 T cells. Additionally it was shown that CD8 cells lyse CD4 cells

that have previously k e n activated in response to SEB in virm.

In contrast to the previous groups, Renno et al. have shown that T cells which undergo

apoptosis in vivo me ones that have proliferated in response to SEB (75). All T cells that

underwent apoptosis had proliferated in response to SEB. Renno suggests that the VP8 cells

that are weakly SEB reactive do not receive a signal that is strong enough to allow

proliferation and then death. However other groups have shown that deletion of CD4 VP8 T

cells can occur in the absence of significant proliferation (67) (69).

The role of IL-2 in SEB mediated deletion has recently k e n addressed. Mice that lack

the IL-2 gene (IL-2 -/-) have normal in vivo expansion of CD4 and CD8 cells in response to

SEI3 (76). However fewer CD4 cells are subsequently deleted. Evidence fkom another group

also points to a role for IL-2 in SEB mediated deletion, Lenardo has s h o w that injection of

an antibody against the IL-2 receptor a chain was able to prevent the reduction of lymph node

VP8 cells in response to repeated injections of SE6 (65).

Mouse Models : The advantaoies of scid and transaenic mice Since the immune response to antigen involves the activity of a specific lymphocyte, it is

advantageous to follow that lymphocyte during the course of an immune response. Recent

molecular genetic technology has allowed for the production of TCR transgenic mice

engineered to express a hi$ frequency of T cells specific for an antigen (30). Since the T cells

in TCR transgenic mice express clonotypic TCRs, they cm be followed in vivo md in vitro

with antibodies that specifically bind to the mtigen receptor. Thus the fate of a speciiic

population of T cells during the course of an imm~ine response c m be studied. It is important

to note that although aTCR transgenic mouse expresses a high pescentage of antigen specific

T cells it also carries other T cells since the lack of allelic exclusion at the cx locus results in T

cells that c m y the transgenic P chain with an endogenous cr chain (30). This can result in a

loss of specificity for the antigen. To overcome this problem scid TCR transgenic mice c m be

used (78). Scid rnice are naturally occurring mutant mice that have lost the ability to

successhIly rearrange their antigen receptors by VDJ recombination (79) (80). This inability is

due to a defect in DNA repair that impairs the cells from rejoining the rearranged antigen

receptor DNA. Therefore scid mice are generally devoid of T and B lymphocytes (80). Mice

that bear the TCR transgene and harbor the scid mutation bear the transgenic T cell in absence

of other T or B cells. These mice are a good in vivo system for studying an immune nsponse

of a clonotypic population of T cells in the absence of other T or B cells.

Thesis The irnmune response to superantigens is controlled by a complex interaction of T cells, B ceUs

and ce11 surface molecules. According to one model, the extent of deletion is dependent the

initial proliferative response to SEB (75). Thus TCRs with low afiïnities for SEB do not

proliferate or get deleted, whereas TCRs with high affinities for SEB proliferate and then are

deleted. However evidence from other groups suggest that deletion cm occur in the absence

of significant proliferation (67) (69). Furthemore other groups implicate CD8 T cells as

important in controlling the deletion of CD4 celis in response to SEB (1 3) (74). These reports

are conflicting. Some groups have shown that in the absence of CD8 cells in (32m -1- mice or

in mice treated with an antibody against CD8, the CD4 VP8 T cells are partiaily deleted ( 1 3).

Other groups using an antibody against the CD8 molecule indicate that CD8 cells are

mimimally required in SEB induced deletion (74). The work presented in this thesis focused

on the investigation of the SEB response in a mouse with a clonotypic population of T cells.

All the T cells in this mouse should have the same affinity for SEB. Therefore if they

psoliferate in response to SEI3 they should te deleted. Since the role of CD8 cells in SEI3

induced deletion of CD4 V(38 T cells is controversial, we wanted to investigate whether the

absence of CD8 cells in this system would affect the extent of deletion of CD4 VB8 T cells.

Model svstem The superantigen SEB was used to analyze the immune response in C.B-17 scid mice (80)

canying a transgenic a(3 T cell receptor seactive to an ovalbumin peptide plus 1-A* (81), a T

cell receptor expressing V a 13 and VP 8.2 and reactive with SEB (33) (82). These mice are

hereafter referred to as OVA scid mice. Since OVA scid mice bear the scid mutation, these

mice have no B cells and only have T cells that bear the a(3 TCR transgene. Therefore the

response of a clonotypic population of SEB reactive T cells can be studied in the absence of al1

other T or B cells. By adoptively transferring different T lymphocyte subsets into this mouse

model, the role that T cells play in the SEB response can be examined. The role of CD8 T ceIls

in SEB induced CD4 VB8 T ceil deletion was investigated by adoptively transfering different T

lymphocytes subsets into these rnice. To overcorne the problem of the high percentage of

superantigen reactive T cells present in the OVA scid mouse, adoptive transfers of OVA scid

splenocytes dong with CD4 or CD8 cells were done into a C.B-17 scid mouse. The results

indicate that a clonotypic population of T cells in the absence of any other T or B cells is able

to mount a response to SEB in the spleen by increasing in transgenic T cell number. These

transgenic T cells are able to undergo superantigen induced deletion. The deletion seen rnay be

due to many factors including the migration of T cells from the spleen to lymph nodes or liver.

The deletion seen may also be due to death of T cells by apoptosis or a combination of

migration and apoptosis. IL-2 R a staining suggests that the T cells in the OVA scid were

activated in response to SEB. Upon SEB injection a population of CD4 TCR+ cells was

detected in the spleens of SEB injected OVA scid nuce. A similar population was seen in the

thymus but not in the spleen of control mice.

Materials and Methods

Mice: BALBlc mice were obtained from the Jackson Laboratories (Bar Harbor, ME). DO-1 1.10

TCR transgenic mice, hereafter referred to as OVA mice (83) were kindly provided by Dr.

Demis Y. Loh (Washington University School of Medicine, St. Louis, Mo.). These mice

express a transgenic aQ T ce11 receptor reactive to an ovalbumin peptide (ova 326-336) plus 1-

A" (8 1) (83). The rnice were produced by Loh et al as follows. A genomic library of the

DO. 1 1.10 hybridoma (84) was made. Probes specific for the remanged DO. I 1.10 a and P

genes were used to isolate the productively rearranged a and P genes. A 13 kb fragment

containing the correct V a rearrangement and a 12 kb fragment containing 5 kb of DNA

upstream of the rearranged VP8.2 including Vfl5.1 and the complete CB1 gene were obtained

and verified through sequencing and restriction mapping. The 13 kb fragment with the correct

V a rearrangement was ligated into the cosmid CaBS2. This cosmid contained the constant

region of the CI chain. The 12 kb b chah tiagment was Iigated to a DNA fragment containing

the entire C92 gene, the Cb enhancer and VQ14 gene and the resulting construct was ligated

into a PCK-X vector. The final fiinctional cx and g constructs used for injection were cut out

of their respective vectors and were 47 kb and 38 kb respectively. Fertilized ernbryos were

obtained from 3-4 week old superovulated (C3HlHeJ X C57BY6) FI females that were mated

to BALBIc males. The rearranged cc. and P gene constructs were injected into the embryo

pronuclei. The first transgenic progeny contained 4 copies of the chah and two copies of the

a chain and was of the H-2" haplotype. This transgenic male was mated to BALBIc H-2*

females to produce H-2d transgenic mice used for breeding. The OVA mice express the Va13

and Vp 8.2 chains of the TCR. The Va13 VP8.2 TCR is SEB reactive (33) (82). In the

