Memory-Effector T cell Retention in Experimental Autoimmune Encephalomyelitis

1. Journal of Neuroimmunology, 40 (1992) 57-70 57
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00
JNI 02223
Direct demonstration of the infiltration of murine central nervous
system by P g p - 1 / C D 4 4 high CD45RB l°w CD4 + T cells
that induce experimental allergic encephalomyelitis
R a n a Zeine a and Trevor Owens a,b
Departments of Medicine, and b Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill Unicersity,
Montreal, Quebec, Canada
(Received 22 January 1992)
(Revised, received 26 March 1992)
(Accepted 27 March 1992)
Key words: Central nervous system infiltration; Memory/effector T cells; Experimental allergic encephalomyelitis
Summary
In experimental allergic encephalomyelitis (EAE), autoimmune T cells infiltrate the central nervous
system (CNS) and initiate demyelinating pathology. We have used flow cytometry to directly analyse the
migration to the CNS of MBP-reactive CD4 + T cells labelled with a lipophilic fluorescent dye (PKH2), in
S J L / J mice with passively transferred EAE. Labelled cells constituted about 45% of the CNS CD4 ÷
population at the time of E A E onset. Almost all ( > 90%) of the PKH2-1abelled CD4 + T cells from E A E
CNS were blasts and were a / ~ T cell receptor (TCR) +, CD44(Pgp-1) high, and the majority were
CD45RW °w. By contrast, most PKH2-1abelled CD4 ÷ T cells in lymph nodes, although CD44 high, were
CD45RB high cells. The cells that were transferred to induce E A E were essentially similar to antigen-
primed lymph node cell populations, containing less than 15% CD44 high cells, and most of them were
CD45RB high. The CD44 high CD45RB l°w phenotype is characteristic of m e m o r y / e f f e c t o r T cells that have
been activated by antigen recognition. The difference in CD45RB expression between CNS and LN
could therefore reflect differential exposure a n d / o r response to antigen. Consistent with this, PKH2-
labelled CD4 + cells isolated from the CNS were responsive to MBP in vitro, whereas PKH2 + CD4 ÷
cells from lymph nodes showed almost undetectable responses. In control experiments in which
ovalbumin (OVA)-reactive T cells were transferred, a small number of fluorescent-labelled CD4 + T cells
were also detected in CNS, but there were very few blasts, and these remained CD45RB high. These
results argue for induction of the m e m o r y / e f f e c t o r phenotype of CD4 + T cells, and their selective
retention in the CNS, as a consequence of antigen recognition.
Correspondence to: T. Owens, Neuroimmunology, Montreal
Neurological Institute, 3801 University Street, Montreal, Que-
bec, Canada H3A 2B4.

2. 5s
Introduction gcn recognition. In the human this corresponds
to CD45RA (MM 21()-220 kDa) to CD45RO
Experimental allergic encephalomyelitis (EAE) (MM 170-180 kDa) transition, as defined by iso-
is an autoimmune disease of the central nervous form-specific mAbs (Akbar ct al., 1988: Kristcn-
system (CNS) which is induced by autoreaetive son et al., 199(t). In the mouse, monoclonal anti-
CD4 + T cells (Raine, 1985). The histopathology bodies (mAbs) that recognize the high MM
of demyelinated lesions and the overall progres- CD45RB isoform have been used to show that
sion of E A E are reminiscent of the human de- prolonged activation of CD4 ~ T cells in vitro
myelinating disease multiple sclerosis (MS) (Mc- leads to a reduction in the level of high MM
Farlin and McFarland, lg82). The mechanism by CD45R expression (Birkeland et al., 1989).
which disease-inducing T cells enter the CNS is Murinc CD44 higl~ CD4.,~RB h,,~ T cells are potent
poorly understood, but the process is probably cytokine producers (Budd et al., 1987; BottomIy
analogous to the infiltration of other tissues by ct al., 1989; Lee et a[., 1990; Swain et al., 199(I;
inflammatory T cells. Studies using radiolabelled Weinberg et al., 1990). This phenotype can thcre-
MBP-specific T cells have shown the accumula- fl)re be considered a marker fl)r m e m o r y / c f f c c t o r
tion of CD4* cells in the brains of mice in which 1" cells.
