ADDITIONAL DATA - Variations among Sublines of Inbred AKR Mice

Variations among Sublines of Inbred AKR Mice

IV. ADDITIONAL DATA

A d d i t i o n a l s t u d i e s of t h e i n t e r - and i n t r a s p e c i f i c d i s t r i b u t i o n of Thy-1-like a n t i g e n s have been undertaken. Absorption experiments i n d i c a t e d an absence from guinea p i g b r a i n t i s s u e o f a n t i g e n s c r o s s - r e a c t i v e with e i t h e r Thy-1.1 o r Thy-1.2. The thymocytes and b r a i n of t h e degu ( ~ c t o d o n degus,

a

South American r o d e n t ) were a l s o t e s t e d 1 f o r t h e presence of Thy-1.1 by a b s o r p t i o n , The r e s u l t s were n e g a t i v e eyen though

an

amount o f Thy-1.1 equal t o one-fourth t h a t expressed by mouse t i s s u e s would have been r e a d i l y d e t e c t e d ,

It

has n o t y e t been p o s s i b l e t o e x p l o i t t h e 8-R system

as a

g e n e t i c marker i n t h e

rat

because no g e n e t i c polymorphism

i n

t h i s a n t i g e n has been d i s c o v e r e d , I n an attempt t o f i n d such a polymorphism, m i c r o c y t o t o x i c i t y t e s t s were performed u s i n g t h e thymocytes of f i v e

i n b r e d s t r a i n s of r a t s not i n c l u d e d i n e a r l i e r experiments (WF/fz, COP, A/2, AUG,

B N . B ~ ) .*

^{A s}

^{i n}

t h e c a s e s p r e v i o u s l y d e s c r i b e d , a l l f i v e of t h e s e s t r a i n s were found t o express 8-R. No c r o s s - r e a c t i v i t y w i t h Thy-1.2 was found. The number of thymocytes from a given r a t s t r a i n r e q u i r e d t o absorb a s t a n d a r d m o u n t of anti-Thy-1.1 antibody v a r i e d from 2.2

x

1 0 t o

5 4.3 x lo5,

s u g g e s t i n g t h a t no l a r g e q u a n t i t a - t i v e d i f f e r e n c e s i n 8-R e x p r e s s i o n e x i s t among t h e s e f i v e s t r a i n s , These values a r e comparable with

2.8 x

5

which was observed f o r thymocytes of s t r a i n WE',

l ~ e ~ u

t i s s u e s were a g i f t from D r . David Boraker, U n i v e r s i t y of Vermont School

of Medicine,

2 ~ h e s e r a t s were a g i f t from Dr, Joy Palm, Wistar I n s t i t u t e , P h i l a d e l p h i a .

29 I f a g e n e t i c polymorphism f o r 8-R

i s

e v e n t u a l l y found, t h e most probable c a n d i d a t e s f o r l i n k a g e s t u d i e s w i l l h e t h o s e markers a l r e a d y known t o be l i n k e d t o Thy-1 i n t h e mouse, S e v e r a l g e n e t i c a l l y con- t r o l l e d t r a n s f e r r i n v a r i a n t s have been r e p o r t e d i n R a t t u s r a t t u s ( 2 2 ) --

b u t no s i m i l a r v a r i a t i o n has been found i n R a t t u s norvegicus,

- 7 , . .

.

~ l e c t r o p h o r e s i s ~ of serum samples from t h e 1 3 inbred and

random-bred R a t t u s norvegicus p o p u l a t i o n s r e f e r r e d t o above r e v e a l e d no v a r i a t i o n i n t h e i r serum t r a n s f e r r i n s ,

E x t r a c t s of kidneys from t h e same r a t s were screened f o r e l e c t r o p h o r e t i c v a r i a n t s of t h e cytoplasmic malic enzyme. Using t h e b u f f e r system d e s c r i b e d i n c h a p t e r

11, t h e r a t enzyme migrated anodally

while t h e products of ~ o d - l a and Mod-1 -- b of t h e mouse migrated cathod-

a l l y a t slower r a t e s . No v a r i a t i o n i n t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e enzyme was observed among t h e 1 5 r a t populations t e s t e d . A s an

a d d i t i o n a l

check f o r p o s s i b l e v a r i a t i o n , t h e b u f f e r system o f

Richmond ( 2 3 ) was a l s o t r i e d . Under t h e s e conditions of e l e c t r o p h o r e s i s both r a t and mouse enzynes migrated a n o d a l l y , t h e l a t t e r moving r e l a - t i v e l y f a s t e r . No d i f f e r e n c e s were observed among t h e following s t r a i n s :

A / 2 , AUG, BN,

BN.B2,

COP, LEN, WF,

WF/fz.