Miller lab, the OVA mice were were backcrossed to BALBIc mice for nine generations. Mice

were screened for the presence of the transgene by flow cytometry using a FITC conjugated

MR 5-2 (anti VP 8.1,8.2) antibody (Pharmingen, San Diego CA). C.B- 17 rnice are congenic

mice originally made by mating BALB/c mice with B6 mice for 17 generations and selecting

for the IgH locus of B6 mice on a H-2d background (85). C.B-17 scitl mice (80) are C.B. 17

mice which have a mutation in the scici gene (86). These mice lack T or B cells due to the scid

defect and have the H-2d haplotype. The OVA scid mice were made in the Miller lab by

mating OVA mice to C.B- 17 scid mice for 6 generations. Mice were screened for rhe presence

of the transgene and absence of any other T or B cells by flow cytometry using a PE

conjugated 53-2.1 (Thy 1.2) antibody (Pharmingen, San Diego CA) and FITC conjugated Ml?

5-2 (anti VP 8.1,8.2) antibody (Pharmingen, San Diego CA). Thereafter the mice were

maintained by brother sister rnating. Al1 mice were bred and maintained in the defined flora

animal colony at the OC1 (Ontario Cancer Institute).

Antibodies and Reaqents: Staphylococcal enterotoxin B (SEB) was purchased fiom Sigma Immunochemicals (St. Louis,

Mo). Monoclonal antibody KJ 1 26.1 ( mouse IgG2a) is specific for the transgenic TCR

expressed by the OVA scid mouse (87). KJI 26.1 culture supernatant was collected from the

KJI 26.1 hybridoma (kindly psovided by Kappler and Mai~ack). The antibody was purified on

Protein A sepharose by affinity chromatography (88) and FITC labeled according to the

Current Protocols in Imrnunology (The Gold Book Bulletin: Supplement 1 June 1991). PE

conjugated H 129.19 (anti-CD4) (89) and PE conjugated 53-6.7 (anti CD8) (90), PE

conjugated RA3-6B2 (anti B220) (91), FITC conjugnted 298 (anti CD3) (92), Steptavidin

Quantam Red and 7AAD (a DNA intercalating dye) (93) were purchased fiom Sigma

Immunochemicals ( St. Louis, Mo.). FITC conjugated MR 10-2 (anti Vp9) (94) was a

generous gift fiom Dr Danska (University of Toronto, Toronto, ON). Biotin conjugated 7D4

(anti IL-2 receptor a) (95) purchased li-om Pharmingen (San Diego, CA) was a generous gift

fiom Dr Ohashi (University of Toronto, Toronto,ON). FITC conjugated MR 5-2 (anti VP

8.1,8.2) (96), FITC conjugated RR4-7 (anti-Vp6) (97) and PE conjugated 53-2.1 (Thy 1.2)

(90) were purchased from Pharmingen (San Diego, CA). Anti-FCR y II supernatant was

produced h m the hybridorna 2.462 (98) purchased from the American Type Culture

Collection (Rockville, MD). Mouse serum was purchased fiom Jackson Imrnuno Research

laboratories (BayHarbor, ME). Anti-CD4 ascites was produced fiom the hybridoma GK 1.5

(99), anti-CD8 ascites was produced from the hybridoma 53-6.72 (90). Both hybridomas

were purchased fiom the Arnerican Type Culture Collection (Rockville, MD) and raised in

C.B-17 scid rnice. Magnetic beads coated with sheep anti-rat IgG were purchased fiom

Dynal (Oslo, Nonvay). Magnetic beads coated with goat anti-mouse IgG beads were

purchased from BIOMAG (Cedariane, Hornby, ON)

Culture Medium: cr MEM purchased fiom Gibco (Missaugua, ON) was supplemented with 20p.M Hepes pH

7.3,50p.M B2-mercaptoethanol, penicillin, streptomycin plus 10% heat-inactivated fetal calf

serum purchased fiom Biowhittaker (Walkersville, MD). This was the media used for al1 the

splenocyte suspensions and is hereafter referred to as complete media (CM).

Flow cvtometrv: All monoclonal antibodies used have been titrated. The following monoclonal antibodies were

used for staining: 1 pg F'ITC conjugated KI 1 26.1 (anti OVA TCR), 0.5pg PE conjugated

Hl 29.19 (anti-CD4), 0.5pg PE conjugated 33-6.7 (anti CD8), 0Spg PE conjugated RA3-6B2

(anti B220), 0.25pg FITC conjugated 29B (anti CD3), 0.5 pg Steptavidin Quantarn Red and 1

pg 7AAD (a DNA intercalahg dye) 0Spg biotin conjugated 7D4 (anti IL-2 receptor a), 1pg

RTC conjugated MR 5-2 (anti VP 8.1,8.2), lpg F'iTC conjugated RRC7(anti VP 6) and

0.125pg LgTC conjugated MR 10-2 (anti VP9). In some cases the splenocytes were harvested

using Lympholyte separation medium (Cederlane Laboratories, Homsby, ON) to remove dead

ceiis. The cells were then washed three times with CM. 5 x 10' cells were washed with 1 %

BSA in PBS (+ c ~ Z ' and Mg .") and then incubated with 40 pl anti FCR y II supernatant (not

added in figure 3 and table 1 data). 5 p1 mouse serum, titrated Biotin, FITC and PE conjugated

antibodies in a final volume of 150-200 jd for 20 minutes. Stained cells were washed with 1 %

BSA in PBS. The secondary antibody steptavidin quantum red was added (0.5pg) and the

cells were incubated for 20 minutes. Stained cells were then washed with 1 % BSA PBS. For

cells that were stained with FITC and PE only, one wash with 1 % BSA PBS was done

followed by an incubation of 5-20 minutes with i pg 7AAD as an indicator of dead cells. In

cases where the splenocytes were passaged over Lympholyte M to eliminate dead cells no

7AAD was added (figure 3 and table 1). Cells were ,ulalyzed using a FACScan flow cytometer

(Becton Dickinson & Co., Mountain View, CA). Fluorescence data were collected using

Iogarithmic amplification; 10,000 events were collected.

Determination of cell numbers: Spleen suspensions were stained ris desci-ikd above. Forward scatter was plotted on the X

axis and side scatter was plotted of the Y axis of a dot plot. Red blood cells (RBC) were gated

out of the analysis based on their fonvard scatter and side scatter characteristics. To exclude

dead cells forward scatter was plotted on the Y mis of a dot plot and 7-AAD (FL-3) was

plotted on the X a i s . 7-AAD positive cells were gated out of the analysis. For two color

analysis FL-2 was plotted on the Y a i s and FL-1 was plotted on the X axis of a dot plot. The

percentage of cells in the appropriate susbset was determined. This percentage was multiplied

by the total number of eosin excuding splenocytes counted on a haemocytometer.

Iniections: 50 pg of SEB in PBS was injected intravenously in the tail vein of the rnice. Fractionated or

unfractionated splenocytes were resuspended in PBS and injected intravenously in the tail vein

of the mice.

Statistics: Independent T tests were used for the statisrical analysis. Al1 calculations were performed

using the cornputer program Microcal Origin version 3.5 (Microcal Software. Inc.,

Northampton, MA).