EAE was induced by passive transfer (Naparstek The T cells in MS lesions and CSF, and iso-
et aI., 1982; Trotter and Steinman, 1984; Wekerle lated from the spinal cords of animals with EAE,
ct al., 1986, 1987). The distribution of myelin have been shown to predominantly express low
basic protein (MBP)-speeific T ceils into other MM CD45R isoforms (Sobel ct al., 1988; Chof-
organs was similar to that of T ceils with irrele- flon et al., 1989; Salonen et al., 1989: Jensen et
vant specificities (Trotter and Steinman, 1984; al., 1990; Zaffaroni et al., 1990; Zeine and Owens,
Wekerle e t a [ . , 1986, 1987). Occasional radiola- 1991), and T cells in other inflammatory- diseases
belled ovalbumin (OVA)-specific T ceils were are similarly biased towards this phenotypc
observed in the brains of non-diseased mice, but (Potocnik et al., 1990). The accumulation of cells
did not persist there (Wekerle et al., 1986, 1987). of the memory./effector phenotype at inflamma-
The association of neuroautoantigen-specific T tory, sites could in principle result from selection
cells with the CNS suggests a role for antigen of that phenotype, or its induction as a conse-
recognition in their retention (Burns et al., 1984; quence of antigen reactivity. Discrimination be-
Sgroi et at., 1986; Hailer et al., 1987). A greater tween these possibilities requires the ability to
understanding of the mechanism by which T cells track infiltrating cells and to simultaneously ana-
infiltrate and are retained in the CNS is of both lyse their antigen reactivity and surface pheno-
fundamental and clinical interest. One approach type. The former requires an adoptive transfer
to this problem is to characterize infiltrating T system, while the latter is not possible using
cells with regard to their recognition of antigen radiolabelled cells. We have used a lipophilic
and induction of specific cellular function. This fluorescent dye (PKH2) to monitor those T cells
can be accomplished by analysis of surface phe- that migrate to the CNS and to peripheral lymph
notype. nodes (LN) in a passive transfer E A E model and
Two T cell surface antigens whose level of to compare their expression of P g p - I / C D 4 4 and
expression correlates with states of activation are CD45RB. Our results show the selective accumu-
CD44 and CD45R. Memory or recall antigen-re- lation in the CNS of autoreactivc CD4 + cells that
active T ceils are defined by their elevated ex- are C D 4 4 high, CD45RB I'''. We find that although
pression of P g p - 1 / C D 4 4 (Butterfield et al., 1989). non-autoreactivc T cells enter the CNS, they are
Similarly, the differential expression of CD45 iso- not retained there and do not express the mem-
forms defines stages of T cell activation. Resting o r y / effector phenotype,
or naive T cells express high molecular mass These results argue for the preferential reten-
(MM) CD45R isoforms, and there is a gradual tion through antigen recognition of m e m o r y /
transition towards the exclusive expression of low effector T cells in the CNS following infiltration
MM CD45R isoforms as a consequence of anti- from the periphery-.

3. 59
Materials and methods neither did MBP-reactive T cells recognize OVA
(not shown).
Mice
Isolation of mononuclear cells from CNS
Female SJL/J mice (5-8 weeks) were ob-
CNS infiltrating lymphocytes were collected by
tained from Harlan-Sprague Dawley (Indianapo-
discontinuous density gradient centrifugation
lis, IN). Animal care and experimental protocols
(Clatch et al., 1990) once EAE onset within the
were approved by the McGill University Animal
group had been verified. Mice were anaes-
Care Committee, and were in accordance with
thetized with chloral hydrate (3.5 g/kg) and per-
CCAC guidelines.
fused through the heart with PBS. The brains,
spinal cords, and lymph nodes were then col-
EAE and PKH2-labelling of MBP-reactive T cells lected and dissociated by passing through a nylon
Donor mice were immunized s.c. with 400 Ixg or stainless steel mesh, respectively. The nervous
of purified bovine MBP (Sigma, St. Louis, MO) in tissue was centrifuged at 200 x g for 10 min and
complete Freund's adjuvant (CFA) (50 ~g My- then resuspended in 4 ml of 70% Percoll (Phar-
cobacterium tuberculosis H37RA (Difco, Detroit, macia) in RPMI 1640 medium. This was then
MI) per mouse) and boosted 7 days later. Drain- overlaid by equal volumes of 37% and 30% Per-
ing LN were collected at day 14, and LN cells coll and the gradient was centrifuged at 500 x g
(LNC) cultured at 4 × 10 6 cells/ml with MBP (50 for 15 min. Mononuclear cells were collected
~ g / m l ) for 4 days. Responsiveness to MBP was from the 37%:70% interface, washed in medium
assessed in parallel microcultures by [3H]thymi- containing 10% FCS (ICN Biomedicals) and
dine incorporation at 4 days following an counted.
overnight pulse (0.5 ~Ci/well (ICN Biomedicals
Inc., Mississauga, Ontario)). Stimulation indices Flow cytometry
of 10-30-fold were routinely observed. Cells were mAbs included phycoerythrin-coupled anti-
collected by centrifugation on Ficoll-Hypaque CD4 (PE-CD4) (Becton-Dickinson, Mountain
(Pharmacia, Montreal, Quebec) and either recul- View, CA), anti-CD8 (53-6.7) (Ledbetter and
tured for another 10 days in vitro with MBP and Herzenberg, 1979), pan-anti-CD45 (M1/89)
irradiated (3000 R) LNC as antigen-presenting (Springer et al., 1978), anti-TcR,/~ (H57-597)
cells, or labelled with PKH2-GL (Zynaxis, (Kubo et al., 1989), anti-Pgp-1/CD44 (IM781)
Malvern, PA) (Horan ad Slezak, 1989) and imme- (Trowbridge et al., 1982), and anti-CD45RB
diately injected i.v. at 10 7 blasts/mouse. Be- (23G2) (Birkeland et al., 1988). Where indicated,
tween 70 and 100% of animals developed clinical mAbs were purified by Protein G-Sepharose
signs of EAE 10 days later. Mice were monitored affinity chromatography (Pharmacia) and coupled
daily and assigned clinical scores as follows: 0 (no with biotin by incubation with biotinamido-
symptoms), 1 (flaccid tail, clumsiness), 2 (mod- caproate N-hydroxy succinimide ester (Sigma).