Additional d a t a r e l a t i n g t o t h e expression of

8-R

a n t i g e n by v a r i o u s rat lymphoid c e l l populations have a l s o been gathered.

Comparative r e s u l t s of c y t o t o x i c and a b s o r p t i o n t e s t s performed on thymus, s p l e e n , lymph node, and bone marrow c e l l s of WF r a t s

( 2 4 )

and AKR/J mice a r e p r e s e n t e d i n Table

2 ,

Although ~hy-1.1' c e l l s i n AKR/J

See chapter I1 f o r a d e s c r i p t i o n of experimental d e t a i l s .

mouse spleen and lymph node preparations were killed using the same antiserum, no significant cytolysis of the corresponding rat cells was observed. The differing levels of

8-R

expression suggested by these results were confirmed by the greatly diminished absorptive capacities of rat lymph node and spleen cells relative to thymocytes,

Although the general pattern of decreased 8-R expression on rat lymph node and spleen cells parallels that reported for T h y ~ l in mice (8,25) an important difference between these systems is apparent.

In rats, 8-R expression on peripheral thymus-derived lymphocytes (T-cells) drops below the level of detectability using the absorption procedures described above, Acton (26) has obtained similar results using quantitative absorption and radioimmunoassay techniques, In contrast, the indirect membrane immunofluorescence data of Micheel et al. (27) indicate the presence of

8-R

+ cells in Wistar rat lymph

- --

node and bone marrow. The experiments described in the remainder of this chapter were designed to shed additional light upon the question of 0-R antigen expression by rat peripheral T-cells,

The first approach to this question was based upon the hypothesis that anti-Thy-1.1 antibodies in the presence of complement might inter- fere with rat peripheral T-cell function, even though they did not cause cytolysis. The particular function chosen for study was the cell-mediated immune destruction of EL^ (mouse) leukemia cells.

Ten to sixteen days before cell-mediated imunity was to be

assayed, mice or rats were injected intraperitoneally with 5 x

6 or

x 10 7 EL^ cells, respectively. On the day of the experiment,

p e r i t o n e a l exudate c e l l s (PEC) were c o l l e c t e d i n t o Dulbecco's

Modified Eagle Medium

(DMEM,

Grand I s l a n d B i o l o g i c a l

C O , )

according t o t h e method of Berke e t a l , (28) .

EL^ a s c i t e s Leukemia c e l l s were maintained by s e r i a l i n t r a y p e r i t o n e a l passage i n C57B1/6~ mice. Twenty t o t h i r t y m i l l i o n washed

EL4

c e l l s suspended i n

m l of DMEM were incubated with 100 micro- c u r i e s of ~ a ~ ~ ' ~ r 0 4 f o r one and one-half hours at 37

0 C ,

then washed f i v e times i n 3 m l a l i q u o t s of t h e same medium p r i o r t o use, The r e l a t i v e p r o p o r t i o n of 5 1 ~ r r e l e a s e d from t h e s e p r e l a b e l e d tumor c e l l s can be used a s a q u a n t i t a t i v e measure of c e l l d e a t h , arid hence cell-mediated immune f u n c t i o n , a f t e r a c o r r e c t i o n has been a p p l i e d f o r t h e r e l a t i v e l y slow r a t e of spontaneous 5 1 ~ r r e l e a s e ( 2 9 ) .