Pre~aration of unfractionated splenocvtes and fractionation of 6 de~leted splenocvtes Mice were sacrificed by cervical dislocation and single spleen suspensions made by standard

techniqiies (100). This was the source of unfractionated cells. For the B depleted T cells used

in Table 1 the splenocytes were were harvested using Lyrnpholyte separation medium

(Cederlane Laboratories, Hornsby. ON) and washed three times in CM. For the subsequent

fiactionations no Ippholyte was used. The suspension then was passaged over a nylon wool

colurnn (101). The percentage of cels recovered fiom the nylon wool purification was about

30-40%. Splenocytes were collected and resuspended at 10' celldml in CM. This suspension

was then treated with magnetic beads coated with goat anti-mouse IgG at the ratio of 20: 1

beaddcell. The mixture was continuously mixed at 4 ' ~ for 45-60 minutes. B cells were then

removed magnetically. The purity of the populations was monitored by flow cytometry. Cells

and was 66-93% CD3+ and 0.4%-1.2% B220+.

Preparation of T depleted s~lenocvtes To obtain T depleted spleen, spIenocytes were harvested using Lympholyte separation medium

(Cederlane Labaratories, Hornsby, ON) and treated with 11300 dilution of rat ascites against

CD4 (GK 1.5) and CD8 ( 53-6.72) at 4OC for a 30 minutes. The suspension was washed and

then magnetic beads coated with goat anti-rat IgG were added (ratio of 2: 1 beaddcell). The

mixture was continuously mixed at 4°C for 30-45 minutes. CD4 and CD8 cells were then

removed magnetically. The purity of the populations was monitored by flow cytometry. The

purity of the population was 90% B220+, 4.3% CD3+, 5.7% CD3- B220-.

Preparation of fractionated CD4 or CD8 cells: For the fractionation of CD4 and CD8 cells, cells fiom the nylon wool column sepuation were

treated with 11300 dilution of rat ascites against CD4 (GKI .5) or CD8 (53-6.72) at 4 O C for a

30 minutes. The suspension was washed and then treated with magnetic beads coated with

goat ~ti-mouse IgG at the ratio of 20: 1 beadslcell and magnetic beads coated with sheep anti-

rat IgG at the ratio of 2: 1 beaddcell. The mixture was continuously mixed at 4°C for 45-60

minutes. B cells and CD4 or CD8 ceHs were then removed masneticnlly. The percentage of

cells recovered from the magnetic k a d sort was about 1 8-34% for CD4 cells and 12-20% for

CD8 cells. The purity of the populations was monitored by flow cytometry. The purity of the

CD4 cells was 90% CD4+, 2.0% CD8,2.0% B220,6.0% CD3- B220-. The purity of the

CD8 cells was 79% CD8,2.0% CD4,2.0% B220, 17% CD3- B220-.

Results

A characterization of OVA scid versus BALBlc thvmocvtes In order to compare TCR transgenic scid thymocytes with non transgenic thymocytes,

thymocytes from OVA scid and BALBIc mice were stained with FITC conjugated anti-CD8,

anti-VB8 or anti OVA TCR and PE conjugated anti-CD4 antibodies (figure 1). The OVA scid

thymus (A) had a higher percentage of CD&, CDS+ and CD4- CDS- thymocytes and a lower

percentage of CD& CDS+ ceUs than a BALBIc thymus (B). Al1 the OVA scid thymocytes

expressed VB8 (C). Only a portion of BALBic thymocytes expressed VBS (D). Ali the OVA

scid thymocytes also expressed the transgenic TCR (E). Only a portion of the Vp8+ (C) and

KJ 1 26.1 + (E) thymocytes expressed CD4 and thus there was a population of CD4- KJ 1 26.1 +

and a population of CD4- Vp8+ cells present in the OVA scid thymus.

A characterization of OVA scid versus BALBlc splenocvtes In order to compare TCR transgenic scicl splenocytes with non transgenic splenocytes,

splenocytes fiom OVA scid and BALBlc mice were stained with HTC conjugated anti-CDS,

anti-VOS or anti OVA TCR and PE conjugated anti-CD4 antibodies (figure 1). The OVA scid

spleen (F) had a higher percentage of CD& cells than a BALB/c spleen (G). The OVA scid

spleen had no CDS+ (F) or B220+ cells (1). The CD& cells in the OVA scid spleen expressed

Vp8 (H) and KJ 1 26.1 (L). BALBIc spleen had CD8+ (G) cells and B220+ (K) and bai-ely

detectable levels of CD4+ KJ 1 26.1 + (M) cells. Only a portion of the CD4 cells in the

BALBIc spleen expressed Vp8 (1).

Fiaure 1 : OVA scid and BALBlc thvmus and spleen Splenocytes (F, G, H, 1, J, K, L and M) and thymocytes (A, B, C, D and E) from OVA scid and BALBic mice were stained with FITC conjugated and PE conjugated antibodies. A, B, F and G: PE conjugated anti CD4 and W C conjugated anti CDS. C, D, H and 1: PE conjugated anti CD4 and FITC conjugated anti Vf3 8.1,8.2. E and L: PE conjugated anti CD4 and FITC conjugated K J I 26.1 (anti OVA TCR). J and K: PE conjugated anti B220 and HTC conjugated anti CD3. Lymphocytes were gated on based on fonvard scatter, side scatter parameters. Dead cells were gated out using the DNA dye 7AAD. Both the BALBic and OVA scid mice were 12 weeks old. Splenocytes fiom a BALBlc mouse were stained with anti CD4 and FïïC conjugated KJI 26.1 (anti OVA TCR) (M) in an independent experiment. The percentage of splenocytes or thyrnocytes of the respective populations is shown in the upper right quandrant.

OVA scid thvmus BALBIc thymus

OVA scid thymus

P .7

KJI 26.1 (3) vs CD4 (4)

OVA scid spleen BALBIc spleen

OVA scid spleen

CD4 VB8 T cells res~ofld to SEB In order to ascertain the antigen specificity of the SEB reponse, BALBIc mice were injected

with SEB and assayed 4 or 1 1 days after injection (4 and 1 1 ); control (C) mice were given PBS

and assayed 1 i days after injection (figure 2). After 4 days or 1 1 days, mice were sacrificed

and the number of CD4 Vp8, CD4 VP6 or CD4 VP9 cells in the spleen was enumerated by

flow cytometry. There was an significant increase in the spienic number of CD4 VP8 but not

CD4 VP6 or CD4 VP9 cells 4 days following SEB injection. On day 1 1 there was a decrease

in the splenic number of CD4 Vp8 but not CD4 Vp6 or CD4 Vp9 cells as compared to day 4.

Therefore SEB allowed for an specific increase and decrease in splenic CD4 VP8 T cell

nurnbers.