erate paresis), 3 (severe paresis or unilateral hind Cells (5 x 105-106) were incubated with antibody
limb paralysis), 4 (bilateral hindlimb paralysis), 5 at 4°C for 20 min and then washed and incubated
(moribund). Short-term OVA-reactive T cell lines with either FITC-coupled streptavidin (Bio-Can
were induced by in vivo priming and boosting Scientific, Toronto, Ontario), FITC-sheep anti-
with 400 #g OVA (Calbiochem, San Diego, CA) mouse Ig (Bio-Can Scientific) or FITC-goat anti-
in CFA. LNC were cultured for 4 days with OVA rat Ig (Southern Biotechnology, Birmingham, AL)
(50 p~g/ml), labelled with PKH2 and transferred after blocking with rat Ig (100 /xg/ml) (Bio-Can
by i.v. injection exactly as for MBP-reactive T Scientific). In some experiments biotinylated
cells. There was no reactivity of OVA-reactive mAbs were visualized using Phycoerythrin :Texas
cells to MBP or to the MBP peptide FFKNIVT- Red (Tandem)-streptavidin (Southern Biotech-
PRTPPP (Multiple Peptide Systems, San Diego, nology). Surface staining was analysed using a
CA), corresponding to amino acids 90-102, which FACScan (Becton Dickinson). Dead cells were
is encephalitogenic in SJL/J (Kono et al., 1988), excluded by propidium iodide staining, or by side

4. 6()
s c a t t e r gating. In s o m e e x p e r i m e n t s d e a d ceils e q u a l l y b e t w e e n 6 ( L N C ) or 12 ( C N S ) r o u n d - b o t -
w e r e e x c l u d e d by g a t i n g o u t FL3 t'<ght p r o p i d i u m tomed microwells (Falcon). Preliminary counts
iodide-stained events, without compromising i n d i c a t e d t h e r e to bc less t h a n 2 × 10 4 o f any
Texas Red detection, and the profiles obtained w h o l e p o p u l a t i o n . I r r a d i a t e d (3(100 R) s y n g e n c i c
w e r e s i m i l a r to t h o s e o b t a i n e d using f o r w a r d scat- s p l e e n cells t h a t h a d b e e n d e p l e t e d of T cells by
t e r gating. i n c u b a t i o n with a n t i - T cell m A b s plus c o m p l e -
m e n t ( O w e n s , 1991) w e r e a d d e d (5 × 1 0 S / w e l l ) ,
FACS with or without MBP or purified protein deriva-
P K H 2 - 1 a b e l l e d , M B P - r e a c t i v e T cells w e r e pu- tive ( P P D ) ( C e d a r l a n e , H o r n b y , O n t a r i o ) , b o t h at
rified by F A C S . L N C a n d C N S m o n o n u c l e a r cells 5(t # g / m l . A s e n s i t i v e b i o a s s a y for IL-3 p r o d u c -
w e r e i s o l a t e d at t h e t i m e o f o n s e t o f p a s s i v e l y - i n - lion was u s e d to m e a s u r e T cell a c t i v a t i o n . A f t c r
duced EAE. Cells were stained with PE-CD4 and 2 days c u l t u r e , t h e IL-3 c o n t e n t of s u p e r n a t a n t s
t h e n t h e P K H 2 +, C D 4 + p o p u l a t i o n was i s o l a t e d was m e a s u r e d in a m i c r o - b i o a s s a y in 20 #1 cul-
by F A C S ( F A C S t a r , B e c t o n - D i c k i n s o n ) . t u r e s u s i n g t h e R 6 X - E 4 . 8 . 9 I L - 3 - d e p e n d e n t cell
line ( O w e n s ct al., 1987). T h e n u m b e r o f viable
Microbioassay for antigen specificity of" sorted cell cells was c o u n t e d u s i n g an i n v e r t e d m i c r o s c o p e
populations a f t e r 24 h.
B e t w e e n 10 4 a n d 105 cells w e r e o b t a i n e d w i t h i n
e a c h g r o u p , a n d t h e t o t a l yield o f cells w e r e
c u l t u r e d in R P M 1 1640 m e d i u m ( G i b c o / B R L , Results
B u r l i n g t o n , O n t a r i o ) s u p p l e m e n t e d w i t h 10% F C S
( I C N B i o m e d i c a l s ) , 50 # M 2 - M E ( S i g m a ) a n d 2 Passit,e transfer of in l'itro labelled encephalito-
m M L - g l u t a m i n e ( C a l b i o c h e m ) in 1-ml c u l t u r e genic T cells
wells ( F a l c o n , F i s h e r , M o n t r e a l , Q u e b e c ) w i t h In o r d e r to d i r e c t l y e x a m i n e t h e m i g r a t i o n of T
IL-2 (10 U / m l ) , for 7 days. A f t e r 7 days, cells ceils to t h e C N S , w e passively t r a n s f e r r e d E A E
were collected, washed once and distributed w i t h M B P - r e a c t i v e T cells t h a t h a d b e e n f l u o r c s -
TABLE 1
ISOLATION OF CD4' T CELLS FROM THE CNS OF MICE FOLLOWING INTRAVENOUS TRANSFER OF PKH2-
LABELLED LNC
EAE was induced as described in Materials and methods. Responsiveness of LN T cells to OVA and MBP were assessed betore
transfer. Stimulation indices of 10 30-fold were routinely observed, with no reactivity to irrelevant antigens. CNS infiltrates were
collected once EAE onset within the group had been verified, as described in Materials and methods. The number of mice pcr
group is indicated in parentheses after the mean EAE score. For each experiment, the day on which symptoms were first observed
and the day on which cells were collected are shown. The average number of mononuclear cells isolated per mouse and the
percentage that were CD4 + was calculated from direct cell counts and FACS analysis.