One-tenth

m l

a l i q u o t s of washed PEC a t a c o n c e n t r a t i o n of

2.5

x 10 / m l i n 7 DMEM were placed i n t o 1 0 x 75 mm g l a s s c u l t u r e t u b e s , Following t h e a d d i t i o n of 1 0 m i c r o l i t e r s of whole mouse normal serum o r antiserum t h e t u b e s were mixed, covered w i t h p l a s t i c f i l m , and incubated f o r 1 5 minutes a t room temperature. F o r t y m i c r o l i t e r s of a s u i t a b l e d i l u t i o n of whole guinea p i g serum ( ~ i g u r e 8 ) o r 20 micro- l i t e r s

u n d i l u t e d guinea p i g serum a able 3 ) , which had been s t o r e d a t

-70 0 C

until s h o r t l y b e f o r e u s e , w e r e a d d e d as a source of complement.

The t u b e s were then mixed, covered, and incubated f o r 20 t o

minutes

a t 37'~. A t t h e end of t h i s p e r i o d t h e volume of each t u b e w a s brought

t o

t o t a l of 1 . 0

by t h e a d d i t i o n of

DMEM,

Following c e n t r i f u g a t i o n

a t 360 x

f o r 10 minutes, t h e s u p e r n a t a n t s were c a r e f u l l y a s p i r a t e d

and d i s c a r d e d so a s t o minimize t h e t r a n s f e r of unbound a n t i b o d i e s or

32 o t h e r serum components, The p e l l e t from each t u b e was resuspended by t r i t u r a t i o n i n 0.9

m l

of DMEM supplemented w i t h 10% f e t a l c a l f serum

(DMEM

+ 10%

FCS,

s e e r e f e r e n c e 28) and t r a n s f e r r e d t o one w e l l of a Disposo-Tray ( ~ i n b r o P l a s t i c s ) ,

When a l l of t h e t r e a t e d PEC from a s i n g l e experiment had been t r a n s f e r r e d t o t h e coded w e l l s of a t r a y , 0 . 1

^ml

of " ~ r - l a b e l e d EL^

leukemia c e l l s a t a c o n c e n t r a t i o n of 5 x 1 0 5

/ m l

i n

DMEM

+ 10% FCS was added t o each w e l l . The e n t i r e t r a y was covered w i t h p l a s t i c f i l m and i n c u b a t e d on a r o c k i n g p l a t f o r m ( 3 0 ) under an atmosphere of 94% a i r -

6% C o p f o r 6 hours.

A t

t h i s p o i n t 1 m l o f DMEM + 10% FCS w a s added t o each w e l l . The c o n t e n t s o f each w e l l were l i g h t l y t r i t u r a t e d and t r a n s f e r r e d t o a d i s p o s a b l e 1 0 x 75

^mm

g l a s s c u l t u r e t u b e . A f t e r c e n t r i f u g a t i o n a t 360 x g f o r 1 0 minutes,

m l (one-half t h e t o t a l volume) of t h e s u p e r n a t a n t from each t u b e was removed f o r counting i n a Nuclear-Chicago Model 4218 w e l l t y p e gamma c o u n t e r . The remaining c o n t e n t s of each t u b e w e r e a l s o counted.

The

p e r c e n t a g e of 51~r r e l e a s e d was computed u s i n g t h e f o l l o w i n g formula:

x ( s u p e r n a t a n t - background)

Percent 51cr

⁼

_remainder + s u p e r n a t a n t - ² ^x background x 100 where " s u p e r n a t a n t w = counts/lO minutes i n t h e

1 ml

s u p e r n a t a n t sample,

I t

remainder"

c o u n t s / l 0 minutes i n t h e t u b e c o n t a i n i n g t h e p e l l e t p l u s

t h e o t h e r h a l f of t h e s u p e r n a t a n t , and "background"

counts/lO minutes

w i t h no sample i n t h e counting chamber, The f a c t o r of

i n t h e numerator

of t h i s e x p r e s s i o n t a k e s account of t h e f a c t t h a t o n l y one-half of t h e

counts r e l e a s e d f r m t h e l a b e l e d c e l l s a r e a c t u a l l y sampled f o r counting

i n t h e " s u p e r n a t a n t " t u b e ,

3 3

The results of an experiment using immune AKR/J PEC are shown in Figure 8, Immune PEC pretreated with AKR/J mouse normal serum uniformly caused maximal tumor cell lysis. In contrast, the cytotoxic

activity of PEC treated with serum containing antibodies specific for Thy-1.1 (8-AKR) was completely abolished. The degree of PEC inactiva- tion observed falls off with decreasing complement concentration, strongly suggesting that the inactivation process is complementc dependent.