SEB remonse in OVA scid versus BAlBlc mice According to some groups the extent of proliferation (and deletion in response to SEB is

dependent on the initial proliferative response to SEB (75). According to other groups CD8 T

ceils may control the reduction in the number of CD4 VB8 T cells in response to SEB (1 3)

(74). Since OVA scid mice had only a clonotypic population of T cells in the absence of other

T or B cells, a comparison was made of the SEB response in OVA scid mice versus BALBIc

mice. Mice were injected with SEB and assayed 4 or IO days afier injection (4 and 10); control

(C) mice were given PBS and assayed 10 days after injection. After 4 days or 10 days, mice

were sacrificed and the number of CD4 Vp8 (BALBlc) or the nuniber of CD4 KJ1 26.1, KJ1

26.1 (OVA scid) cells in the spleen was enumerated by flow cytometry (figure 3). Both OVA

scid mice and BALB/c mice were capable of responding to the SEB injection since there was

Fiqure 2: CD4 VI38 T cells respond to SEB Splenocyes f?om SEB injected (4, 1 1) or PBS injected (C) BALBIc were assriyed on day 4 or day 1 1 after injection. BALB/c splenocytes were stained with PE conjugated anti CD4 and FiTC conjugated anti VP8.1, 8.2, PE conjugated anti CD4 and FiTC conjugated anti VP6 or PE conjugated anti CD4 and FITC conjugated anti V(3 9 at day 4 or 1 1 after injection. The numkr of cells per spleen inbthe appropriate subset was enumerated. BALBIc mice were 6 wks at tirne of injection. Dead cells were gated out using the DNA dye 7AAD. Control mice (C) were given PBS. Three mice were used per group. The standard error of the mean is shown.

Fiaure 3: SEB response in OVA scid versus BALBlc mice Splenocytes from SEI3 injected (DAY 4, DAY 10) or PBS injected (C) BALB/c and OVA scid mice were harvested using lyrnpholyte separation medium. BALB/c splenocytes were stained with PE conjugated anti CD4 and FITC conjugated anti VB8.1, 8.2 at day 4 or 10 after injection. The number of CD4 VB8 cells per spleen was enumerated. OVA scid splenocytes were stained with PE conjugated anti CD4 and FITC conjugated KJ1 26.1 (anti OVA TCR) at day 4 or 10 after injection. The number of CD4 VCj8 and CD4 KJ 1 26.1 cells per spleen was enumerated. BALB/c rnice were 12 wks at time of injection and OVA scid mice were 10- 16 weeks old at the t h e of injection. The standard error of the mean is shown. Two (C), four ( DAY 4) and four (DAY IO) mice were used.

BALBlc

1 DAY

OVA scid

C 4 1 O

DAY

an increase in the splenic number of CD4 VP8 (BALBIc) and CD4 KJI 26.1 (OVA scid) in

both mice 4 days following SEB injection. The response of OVA scid and BALBIc mice was

compared on day 10. While the number of CD4 VB8 cells recovered from the BALBIc on day

IO was less than that of PBS injected (C) mice, the number of CD4 KJ1 26.1 or KJ1 26.1 cells

recovered from OVA scid rnice was not significantly less than that of PBS injected (C) mice.

An independent T test comparing the number of CD4 Vp8 T cells recovered fiom control and

day 10 mice suggested that the difference seen in BALBIc mice were statistically signifiant

(p<0.01). The differences seen in the number of CD4 KJ1 26.1 cells recovered from control

versus day I O OVA scid mice was not statistically significant (p>0.01).

Ado~tive transfer of BALBlc s~lenocvtes into OVA scid mice Since the OVA scid rnice lack any T or B cells and CD8 T cells have k e n implicated as king

important in the reduction of CD4 VP8 cells T to below levels found in control mice (1 3), we

wanted to investigate whether the addition of fractionated or unfiactionated BALBIc

splenocytes could affect the number of CD4 KJ 1 26.1 T cells recovered from SEB injected

OVA scid mice. To explore this question a set of adoptive transfer experiments was done

(table 1). To examine the role of other T cells in the SEB response, fractionated B cell

depleted (exp 4), T ceIl depleted (exp 5) or unfractionated BALBIc splenocytes (exp 1-4) were

coinjected with SEB into OVA scid mice. The response of the OVA scid T cells (CD4 KJ1

26.1) was followed. Control mice were given SEB (SEB) or PBS (C) only. SpIenocytes from

the mice were stained with CD4 KJ 1 26.1 antibodies at day 8,9 and 10 in five independent

experiments (exp 1-5). The number of CD4 KJ 1 26.1 cells per spleen was enumerated. The

addition of unfractionated BALBIc spleen (exp 1-4) was unable to reduce the number of CD4

TABLE 1 : Ado~tive transfer of BALBlc s~lenocvtes into OVA scid mice Adoptive transfers of varying numbers of untiactionated (uc) or fractionared (fc) BALBic splenocytes plus SEB were done into an OVA scid. Control mice were given PBS (C) or SEI3 (SEB). The age of the recipient mice for each experirnent is shown. At the appropriate day of assay (day) splenocytes fiom the mice were harvested usin; IyrnphoIyte separation medium. Splenocytes were stained with PE conjugated anti CD4 and FiTC conjugated KJ1 26.1 (anti OVA TCR). In experiment 5 (exp) the splenocytes were also stained with PE conjugated anti B220 The number of CD4 KJ 1 26.1 ceils per spleen was enurnerateci.

# ceils / group / cls 6.6~+071 C 14.2~+06

Exp 1

2

3

4

5

SE0 1 SEB 2 SEB+ uc

cells unfrac

unfrac

unfrac

unfracg frac

9frac4

age 10

12

9

4

11

4.6E+06 7.6E+06 4.OE+07

6.1 E+07

Exp (expriment number), age (age of rnouse in weeks), day (day of assay), cells ( t jpe of cells injected), # cells (number of fractionated or unfractionated BALBlc splenocytes injected), group (group). c l s (number of cells F r spleen). % inj cells (percentage of CD4+ KJ I 26.1- injecied cells recovered) 1. % CD4 KJ 1 26.1 splenocytes recovered 2. % CD4+ KJ 1 26.1- 3. fractionated cells: 92% CD3+, 1.2% B22Qt. 6.8% CD3- B220-

unfractionated cells: 43% CD3+, 52% B220+, 5% CD3- B220- 4.90% B220+, 4.3% CD3+, 5.78 CD3- B220- 5. B22ût

day 8

9

10

9

7.5E+07

C SEB SEB + uc 1 SE0 + uc 2

2.8E+06 9.5E+06 1.2E+07 1.4E+07

C S EB SEB + fc

2.9E+06 4.4E+06 1.9E-i.07

KJ1 26.1 cells recovered to less than that of control (C) mice. In experiments 1 (SEB l), 4

and 5 the nrirnbers of CD4 KJ 1 26.1 recovered kom SEI3 treated mice was less than the

numbers recovered fiom control. However in experiments 1 (SEB 2), 2 and 3 the nurnbers of

CD4 Ki 1 2 1 recovered from SEB treated mice was more than the numbers recovered from

control. The percentage of CD4 KJ1 26.1 cells in control mice varied from 69% (exp 5) to

74% (exp 1 ). The percentage of CD4 KJ I 26.1 cells in SEB injected rnice varied from 36%

(exp 5) to 69% (expt 2). Therefore the addition of unfractionated BALB/c splenocytes was

unable to reduce the numbers of OVA scid T cells recovered from SEB injected mice to less

than that recovered from PBS injected mice.

We postulated that since the percentage of CD4 Vp8 T cells is high in the transgenic

system the adoptively transferred cells may be unable to fùrther reduce the numbers of CD4

Vp8 T cells recovered. We tried adoptive transfer experiments using fnctionated splenocytes

fiom BALBIc mice, congenic to C.B- 17 differing only at the IgH locus, to answer the above

question. Adoptive transfer of OVA scid splenocytes plus BALBk T cells into a C.B- 17 scid

allowed us to get over the problem of having a high percentage of CD4 KJ 1 26.1tCD4 Vfi 8 T

cells. It also allowed us to use titration experiments to enhance the conditions required for the

reduction CD4 Vb8 T ce11 number.