Treatment Day of onset Day of study Mean EAE score Number of CD4 + cells/mouse CNS ( × 111 x)
PKH2 - PKH2 Total
Normal NA ~' NA NA NA NA 0.90
NA NA HA NA NA 0.84
EAE 7 10 2.1 (7) 11.4 15.1 26.5
8 11 2 (10) 8.0 17.3 25.3
9 11 1.2 (5) NC h NC 8.2
6 12 0.5 (10) 2.2 1.3 3.5
OVA NA 10 (1 (5) 1.5 2.5 4.11
NA 10 0 (6) NC NC 8.4
~' Not applicable.
b Not calculated, data not available. In these experiments, only CD4 + PKH2 + events were acquired and analysed. Their
proportion within the total CD4 + population was not determined.

5. 61
cently labelled in vitro before transfer. For pas-
A
sive transfer of E A E , T cells must be re-activated
in vitro (Pettinelli and McFarlin, 1981). The in
vitro manipulation of these T cells before la-
10 3
belling and transfer was limited to a brief culture
PKH2
with antigen, without any other addition to cul-
ture or cell fractionation. The lipophilic dye 10 2
P K H 2 - G L labels all cells, and is retained in mem-
branes over 2-week periods in culture or in vivo
101
A
.... i f,'~'--~* "' ,I, I , ,' , J ....
10 3 200 400 600 800
CD4 Forward scatter
m
B
102 ' ~:~e~'~d:i:.;: ...... .. e,J
, ,,(~{ .......... : E
,.!
!4~ ...~.~,'~:(:.':'..,'-._. :
~ e,.
i ' :-.~,.~;~;?.~ !:.. ; .... .
0~ ~ I .:"..;)i~-~i:,£;!:: : 0
I ." ",,5~;.~ !".:'."--';'.; " " ' " 0
" L " :~'-;@~:,'':":
>
. ! ~'(:t4~~:"V"2:i:;'::-: : " , , m
101 10 2 10 3 ee
''''J''''l''''l'''']''''
PKH2
200 400 600 800
Forward scatter
B Fig. 2. Size and fluorescence of PKH2+-labelled CD4 + T
cells from CNS and lymph nodes after passive transfer of
E EAE. MBP-reactive T cells were labelled in vitro with PKH2-
c GL, and injected i.v. to induce E A E as described in Materials
tl
and methods. Following isolation at 10 days post-transfer,
o cells were stained with PE-CD4. PKH2 fluorescence was
analysed in the FL1 channel. Dead cells were excluded by
>
°m propidium iodide staining. A. CNS CD4 + cells, showing
o.e
PKH2-fluorescence plotted against forward scatter. B. CD4 +,
J
' L%
PKH2-fluorescent (FL1 gated as indicated in (A)) lymph node
n- / % ,,,
cells.
101 10 2 10 3
(Horan and Slezak, 1989). Calibration experi-
PKH2 ments showed that even after a 65-fold increase
Fig. 1. PKH2 fluorescence and CD4 expression by MBP-reac- in cell number, the fluorescence level of T cells in
tire cells that were transferred to induce EAE. MBP-reactive culture was still distinguishable from that of con-
LNC were collected from culture, labelled with the lipophilic trols, despite reduction of the per cell fluores-
dye PKH2 and stained with PE-CD4 as described in Materials cence intensity t h r o u g h proliferation. The trans-
and methods. A. Input cells, showing CD4 expression plotted
against PIQ-I2 fluorescence. B. Histogram of fluorescence
ferred populations were all P K H 2 positive (Fig.
before and after labelling with PKH2; ( ): labelled cells; 1), and contained about 55% CD4 + cells (Fig.
( . . . . . . ), unlabelled cells. 1A).

6. ~2
Isolation of mononuclear cells .l}'om CNS fluorescence. Figure 2A shows that the C D 4 T
We isolated mononuclear cells by discontinu- cells in the CNS consisted of two major subpopu-
ous density gradient centrifugation (Clatch et al., lations distinguishable by size and fluorescence.
1990) from the pooled brains and spinal cords of The majority of PKH2 cells were small, while
S J L / J female mice, within 2-3 days of verifica- most of the PKH2 + cells were blasts (Fig. 2A). By
tion of E A E within the group. About 105 contrast, the majority ( > 75%) of fluorescent-
mononuclear cells were recovered per mouse labelled CD4* cells in LN were small cells (Fig.
(mean 1.3 _+0.6 x 105), compared to less than 2B). The fluorescence level in most PKH2 + CD4 ~
5 X 1(14 cells from normal animals (4.5 _+ 0.5 × CNS T cells was reduced by an order of magni-
104). Most ( > 95%) of the isolated cells were tude from that in thc input, transferred popula-
stained with the pan-reactive anti-CD45 mAb tion (compare Fig. IA with Fig. 2A). Similar
M 1 / 8 9 (not shown), and were therefore leuko- reductions in PKH2 levels occur in vitro as a
cytes. The number of CD4 ~ cells was significantly consequence of proliferation, so this is consistent
higher in E A E compared to control CNS, and with proliferative activation of these cells in CNS.