Data from a similar experiment with immune

rat PEC are p r e ~ sented in Table 3. Each sample of immune PEC was pretreated with

either AKR/Cum normal serum, A K R / C ~ ~ anti-AKR/J thymocyte serum (con- taining anti-Thy-1.1 antibodies), or AKR/Cum anti-BN rat spleen serum, 4

Rat PEC pretreated with AXR/cum anti-AKR/J thymocyte serum showed anti-tumor activity equivalent to those treated with normal serum, but pretreatment of these cells with AKR/Cum anti-BN spleen serum resulted in a complete inhibition of their cytotoxic activity.

Thus, the ability of these antisera to interfere with the function of

rat peritoneal cytotoxic cells is correlated with the presence of species antibodies, and not of those directed against Thy-1.1, This is quite different from the pattern observed for AKR/J immune PEC, which are strongly inactivated by either AKR/Cum anti-AKR/J thymocyte

serum or AKR/Cum anti-BN rat spleen serum, but not by AKR/Cum normal serum.

4 This serum also contains anti-Thy-1.1 antibodies in addition to anti-

bodies directed against rat species-specific antigens. The properties of

this antiserum are discussed further in connection with Figure 9.

3 4

The effect of a similar set of antisera on

rat PEC viability was determined in a separate experiment, One-tenth

m l

aliquots of PEC were pretreated with antibody and complement as described above, The viability of the cells in each group was then determined by the Trypan Blue exclusion method, It is apparent from the data presented in Table 4 that the inactivation of rat PEC by AKR/Cum anti-BN rat spleen serum is the result of complement-mediated cytolysis, The partial decline in the viability of cells treated with either AKR/Cm normal serum or anti-Thy-1.1 antibody5 also correlates well with the partial loss of effector cell activity observed in similarly treated cell populations

able 3). On the basis of these data plus those presented in Table 2, it seems unlikely that anti-Thy-1.1 reagents can be used for the selective elimination of rat peripheral T-cells from mixed lymphoid cell populations, as has been done successfully in the mouse,

The final approach taken

in search of 8-R

on rat peripheral T- cells was based upon the hypothesis that small amounts of this material might be immunogenic even though they did not present an adequate

target for complement-mediated cell damage, In order to evaluate this possibility, AKR/Cum mice were injected with

rat spleen cells

according to the previously described protocol for raising anti-Thy-l,l antibodies. The results of direct complement-dependent cytotoxic tests of these sera against thymocytes from mice of strains A K R / C ~ ~ , AKR/J, and C3H/HeJ are shown in Figure 9. The specific killing of Thy-1.1 +

5 ~ 3 ~ / ~ e ~ anti-AKR/J thymocyte serum rather than AKR-Cum anti&m/J thy-

mocyte serum was used in this experiment because of its higher titer.

thymocytes i n d i c a t e s t h e p r e s e n c e of anti-Thy-1.1 a n t i b o d i e s i n t h i s a n t i s e r u m , and hence t h e p r e s e n c e o r 0-R on r a t spleen c e l l s ,

A s a t e s t f o r t h e presence o f emR on o t h e r p e r i p h e r a l T - c e l l s of t h e r a t , groups of

AKR/Cum

mice were immunized w i t h

r a t c e l l s t a k e n from t h e m e s e n t e r i c lymph node, bone marrow, P e y e r t s p a t c h e s , p e r i - t o n e a l c a v i t y , o r blood l e u k o c y t e f r a c t i o n o f

r a t s , Thymocytes and s p l e e n c e l l s were used a s p o s i t i v e c o n t r o l immunogens, P r e l i m i n a r y t e s t s i n d i c a t e d t h e presence of anti-Thy-1.1 a n t i b o d i e s i n a l l groups, 6