OVA scid'h cells are detectable in C.B-17 scid mice mon ado~tive transfer In order to determine whether OVA scid T cells could be transferred and detected in a C.B- 17

scid mouse an adoptive transfer titration experiment was done. Adoptive transfen of OVA

scid splenocytes plus SEB were performed in a C.B- 17 scid. Vnrying numbers of OVA scid

splenocytes were injected into C.B- 17 scid mice. Control mice (O.OOE+OO) were given PBS

Fiqure 4: OVA scid T cells are detectable in C.B-17 scid mice upon adoptive transfer

Adoptive transfers of OVA scid splenocytes was done into C.B- 17 scid mice(l2 weeks old). Varying numbers of OVA scid spienocyies (65% CD& KJ1 26.1 +) were injected into C.B-17 scid mice. Control mice (O.OûE+O.OO) were given PBS. Splenocytes from the C.B- 17 scid recipients were stained with PE conjugated anti CD4 and FiTC conjugated KJ1 26.1 (anti OVA TCR) four days after injection. The number of CD4 KJ 1 26.1 cells per spleen was enumerated. Dead ceils were gated out uçing the DNA dye 7AAD. One mouse was used per group.

0.00E+00 3.00E+06 6.00E+06 1.00E+07

number of OVA scid cells injected

1 # OVA scid cells injected 1 celldspleen 1 % CD4+ KI 1 26. l + 1

(figure 4). Splenocytes from the mice were stained with CD4 KJ1 26.1 antibodies 4 days afier

injection. The number of CD4 KJ 1 26.1 cells per spleen was enumerated. OVA scid T cells

were detectable after four days of transfer in the C.B- 17 scici in a dose dependent manner.

OVA scid T cells are capable of mountinci a remonse to SEB w o n ado~tive transfer To determine whether the adoptively transferred cells could respond to SEB in the C.B-17

scid, OVA scid splenocytes plus SEB were transferred into a C.B- 17 scid (table 2). Varying

numbers of OVA scid splenocytes were injected with SEB into C.B- 17 scid mice in three

independent experiments (A-C). Control mice were given OVA scid splenocytes only lo7

(expt A), 5 x 1 o6 (exp B) or 3 x 1 o6 (expt C), and assayed on day 4 after injection (table 2).

Splenocytes from the mice were stained with CD4 KJ1 26.1 antibodies at 4 (expt A-C), 7

(expt A), and 1 1 (expt B and C) days after injection. The number of CD4 KJ1 26.1 cells per

spleen was enumerated. The adoptively tmsferred OVA scid T cells (CD4 KJ 1 26.1)

recovered from mice given SEB plus OVA scid splenocytes were higher than C.B- 17 scid mice

given OVA scid splenocytes alone (C versus day 4 experiments A-C). C.B- 17 scid mice given

OVA scid splenocytes plus SEB and assayed on day 7 (exp A) or day 1 1 (expt B and C) had a

higher number of OVA scid T celIs (CD4 KJ 1 26.1 ) recovered than control (C) mice given

OVA scicl splenocytes ( C versus day 7 expt A, C versus day 1 1 expt B and C) of OVA scici T

cells (CD4 KJI 26.1). However, independent experiments (data not shown) in which OVA

scid cells were injected into C.B- 17 scici mice without SEB and assayed on day 12 or 13

showed that in the absence of SEB the CD4 KJ1 26.1 T cell were barely detectable (0.03%-

0.06%). Since the OVA scid T cells was were barely detectable in the absence of SEB, the

TABLE 2: OVA scid T cells are ca~able of mountina an immune remonse won adoptive transfer

Adoptive transfers of OVA scid splenocytes plus SEB were done into C.B- 17 scid rnice in 3 independent experiments (A-C). 1 x 10' (68% CD4+ KJ 1 26.1 +) A, 5 x 1 o6 (16% CD4 KJI 26.1) (B) or 3 x 106 (28% CD& KJ1 26.1+) C, OVA scid splenocytes were injected with SEB into C.B-17 scid mice. Control mice (C) were given OVA scid splenocytes only and assayed on day 4. Splenocytes from the C.B-17 scid mice were stained with PE conjugated anti CD4 and FITC conjugated KJ 1 26.1 (anti OVA TCR). The number of CD4 KJ1 26.1 cells per spleen was enumerated. Dead cells were gated out using the DNA dye 7AAD. One mouse was used per group.

Experirnent A

group C

Experirnent B

day 4 ) 7.2Ei-07 day 7 1 6.5E-I-07

Experiment C

celidspleen 1 .1E+07

1 group 1 cellsispleen 1 % CD4 KJ 1 26.1 1 # CD4 KT 1 26.1 1

1.9% 1.5%

%CD4 KJ 1 26.1 2.4%

1.4Ei-06 9.8E+05

1 day I I 1 7.8E-47 1 1.2% 1 9.4E+05

# CD4 K. 1 26.1 2.6EG5

C day 4

2.6EG7 1.OE-I-08

O. 19% 3.9%

4.9E+04 3.9E+06

reduction in splenic cell number of CD4 VB8 T cells in response to SEB cannot be compared

to mice given OVA scid T cells alone.

CD4 and CD8 T cells from BALBic reduce the number of OVA scid T cells recovered

Since CD8 T ceUs can influence the fate of CD4 T cells during an immune response (2) (3)

(3 l), we wanted to examine whether the addition of CD4 or CD8 cells could influence the

numbers of CD4 KJ 1 26.1 ceils recovered fiom a C.B-17 scid mouse injected with OVA scid

splenocytes plus SEB, Since OVA scid T cells (CD4 KJ 1 26.1) were not detectable upon

adoptive transfer into C.B- 17 scid mice in the absence of SEB on day 12 or 13, a comparison

was made between mice given OVA scid splenocytes pliis SEB versus OVA scid splenocytes

plus SEB and CD4 or CD8 T cells. The role of the added CD4 or CD8 cells in influencing

the number of CD4 KJI 26.1 recovered was investigated. It is important to note that since

there was no control (without SEB) in this experiment the issue of reduction in CD4 Vb8 T

cell number in comparison to rnice not given SEB was not addressed. CD4 and CD8 cells

from BALBic spleen were transferred with OVA scid splenocytes plus SEB into a C.B-17 scid

(figure 5). Control mice were given 5 x 1 o6 OVA scid splenocytes plus SEB only. 1 x IO'

fractionated BALBlc and 5 x 106 OVA scid splenocytes were injected with SEB into C.B- 17

scid mice. Splenocytes from the mice were stained with CD4 KJ 1 26.1 , CD4 Vg8 or CD3

B220 antibodies on day 1 I after injection. The numbers of CD4 KJ 1 26.1, CD4 Vp8 and CD3

cells per spleen were enurnerated. The number of CD4 Vp8 T cells recovered fiom mice given