correlated well with the severity of disease (3.5- The PKH2 CD4 + T cells in the CNS repre-
26.5 × 10 ~ in EAE, compared to 0.9 × 103 in sent endogenous]y derived migrants from the
normal mice) (Table 1). All of the CD4 + cells periphery. Our data show that these make up
were CD3 +, a//3 TCR + T ceils (not shown). The 40-70c)b of the CNS CD4 + population. This
CD4 CD8 cells were all CD3 . The propor- agrees with other estimates of endogenously re-
tions of CD4 + and CD8 + T cells in these CNS cruited host T cells, obtained using [~4C]labelled
mononuclear populations isolated from early on- transferred lymphocytes (Trotter and Steinman,
set E A E are consistent with values from in situ 1984; Wekerle et aI., 1986, 1987; Cross et al..
immunopathological studies (Sriram et al., 1982; 1990). Correction for the very small numbers of
Traugott et al., 1985) and previous studies of CD4 + T cells that were isolated from unprimed
isolated CNS cells (Clatch et al., 1990; Williamson animals does not significantly affect this interpre-
et al., 1991). tation. We focussed on the fluorescent-labelled,
PKH2-1abelled CD4 + T cells were easily dis- transferred CD4 + cells in our analysis of the
tinguishable from unlabelled cells by their green induction of effector phenotypes.
i A I ; B
.Q
103 E
CD44 31 i • == C
- - %!~'i",-'%? .,
102 ' ' !
o 2 - - - ;:G - -
I
]
l
/
• • [
I
101 " I 3
I o
C¢
Lf i i i I;,lll
200 400 600 800 200 400 600 800 I01 102 103
Forward scatter Forward scatter CD44
Fig. 3. Pgp-1/CD44 expression on PKH2+-labelled CD4" cells after passive transfer of EAE. ('ells were isolated from CNS and
lymph nodes after onset of EAE, and stained with PE-CD4. Biotinylated IM7.8.1 binding was visualized using Phycoerythrin : Texas
red (Tandern)-streptavidin. Dead cells were excluded by side scatter gating. A. CNS. B. Lymph nodes. Profiles show CD44 plotted
against forward scatter for PKH2-fluorescent (gated as in Fig. 2) CD4 + cells. C. CD44 distribution on CD4 + cells within the donor.
EAE-inducing MBP-reactive population. Input cells were 100% PKH2-fluorescent.

7. 63
C D 4 + T cells that migrated to C N S were C D 4 4 m~h
,=, 200 -
C D 4 5 R B I°~
About 75% (78.3 +_4.2) of the transferred _~ • PKH2 + LNC
(PKH2 +) CD4 + T cells in the CNS were CD44 high Z I IB PKH2+CNS
(Fig. 3A). These cells differ from the original > 100
input population from which they derive both in =
c
their forward scatter profile, and in their CD44
distribution. The MBP-reactive population of
CD4 + T ceils that were transferred to induce ~ 0
-- Medium MBP PPD
E A E were essentially similar to antigen-primed Fig. 5. Antigen-specificity of populations sorted from CNS.
LNC populations, containing less than 15% EAE was induced by passive transfer with PKH2-1abelled,
CD44 high ceils (Fig. 3C). Very few (between 1 and MBP-reactive T cells, and mononuclear cells isolated from the
CNS at the time of onset. Cells were stained with PE-CD4
and PKH2 +, CD4 + cells were isolated by fluorescence-
activated cell sorting. 9 × 104 PKH2 + CD4 + lymph node cells
.Q A and 3.5 × 104 PKH2 +, CD4 + CNS cells were cultured in IL-2
E (10 U / m l ) , 1 ml/culture, for 7 days. After 7 days, cells were
distributed equally between 6 (LNC) or 12 (CNS) round-bot-
tomed microwells. Preliminary counts indicated there to be
G) less than 2× 104 of any whole population. Irradiated (3000R)
0
T-depleted syngeneic spleen cells were added (5 × ll)5/well),
~)
with or without MBP or PPD. After 2 days culture, the IL-3
content of supernatants was measured in a micro-bioassay as
~) described in Materials and methods. The number of viable
O: cells was counted after 24 h. The assay background was 7 + 2
,
l "i'ii11,I l ' ' ''''"I , ,,,i, H i
cells, and 314 + 35 viable cells were counted in the presence of
101 102 103 1 U / m l IL-3.
CD45RB
i B 4%) of LN CD4 + T cells were PKH2-1abelled. Of
I these, only a small proportion were either
I CD44 high (13.1% +- 2.9) or blasts (16.0% +_ 6.2)
10 3 I (Fig. 3B).
CD45RB Figure 4 shows that CNS CD4 + PKH2 + cells
•: L i~ ,i~:i!'i,'~:L'~
expressed significantly lower levels of CD45RB as
10 2
a population than PKH2 + CD4 + blasts in LN.
The majority of transferred, memory (CD44 high)
101 • ' :~i"/~'~:.I'" ' i= CD4 + T cells that had infiltrated the CNS were
, . : . ,
in this way identified as activated effector cells.
• " ,I. By contrast, most of the PKH2 + CD4 + blasts in
LN were CD45RB high (Fig. 4A), showing a bias
i , ] F ~ ~ i"1 r l'l'l T i [ , , ] , i r z I i
200 400 600 800 towards the activated effector phenotype in the
Forward scatter CNS. The CD4 + LN T cells that were transferred
did not contain as substantial a proportion of
Fig. 4. CD45RB expression on PKH2+-labelled CD4 ÷ cells
after passive transfer of EAE. Cell preparation, staining with
either large blasts or of CD44 high, CD45RB j''w
PE-CD4 and 23G2 and fluorescence analysis was carried out cells (Fig. 4B) as were found in both CNS and LN
as for other figures. In lymph nodes, about 25% of PKH2 + 10 days post-transfer (see Fig. 3).