The p r i n c i p a l o b j e c t i o n t o t h i s t y p e of experiment has been r a i s e d by Komuro -- e t a l . ( 3 1 ) . These a u t h o r s found t h a t mice l a c k i n g t h e TL a n t i g e n sometimes produced anti-TL a n t i b o d i e s f o l l o w i n g irnrnuni- z a t i o n w i t h s p l e e n c e l l s from TL + donors. This o b s e r v a t i o n was p u z z l i n g because TL has been r e p o r t e d t o be p r e s e n t only on thymocytes, and not on p e r i p h e r a l mouse T - c e l l s ( 7 ) . Mice thymectomized a s a d u l t s f a i l e d t o g i v e

t h e a.?n,nmalous

response t o TL although t h e y responded normally t o Thy-1. This f i n d i n g was i n t e r p r e t e d t o mean t h a t t h e o r i g i n a l s p l e e n c e l l inoculum c o n t a i n e d (TL-, Thy-1 +

)

stem c e l l s which d i f f e r e n t i a t e d i n t o TL + c e l l s under t h e i n f l u e n c e of t h e h o s t ' s thymus, These d i f - f e r e n t i a t e d c e l l s t h e n evoked an immunological response t o t h e TL

a n t i g e n . Experiments designed t o e v a l u a t e t h e p o s s i b i l i t y t h a t t h i s i s happening i n t h e

8-R

system a r e c u r r e n t l y b e i n g pursued i n c o l l a b o r a t i o n w i t h Andrew Dowsett, 7

6 Andrew Dowsett, p e r s o n a l communication

7~ndergradurvte, C a l i f o r n i a I n s t i t u t e of Technology

TABLE

Expression of Thy-1.1 Antigen by WF Rat and AKR/J Mouse Lymphoid Cells WF Rat cellsa

AKR/J

Mouse Cells

Cytotoxic Absorptive Cytotoxic Absorptive

b

-1,ndex Capacity Index Capacity

Cell Type -

Thymus 9

100 97 100

Spleen

< 3 5 7 5 .O

Lymph Node 10 < 4 69 597

Bone Marrow 8 ND' ND <1,5

a Data from reference (24), which is included as Appendix I b~elative to an equivalent number of thymocytes

Unpublished data

d~etermined by Reif and Allen (8), using a somewhat different absorption procedure

e Not done

TABLE 3

I n V i t r o K i l l i n g of EL^ Leukemia C e l l s by Immune WF Rat P e r i t o n e a l

--

Exudate C e l l s

( $

51~r ~ e l e a s e d ) Experiment Number

Pretreatment of PEC --- ¹ ² ---- ³ ⁴ ⁵ ⁶ ^Average

AKR/Cum

Normal Serum 35.3 36,4 32.5 36.2

30.7

28.2 33.2

AKR/Cum anti-AKR/J 29.0 34,2 35.5 35*1 30.4 32.9 32,9

Thymocyte Serum AKR/Cum anti-BN R a t

Spleen Serum None

Background 51~r

Release

TABLE

4 Complement - Dependent C y t o l y s i s

o f

WF R a t P e r i t o n e a l Exudate C e l l s by Mouse Antiserum

P r e t r e a t m e n t of PEC V i a b i l i t y (%)

31*_

AKR/CU

Normal Serum '71

c3H/HeJ anti-AKR/J 73

Thymocyte Serum

A K R / C U ~ anti-BN Rat 3

Spleen Serum

None 9

39 FIGURE LEGENDS

Figure 8. -- I n v i t r o k i l l i n g o f EL^ leukemia c e l l s by immune AKR/J mouse p e r i t o n e a l exudate c e l l s . The percentage of 5 1 ~ r r e l e a s e d from l a b e l e d EL^ c e l l s by immune PEC p r e t r e a t e d w i t h e i t h e r AKR/J normal serum ( 0 ) o r AKR/Cum anti-AKR/J serum (0

)

w a s measured,

The

amount of l a b e l r e l e a s e d following i n c u b a t i o n with no PEC (background r e l e a s e ) o r u n t r e a t e d

PEC

i s s h o w n b y ( ~ ) and

( A ) ,

r e s p e c t i v e l y , Each p o i n t r e p r e - s e n t s t h e average of t h r e e d e t e r m i n a t i o n s , except t h o s e marked

, f o r which only

measurements were made, E r r o r b a r s show t h e range f o r each v a l u e . "Complement d i l u t i o n " r e f e r s t o t h e c o n d i t i o n s under which immune PEC were p r e t r e a t e d ( s e e t e x t ) ,