BALBIc CD4T cells (CD4) was more than the number recovered from SEB (SEB) or

Figure 5: CD4 and CD8 T cells from BALBIc reduce the number of OVA scidT cells recovered Adoptive transfers of 1 X lo7 CD4 (cD~) ' or CD8 ( c D ~ ) ~ cells from BALB/c spleen and 5 X 106(54% CD4 + KJ1 26.1 +) OVA scid splenocytes plus SEB was done into C.B- 17 scid ( 1 2 weeks old). Control mice were given 5 x 106 OVA scid splenocytes and SEB (SEB). Splenocytes from the C.B-17 scid mice were stained with PE conjugated anti CD4 and FiTC conjugated KJ1 26.1 (anti OVA TCR), PE conjugated anti CD4 and FITC conjugated anti Vp8.1 8.2 or PE conjugated anti B220 and FITC conjugated anti CD3 at day 1 1 following the injections. The number of CD& KJ1 26.l+ cells, CD4-t Vp8+, CD3+ B220- ceiis per spleen was enurnerated. Dead cells were gated out using the DNA dye 7AAD. Three rnice were used per group. The standard error of the mean is shown.

group SEB CD4 CD8

ceIlsispleen 8.8E+M 1.6E+07 9.lE+06

#CD4 KJ 1 26.1 1,9E+05 4.5E+04 3.5E+04

# CD4 VP8 2.2E+05 7.7E+05 9.00Ei.04

# CD3 I.9Et0.5 1.6E+06 5.6Et05

O/o CD4 KJ 1 26.1 2.2 0.28 0.39

F/c CD4 VP8 2.5 4.8 0.99

70 CD3 2.2 1 O 6.2

BALB/c CD8 T cell (CD8) injected mice. This difference probably reflects BALBIc derived

CD4 VB8 T cells that respond to SEB. It is interesting to note that as compared to the mouse

given OVA scid splenocytes and SEB the addition of either CD4 or CD8 cells reduced the

numkr of CD4 KJ1 26.1 cells recovered. However, in the three groups it seemed that a

different nurnber of T cells (CD3+) were detected in the spleen. Mice given BALBic CD4

cells had more T cells (CD3+) in the spleen than mice given BALBIc CD8 cells. Therefore it is

possible that the BALB/c CD4 or CD8 cells occupied the space in the spleen that would

othenvise be occupied by CD4 KJ 1 26.1 cells. Although it seems that either CD4 or CD8 T

cells were able ta reduce the number of CD4 KJ 1 26.1 cells recovered; it is not clear whether

this result points to a direct role for CD4 or CD8 cells. This reduction in CD4 KJ i 26.1 cell

number could be non specific since both populations were able to reduce the numbers of CD4

KJ 1 26. I cells recovered.

Ado~tive transfer of BALBlc sdenocvtes into OVA scid mice

Since the experiments in table 1 had variable numbers of splenocytes recovered fiom control

OVA scid spleens ( 1 SOX 10'- 4 .70~ 1 06) it was possible that the lympholyte separation

medium was causing non specific loss of splenocytes in the OVA scid spleen. Therefore we

decided to repeat the observations seen in table 1 without the lynipholyte sepmation medium.

Adoptive trmsfers of fiactionated (B depleted) or unfractionated (unfrac) BALBtc splenocytes

were coinjected with SEB into OVA scid mice (figure 6). The response of the OVA scid T

cells (CD4 KJ1 26.1) was followed. Control mice were given SEB only (SEB). Splenocytes

from the mice were stained witli CD4 KJ 1 26.1 antibodies 1 1 days after injection. The nurnkr

of CD4 KJ1 26.1 cells per spleen was enumerated. The addition of B depleted or

Fiuure 6: Adoptive transfer of BALBlc s~lenocvtes into OVA scid mice Adoptive transfers of B depleted BALB/c spleen plus SEB were done into an OVA scid 1 x 10' B depleted splenocytes (B depleted)' or 1 x 10' unfi.actionated (unf~üc)' BALBlc splenocytes were injected with SEB into OVA scid mice. Splenocytes from the mice were stained with PE conjugated anti CD4 and FiTC conjugated KJ 1 26.1 (anti OVA TCR), PE conjugated anti CD4 and FITC conjugated anti Vp8.1 8.2 or PE conjugated anti B220 and FITC conjugated anti CD3 at day 1 1 following the injections. The number of CD4+ KJ1 26.1 + cells, CD4+ Vp8+, CD3+ B220- and B220+ cells per spleen was enumerated. Dead cells were gated out using the DNA dye 7AAD. Three mice were used per group. The standard error of the mean is shown. Control mice were given SE8 alone (SEB). OVA scid mice were 8 weeks old at the time of injection.

S B B depleted uni rac

El# CD4 KJl 26.1

.# Co4 Vb8

W U P

SEB B dcplctcd unhc

ccllskpleen 1.5EtO7 1.4E+07 1.8E+07

#CD4 KJ 1 26.1 ?.?Et06 1.4E+06 4.1 E+06

# CD4 Vp8 2.4E+06 1.8E+06 4.5E+06

# CD3 1.9E+06 4.8E+O6 2.6E+O6

# B72O O O 6.GE+O5

% CD4 KI I 76.1 15% 1 O%, 239

% CD1 VPS 1 6% 13% 25%

%B?ZO O O 3.7%

unfractionated (unfrac) BALBIc spleen was unable to significantly rediice the number of CD4

KJl 26.1 ceIls recovered from an SEB (SEB) injected OVA scid rnouse.

SEB response in OVA scid versus BALBlc mice Since figure 3 was done with a low number of mice two (control) four (day 4) and four (day

IO), and experiments in table 1 had variable numkrs of splenocytes recovered from control

OVA scid spleens (1 50x1 0% 4.70~1 06) it was possible that the lympholyte separation medium

was causing non specific loss of splenocytes in the OVA scid spleen. Therefore the experirnent

shown in figure 3 was repeated with a larger number of mice and without lympholyte (figure

7). BALBIc or OVA scid mice were injected with SEB (4 and I 1 ) ; control mice (C) were

given PBS. After 4 days or 1 1 days, mice were sacrificed and the number of CD4 VB8

( B A W c and OVA scid) and CD4 KJ1 26.1 (OVA scid) in the spleen was enumerated by flow

cytometry (figure 7). Both OVA scid mice and BALB/c mice seemed to be capable of

responding to the SEB injection since there was an increase in the number of CD4 VB8 in both

mice at 4 days following SEB injection. The response of OVA scid and BALBIc mice on day

1 1 was compared. The number of CD4 VpS T cells recovered from both the BALBlc and

OVA scid mice on day 1 1 was less than that of PBS injected mice. An independent T test

comparing the number of CD4 VP8 T cells recovered From control and day 1 1 mice suggested

that the differences between BALB/c control and day 1 1 SEB injected were significant

(pc0.05), and the differences seen between OVA scid control versus day 1 1 were not

significantly different (p>0.05). However at the the 2% significance level neither difference

was statistically significant. Therefore any differences seen between the BALBlc mice versus

the OVA scid micc were mimimal.

Fiuure 7: SEB rewonse in OVA scid versus BALBIc mice Splenocytes from SEB injected ( DAY 4, DAY 1 1) or PJ3S injected (C) BALB/c and OVA scid were assayed on day 4 or day 1 1 after injection. BALBic splenocytes were stained with PE conjugated anti CD4 and FiTC conjugated anti Vg8.1, 8.2 at day 4 or 1 I after injection. The number of CD4 VP8 cells per spleen was enumerated. OVA scid splenocytes were stained with PE conjugated anti CD4 and FITC conjugated anti VpS. 1, 8.2 or PE conjugated anti CD4 and FiTC conjugated KJ 1 26.1 (anti OVA TCR) at day 4 or 1 1 after injection. The number of CD4 Vp8 and CD4 KJ 1 26.1 celis per spleen was enumerated. BALB/c rnice were 12 wks at time of injection and OVA scid mice were 14 weeks old at the time of injection. Dead cells were gated out usin$ the DNA dye 7AAD. Control mice (Cj were given PBS. Four mice were used per group. The standard error of the mean is .. - shown. - .- -

BAL wc

- ....................