CD4 + cells were blasts (defined by forward scatter as for Fig.
1). Greater than 90% of CNS CD4 + PKH2 + cells were
PKH2-labelled C D 4 + cells in C N S were M B P - r e a c -
blasts. A. ( ): CNS CD4 + PKH2 + cells; ( . . . . . . ), lymph
node CD4 + PKH2 + blasts. B. CD45RB expression and For-
tiue
ward light scatter by in vitro-cultured MBP-reactive CD4 + T To verify that the effector CD4 + phenotype in
cells. CNS reflected the selective retention of autoreac-

8. tive T cells, we purified PKH2 + CD4 * cells from Phenotype of MBP-reactice LNC following pro-
CNS and LN by FACS. The antigen specificity of longed s'timulation m citro
these cells was measured following culture in Because of the remarkable contrast in the pro-
IL-2 (Whitham et al., 1991). Between 10 4 and portions of memory-effector cells found within
1.5 × 105 cells were recovered after sorting. Be- the CNS and LNC populations 10 days after
cause of the low number of cells recovered after transfer, it was of interest to know the phenotype
sorting and 7 days in vitro culture, we used a of LNC that had been re-stimulated in vitro for
sensitive bioassay for IL-3 to amplify responses to 10 days. We cultured MBP-reactive LNC with
antigen. The R6X-E4.8.9 cell line dies quickly in MBP in the presence of irradiated syngeneie LNC
the absence of IL-3, and both survives and prolif- for a period of 10 days. The cells were then
erates in response to very small IL-3 titers. This collected, centrifuged on Ficoll and double
microbioassay has been used for analysis of the stained for CD4 and either CD44 or CD45RB.
activation of single, isolated T cells (Kelso and All of these in vitro activated cells were now
Owens, 1988) and was therefore best suited for CD44 high (not shown) and the majority were
the requirements of our experiment. In the exper- CD45RB b~' (Fig. 6). It was therefore possible to
iment shown in Fig. 5, CNS CD4 + T cells re- generate large numbers of mcmory-effectors upon
sponded to MBP. No response was detectable prolonged stimulation of LNC with the appropri-
from LN PKH2-1abelled CD4 + cells, although ate antigen.
these cells responded to PPD as strongly as did
CNS CD4 ~ cells. In other experiments, using Migration of OVA-reactice T cells' to CNS
greater numbers of PKH2 + LNC per well, re- The above results demonstrated the retention
sponse to MBP was detectable, but CNS cells at in CNS of m e m o r y / e f f e c t o r CD4 + cells that
comparable numbers generated at least five-fold were reactive to antigen in the CNS. They also
stronger responses. showed the presence of PPD-reactivc CD4 + T
A
E
:3
¢- iI
= m
re
- ° .
101 102 103 101 102 103
CD45RB CD45RB
Fig. 6. CD45RB expression by MBP-reactive C D 4 ~ LNC following in vitro culture for 4 days (A), and for a further 10 days (B). ~
Lymph node cells were isolated from mice that had been primed and boosted with M B P / C F A . The cells were cultured with MBP
for 4 days and then collected, centrifuged on Ficoll, ad recultured for a further 10 days with added MBP and irradiated LNC, after
which they were again centrifuged on Ficoll and double-stained with PE-CD4 and 23G2 supernatant. 23G2 was visualized using
FITC-goat-anti-rat Ig. Dead cells were excluded by side scatter gating.

9. 65
PKH2 + CD4 + cells that entered the CNS in
O V A transfers, nor was their CD45RB expres-
10 3` sion reduced (Fig. 7). These data argue against
: .. • .-,
the possibility that T cells with the CD45RB ~°w
.
CD45RB .. : .,:.: ~; }: (vv. Lymph Node
=-".: ." :'..:,~r:~.,:,:~:.'.L~,--%d<:.:.,.: phenotype were selectively retained in CNS, and
'.... :. ?i...y~.]:~i~,i~j'~.~,:~:::~: .1...
10 2-
.... + +,
.
suggest instead that their retention was a conse-
...:;~!,z:,.:.2h~;,:::.". ..-
'.~:: t quence of antigen recognition.
101 "
Discussion
200 400 600 800
Forward scatter
We have been able to track infiltrating CD4 +
T cells from the periphery into the CNS in a
passive transfer E A E model. By using a fluores-
CD45RB
103] 1 CNS
cent dye to label cells, we could examine the
phenotype of the CD4 + T cells that accumulated
in the CNS. Our results show that the CNS-in-
..