.. -. - -- - --

4 DAY

OVA scid

4 DAY

IL-2 receptor a expression on OVA scid versus BALBlc CD4 VB8 T cells Since IL-2 has been implicated in SEB rnedinted deletion (76), (65) , the expression of the IL-2

R on OVA scid T cells upon SEB injection was investigated. The levels of IL-2 receptor

were assayed in OVA scid and BALBIc mice 20 hours after SEB injection (B and D). Control

mice were given PBS (A and C). Both BALBIc and OVA scid mice had elevated levels of IL-

2 receptor a 20 hours following SEB injection (figure 8). Since OVA scid splenocytes

expressed detectable levels of IL-2 receptor c i upon SEB injection, they seemed to be activated

in response to SEB.

CD4- TCR+ cells are seen u ~ o n SEB iniection Upon SEB injection a population of cells that do not express CD4 but do express TCR were

seen. Splenocytes from SEB injected (B and D) or PBS injected (A and C) OVA scid mice

were assayed on day 1 1 after injection (figure 9). The SEB injected rnice had a splenic

population of CD4- VB8+ (B) and CD4- KJ1 26.1 (D) cells. This population was barely

detectable in PBS injected mice (panel A, C). In terms of number of cells per spleen the SEB

injected mice had the following nurnbers: CD4- VB8+ ( 1.8 x 1 o6 + 1.6 x 1 0') md CD4- KJ 1

26.1 ( 2.0 x 1 o6 k1,8 x 1 05). The PBS injected mice had CD4- VB8+ ( 9.9 x 1 0' $2.8 x 10')

and CD4- KJ 1 26.1 ( 5.9 x 1 0' k 2.1 x 1 04).

Fiqure 8: 11-2 receptor a expression on OVA seidversus BALBlc CD4 VB8 T cetls -

Splenocytes from OVA scid and BALBIc mice injected with SEI3 (B, D) or PBS (A, C ) were stained with PE conjugated anti CD4, FiTC conjugated anti V(38.1,8.2 and biotin conjugated anti IL-2 Ra at 20 hours after injection. CD4 VB8 cells were gated for their IL-2 R a. Data shown is representative of 3 SEB injected rnice and 2 control rnice. Mice were 10 weeks at the time of injection. The MFI is shown.

OVA scid

Fisure 9: CD4 TCR+ cells are seen won SEB iniection Splenocytes from SEB injected (B and D) or PBS injected (A and C) OVA scid mice were assayed on day 1 1 after injection. OVA scirf splenocytes were stained with PE conjugated anti CD4 and FITC conjugated anti VP8.1, 8.2 or PE conjugated anti CD4 and FITC conjugated KI 1 26.1 (anti OVA TCR) at 1 1 afier injection. OVA scid mice were 14 weeks old at the time of injection. Dead cells were gated out using the DNA dye 7AAD. Lymphocytes were gated on based on fonvard scatter and side scatter parameters. The percentage of splenocytes of the respective populations is shown in the upper right quandrant.

PBS iniected SEB iniected

O YB 8 (3) YS CD4 (4)

O 7

Discussion The CD4 VP8 response to superantigens in vivo is characterized by a burst of proliferation that

peaks on day 3-4 and a subsequent drop in ceil nurnber (day 8-1 1) that results in the deletion of

the responding cells to below the levels found in PBS injected control rnice (50) (54).

According to some groups the extent of deletion seen in response to SEB is dependent on the

extent of proliferation (75). According to other groups CD8 cells may play a rale in reducing

the nurnber of CD4 Vp8 T ceiis to below levels found in PBS injected controls (1 3). The

superantigen SEB was used to analyze the immune response in a C.B- 17 scid mouse

expressing a transgenic a$ T cell receptor reactive to a class II restricted 1-A~ ovalbumin

peptide. This TCR receptor expresses Va 13 and VB8.2 and is reactive with SEB. Since

C.B-17 mice bear the scid mutation, OVA scid mice have no B cells and the only population of

T cells that they wiii have will bear the transgenic TCR. Therefore using this rnouse the

response of a single clonotypic population of SEB reactive T celIs can be studied in the absence

of al1 other T or B ceils. The monoclonal antibody KJ1 26.1 specific for the transgenic TCR

expressed by the OVA mouse (87) was used to follow the course of these cells during the

immune response.

In order to examine the SEB response in the OVA scid mouse, the response of OVA

scid and BALBIc mice at 4 and 1 1 days after SEB injection was compared. Both these mice

have the H-2* haplotype and have TCRs bearing Vp8 chains in varying proportions. Both

mice seemed to be capable of responding to the SEB injection since there was an increase in

the nurnber of CD4 VB8 in mice 4 days following SEB injection. Since the OVA scid mouse

mounted a comparable response to SEB lit vivo, a clonotypic T cell is able to mount an SEB

induced immune response in the absence or presence of other T cells or B cells. In order to

furthur study the response to SEB in these mice, the response of OVA scid and BALB/c mice

was compared on day 1 1. The number of CD4 V(38 cells recovered from both the BALBlc

and the OVA scid mouse on day 1 1 was less than that of PBS injected mice. Although the

OVA scid mouse has only the clonotypic population of SEB reactive T cells in the absence of

other T cells, it is stilI capable of responding to SEB in the absence of other lymphocytes.

Therefore a clonotypic population of T cells is able to mount an SEB response in the spleen

from proliferation to deletion. It is important to note that deletion is defined as the loss of CD4

KJI 26.1 ceUs from the spleens of mice after day 4 of SEB injection.

In order to examine the role for other T cells particularly CD4 and CD8 T cells in the

response to SEB, we decided to investigate whether the addition of other CD4 and CD8 T

cells could influence the number of CD4 KJ1 26.1 T cells recovered fiom SEB injected OVA

scid mice. The roie of other CD4 cells or CD8 cells in the SEB response is not clearly defined

(13 (74). There are two reports that discuss the role of CD8 T cells in reducing the number of

CD4 VP8 T cells recovered fiom SEB injected mice. One report using CD8 depleted mice

suggests that the role for CD8 celis is minimal if any, since CD8 depleted mice seem to have

less CD4 VB8 T cells recovered from SEB injected mice than from PBS injected mice (74).

Another report using both CD8 depleted and (32m knockout mice (lack class 1 MHC) suggests

that CD8 celis are needed to reduce the numbers of CD4 V(38 T cells recovered to less than

that in PBS injected mice (13). They suggest that CD8 cells lyse CD4 cells that have

previously ken activated in response to SEB. It is important to note that the latter report

defined CD8 cytotoxic function entirely itz vitro and presented no evidence that this function

exists irl vivo. The role for y6 T cells and CD4 cells other than the ones that c q the SEB

reactive TCR in the SEB response is unknown.