10 2 ,-:" • : . filtrating CD4 + T cells in E A E are of an acti-
i ::4d," -' vated or m e m o r y / e f f e c t o r phenotype that is rela-
,o,? :flf tively poorly represented in LN or in the in
vitro-activated autoreactive populations that
transfer EAE. The accumulation of cells of a
',1 . . . . i . . . . i , ~ i i i . . . . i . . . .
defined phenotype in a organ or tissue could
200 400 600 800
result from selective migration, or selective reten-
Forward scatter
tion. The fact that PPD-reactive CD4 + T cells
Fig. 7. C D 4 5 R B expression by PKH2-labelled OVA-reactive
are found in the CNS argues against there being
C D 4 + cells in CNS. Cells were isolated from CNS and lymph
nodes 10 days after transfer of PKH2-1abelled OVA-reactive
an absolute prohibition of entry of non-CNS-re-
cells, and C D 4 + cells identified with PE-CD4. Biotinylated active T cells. This is consistent with other re-
23G2 binding was visualized using P h y c o e r y t h r i n : T e x a s Red ports of entry of non-CNS-reactive T cells to the
(Tandem)-streptavidin. D e a d cells were excluded by side scat- CNS (Trotter and Steinman, 1984; Wekerle et al.,
ter gating. 1986, 1987), and with the lack of antigen speci-
ficity of either the expression or function of adhe-
sion molecules that are implicated in the interac-
cells. To further examine the role of CNS-anti- tion of T cells with endothelia (Picker et al., 1991;
gen-reactivity we transferred PKH2-1abelled Shimizu et al., 1991). However, the number of
OVA-reactive T cells into S J L / J mice. The mice CD4 + T cells in the CNS was very much less
did not develop EAE. The absolute number of following the transfer of OVA-reactive LNC than
CD4 + T cells recovered from the CNS was about that of MBP-reactive T cells. This points to a role
10-fold less than that obtained from animals with for CNS-antigen reactivity in the accumulation of
E A E (Table 1). 26% of CD4 + T cells from CNS CD4 + T cells in the CNS, and this could result
in the O V A transfer were PKH2 + , but only 6% from one of two processes, which are not mutu-
were blasts (not shown). The proportion of LN ally exclusive. Either MBP-reactive T cells are
CD4 + T cells that were PKH2-1abelled was simi- favored for entry to the CNS, or they are prefer-
lar in O V A transfers to that in MBP transfers. entially retained without there being any specific
However, in O V A transfers there was a greater barrier to T cell entry. A role for antigen recogni-
accumulation of PKH2 + blasts in lymph nodes tion is strongly favoured by the dramatic bias
(about 30%) (Fig. 7). These included cells show- among infiltrating MBP-reactive cells towards the
ing reduced levels of expression of CD45RB (Fig. CD44 high CD45RW °w phenotype. The homoge-
7). These were almost no blasts among the neous expression of high CD44 levels by CD4 +

10. MBP-reactive cells in the CNS contrasts with cclls dcscribed in Fig. 5. Secondary activation in
activated LN populations where at most 15% of thc CNS may be accompanied by other pheno-
CD4 + T cells are CD44 "igh (Fig. 3). Recall anti- typic changes that lead to T cell retention in thc
gen reactivity is associated with this phenotype tissue. Prolonged activation of T cells in vitro
(Butterfield et al., 1989), and prolonged activa- induces down-regulation of CD45RB expression
tion of T cell populations in vitro (e.g. to generate (Fig. 6; Birkcland et al., 1989). Our data arc
cell lines) induces homogeneously high levels of consistent with proliferative activation in the CNS
CD44. This argues that thc CNS-infiltrating that induces the CD45RB I°'~ phenotypc. Acti-
CD4 + T cells were all activated. Because vated T cells have been shown to preferentially
CD45RB big" cells were also found in the CNS, traffic in the blood (Mackay et al., 1990), and this
the CD45RB I°'~ phenotype cannot bc a require- accounts in part for the dominance of the
ment for extravasation. The fact that C D 4 5 R B " ' ' CD45RB high phenotype even in primed LN popu-
blasts were not found in the CNS following trans- lations. The significance of our observations is
fer of OVA-reactive cells but were present in LN further enhanced by the fact that these pheno-
shows that cells with this phenotype are not com- type changes are dynamic. Reverse transitions
mitted to migrate into the CNS. The CD45RB "'w from low to high MW CD45R expression can
phenotype is induced in vitro by activation occur and have been described for CD45RB in
through T C R / C D 3 ligation (Birkeland et al., rat (Bell et al., 1991)) and for CD45RO to
1989). The relative absence of blasts and the high CD45RA in human T cell lines (Lasalle and
level of CD45RB expression among the few CNS Hafler, 1991; Rothstein et al., 1991), Such reverse
infiltrating T cells in O V A transfers, in contrast transitions must contribute to the heterogeneity
to the CD45RB h'' blasts in MBP-induced E A E of LN populations. In this regard, the near-homo-
argues that retention of MBP-reactive T cells in geneity of CD45R expression by MBP-reactive
CNS was a consequence of their activation CD4 ~ T cells further supports our view that
through antigen recognition. This conclusion is these cells are actively responding to antigen. In
further supported by our data showing the gener- fact, it was possible to generate homogeneous
ation of CD45RB >w cells by antigenic stimulation populations of CD45RB l°' cells following pro-
for 10 days in vitro. longed in vitro culture of LNC although freshly
The remarkable correlation between seques- isolated cells from LN were always heteroge-
tration and the effector phenotype seems to us neous. The implication would be that cells with
compatible with the following model for the in- the memory-effector phenotype that are gener-
duction of E A E in an antigen-primed mouse. T ated within the LN migrate to the CNS where
cell activation in primed LN induces a m e m o r y / they accumulate within that antigenically rich en-
effector (CD44 high CD45RB I°~') phenotype, which vironment.
predisposes T cells to migrate from LN and to The CD44 high, CD45RB "~w phenotype has been
infiltrate tissues (Mackay et al., 1990; Mackay, implicated as a m e m o r y / e f f e c t o r subset largely
1990). Antigen-specificity may influence entry to on the basis of in vitro analysis. That this pheno-
certain tissues but the effect is not absolute. The type is predominantly represented at a site of
major influence on accumulation is at the level of autoimmune pathology supports its designation
retention as a consequence of antigen recogni- as an effector T cell subset. In a separate study,
tion. The selective accumulation in the CNS of we have shown that the T cells in the CNS of
CD45RB ~'''', CD4 + T cells then reflects the sec- animals with E A E induced by immunization with
ondary activation of CNS-reactive cells through spinal cord in adjuvant were also of a m e m o r y /
antigen recognition. Whether non-CNS-reactivc effector phenotype. The frequency of cytokine-
cells that have co-infiltrated are also induced to secreting CD4 + T cells in perivascular infiltrates
express the CD45RB l°w phenotype (e.g. through in the brains of those mice were 10-fold higher
bystander cytokine activation) remains to be de- than in primed LN or in in vitro-activated T cell
termincd. For instance, we do not know the populations (Renno et al., submitted). This is
CD45RB phenotype of the PPD-reactive CD4 + T entirely consistent with the results presented here.

11. 67
By studying the migration of PKH2-1abelled (1989) Changes in CD45 isoform expression accompany
antigen-primed T cells, we have been able to antigen-induced murine T-cell activation. Proc. Natl. Acad.
Sci. USA 86, 6734-6738.
assess the dynamics of effector selection and re- Bottomly, K., Luqman, M., Greenbaum, L., Carding, S., West,
tention in vivo, with a minimum of perturbation J., Pasqualini, T. and Murphy, D.B. (1989) A monoclonal
of the cells being analysed. Our experimental antibody to murine CD45R distinguishes CD4 T cell popu-
approach has therefore allowed us to identify lations that produce different cytokines. Eur. J. Immunol.
activated effectors within the CNS, and by use of 19, 617-623.
Budd, R.C., Cerottini, J.-C. and MacDonald, H.R. (1987)
cell sorted fluorescent-labelled populations from Selectively increased production of interferon-y by subsets
the CNS, to ascertain their antigen specificity. of Lyt-2 + and L3T4 + T cells identified by expression of
These results confirm a role for antigen recogni- Pgp-1. J. Immunol. 138, 3583-3586.
tion in the CNS in EAE, and point to its media- Burns, J., Rosenzweig, A., Zweiman, B., Moskovitz, A. and
tion of effector cell retention at this site. Lisak, R. (1984) Recovery of myelin basic protein reactive
T cells from spinal cords of Lewis rats with autoimmune
encephalomyelitis. J. Immunol. 132, 2690-2692.
Butterfield, K., Fathman, C.G. and Budd, R.C. (1989) A
Acknowledgements subset of memory CD4 + helper T lymphocytes identified
by expression of Pgp-1. J. Exp. Med. 169, 1461-1466.
We thank Diane Heath for technical assistance Chofilon, M., Weiner, H.L., Morimoto, C. and Hailer, D.A.
and preparation of reagents, and Claude Cantin (1989) Decrease of suppressor inducer (CD4 + 2 H 4 + ) T
cells in multiple sclerosis cerebrospinal fluid. Ann. Neurol.
(IRCM, Montreal) for FACS. We are grateful to
25, 494-499.
Dr. R. Kubo (Denver) for provision of the H57- Clatch, R.J., Miller, S.D., Metzner, R., Dal Canto, M.C. and
597 hybridoma and to Dr. Michael Julius (McGill Lipton, H.L. (1990) Monocytes/macrophages isolated from
University) for M1/89 antibody. We thank Dr. K. the mouse central nervous system contain infectious
Bottomly (Yale University) for generously provid- Theiler's Murine Encephalomyelitis Virus (TMEV). Virol-
ogy 176, 244-254.
ing purified anti-CD45RB mAb (16A) which was
Cross, A.H., Cannella, B., Brosnan, C.F. and Raine, C.S.
used for the initiation of these studies. We thank (1990) Homing to central nervous system vasculature by
Drs. Michael Ratcliffe, Neil Cashman, and Jack antigen-specific lymphocytes. I. Localization of 14C-labeled
Antel for discussions throughout this study, and cells during acute, chronic, and relapsing experimental
for review of the manuscript. This work was sup- allergic encephalomyelitis. Lab. Invest. 63, 162-170.
Hailer, D.A., Benjamin, D.S., Burks, J. and Weiner, H. (1987)
ported by the Multiple Sclerosis Society of
Myelin basic protein and proteolipid protein reactivity of
Canada, and by personal support awards from brain and cerebrospinal fluid-derived T cell clones in
MRC-Canada (T.O.) and Fonds pour la Forma- multiple sclerosis and postinfectious encephalomyelitis. J.
tion de Chercheurs et l'Aide ?t Ia Recherche Immunol. 139, 68-72.
(FCAR)-Qudbec and the Multiple Sclerosis Soci- Horan, P.K. and Slezak, S.E. (1989) Stable cell membrane
labelling. Nature 340, 167-168.
ety of Canada (R.Z.).
Jensen, M.A., Noronha, A., Toscas, A. and Arnason, B.G.W.
(1990) Activated T cells in the spinal cord in EAE differ
from activated T cells in draining nodes (Abstract). Neuro-
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