In order to test the role that T ceIIs, specifically CD8 cells, have in reducing the

numbers of CD4 Vp8 T cells in SEB injected mice, a set of adoptive transfer experiments was

done. Fractionated (B depleted) or unfractionated BALBIc splenocytes were coinjected with

SEB and the immune response was followed. The addition of any one of these populations was

unable to affect the numbers of OVA scid T cells recovered. Therefore there was no further

reduction in OVA scid T ce11 numbers seen by the addition of unfractionated or ftactionated

BALBlc splenocytes. It was postulated that perhaps the percentage of CD4 V88 cells was so

high in the transgenic system that the adoptively transferred cells were unable to effectively

reduce them. An alternative adoptive transfer system was investigated. Adoptive trmsfers of

OVA scid cells plus BALBIc cells, congenic to C.B-17 and differing only at the IgH locus, into

a CB. 17 scid mouse was investigated. This aIlowed one to get over the problem of having a

large number of antigen reactive cells, This approach also allowed for titration experiments to

optimize the conditions needed for the reduction in ce11 number. However, in the absence of

SEB, the transferred OVA scid T cells were barely detectable. It is possible that the presence

of the transgene or the presence of the scid mutation in these T cells results in a defect in long

term recirculation ( Dr Ohashi personal communication). Therefore the question of the

reduction in number of CD4 Vp8 T cells as compared to mice not given SEB cannot be

addressed in this system.

Since CD8 T cells c m influence the fate of CD4 T cells during an immune response (2)

(3) (3 11, we wanted to detennine whether the addition of CD8 cells could influence the

numbers of OVA scid T cells recovered in C.B- 17 scid mice injected with OVA scid T cells

plus SEB. Adoptive transfers of CD4 or CD8 T cells and OVA scid splenocytes plus SE3

was done into a C.B-17 scid (figure 5). It is interesting to note that as compared to the mouse

given OVA scid spenocytes and SEB the addition of either CD4 or CD8 cells seemed to

reduce the number of CD4 KJ1 26.1 cells recovered. Since either CD4 or CD8 cells were able

to reduce the number of OVA scid T cells recovered, the reduction in OVA scid T ce11 number

may not be specific to CD4 or CD8 cells. The CD3 data in figure 5 showed that a different

number of T cells were recovered fkom the spleen in the three situations. It is possible that in

the presence of CD4cells more T cells homed to the spleen than in the presence of CD8 cells.

Therefore the CD4 T ceils that homed to the spleen rnay occupy the space of the TCR

transgenic cells,

Our results are consistent with Williams et al's results that showed that CD8 cells alone

do not play an active role in mediating deletion of SEB reactive T cells (74). This result is in

contrast to Jiang et al's work (1 3) that suggested that CD8 cells play an active role in SEB

induced deletion of CD4 V$8 T cells. However since their work primarily dehed the

cytotoxic role of CD8 cells in vitro this phenomenon may not exist in vivo. Our results show

that a clonotypic population of T cells is able to mediate al1 phases of the SEB induced immune

response in the spleen from proliferation to deletion. IL-2 R cx staining suggests that the T cells

in the OVA scid have ken activated in response to SEB. Adoptive transfers of B depleted

BALBIc spleen or unfractionated BALBlc spleen was unable to influence the extent of SEB

induced deletion of CD4 KJI 26.1 celIs. Therefore a minimal role for CD8 T cells in SEB

induced deletion was found in our mode1 system. Adoptive transfers of CD4 or CD8 cells plus

OVA scid T cells into a C.B-17 scid was able to reduce the numkrs of OVA scicl T cells

recovered from a single dose of SEB. However this reduction in the nurnber of OVA scid T

ce11 recovered is not specific to CD4 or CD8 cells, and does not relate to SEB induced CD4

VP8 T ce11 deletion.

Recent evidence frorn several groups has shown that there is heterogeneity in the

interaction of particular SEB reactive VP chains with superantigem. This suggests that there

are other contributing elements to superantigen recognition. For example the a chain has been

irnplicated in influencing the response to SEB (43) (44, as well as MIS (45) (46) (47). Since

there are many factors that influence the SEB response other than the presence of a V(38 chain,

our results do not rule out the possibility of a suboptirnal SEB response in these mice. The

evidence from the SEB, MHC, TCR interactions suggest there is a gradient in reactivity

towards SEB (34) (43) (44) (49). This gradient includes VP8 ceils with differing reactivity

towards SEB From highly reactive to non reactive. Therefore it is forrnally possible that the

Val3 Vp8.2 TCR present in the OVA scid mouse is of a moderate to low or moderate to high

a h i t y towards SEB. It would be interesting to study whether the Va13 VB8.2 TCR present

in the OVA scid mouse has a high or a low affinity for SEB as compared to Vp8.2 TCRs with

other ci chains. This could be studied in the OVA transgenic mouse. Al1 the T cells in this

mouse express Vp8 and only 50% express the transgene combination (82). Therefore the

influence of different a chains in the in vivo response to SEB could be studied using this

mouse. Interestingly both BALBIc and OVA scid mice had elevated levels of IL-2 cx receptor

20 hours following SEB injection (figure 8). This suggests that the transgenic TCR has a

sufficent affinity for SEB to allow for T cell activation in response to SEB.

The adoptive transfer of OVA scid splenocytes into the C.B-17 scid without SEB

suggested that TCR transgenic scid T cells are unable to reckculate for a long tirne in the

absence of stimulation. This is because the TCR transgenic scid T cells were detected earlier

(day 4) in the absence of SEB but were virtually undetectable at day 12 or day 13. Therefore

one cm study whether the scid defect or the presence of a TCR trmsgene results in a defect in

recirculation by following the fate of adoptively transferred TCR transgenic scid T cells versus

T cells from a transgenic rnouse that does not harbour the scid mutation. The fate of these T

cells could be compared to the fate of adoptively transferred T cells from a normal mouse.

Upon SEB injection a novel population of C D 4 TCR+ cells was seen in the spleen of

SEB injected OVA scid mice. A population of CD4 TCR+ (DN T cells) cells was also seen in

the thymus of uninjected OVA scid mice. This population does not express the CD8 molecule.

C D 4 CD8- TCR+ (DN T cells) cells have seen in various systerns (102) including in MRUlpr

mice ( 103) , CD4 knockout mice (1 6) ( 1 04), humm peripheral blood (1 O5), normal mouse

liver (1 03) and TCR transgenic mice (30) (78) (82) ( 1 06). In some cases this population is

thymically derived (30) (78) (1 02). These DN T cells can secrete cytokines (104) (106). In

some cases these cells are cytotoxic towards class II bearing antigen presenting cells ( 106). It

would be interesting to investigate the role of these cells in the OVA scid system It is possible

that in the absence of CD8 cells these cells are responsible for cytotoxicity (1 06).

References

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2. Koh, D.R., W.P. Fung-Leung, A. Ho, D. Gray, H. Acha-Orbea, and T.K. Mak. 1992. Less mortality but more relapses in experimental allergic encephalornyelitis in CD8 -1- mice. Science 256: 12 IO.

3. Jiang, H., S. Zhang, and B. Pernis. 1992. Role of CD8 T cells jn r n u ~ e expefiental allergic encephalomyelitis. Science 256: 12 13.

4. Fernandez-Botran, R., V.M. Sanders, T.R. Mosmann, and E.S. Vitetta. 1988. Lymphokine mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 ce&. The Journal of Experimental Medicine 168543.

5. Heinzel, F.P., M.D. Dadick, B.J. Holaday, R.L, Coffman, and R.M. Locksley. 1989. Reciprical expression of FN y or IL-4 during the resolution of murine Leishrnaniasis. The Journal of Experimental Medicine 16959.

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