serological and biological cross-reactivity of class ii antigens between mice and humans in...
TRANSCRIPT
CELLULAR IMMUNOLOGY 87,659-673 (1984)
Serological and Biological Cross-Reactivity of Class II Antigens between Mice and Humans in Antigen-Specific T-Cell
Proliferative Responses
MASUJI YAMAMOTO AND AKIHIKO YANO’
Laboratory of Immunology, Department of Infectious Diseases and Parasitology, Shinshu University School of Medicine, 3-l-l Asahi, Matsumoto 390, Japan
Received March 21, 1984: accepted April 24, 1984
Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from BlO.S(9R) which possess I-A’ and I-E’ molecules serologically cross-reacting with human Class II antigens. On the contrary, BlOS(9R) T cells primed to PPD were stimulated by PPD- pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.
INTRODUCTION
Recently, several investigators have described the serological cross-reactivity between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens (l-3), while Lunney et al. have reported that murine allo-anti-Iak antibody cross- reacts with the shared determinant(s) of human Ia-like molecules (presumably DRa- chain) on B cells (4). Uemura and Yano have shown that human antigen-presenting cells (APC)2 share a serological cross-reactive determinant with murine I-AS molecules, and further have indicated that the cross-reactive determinant can function either as antigen-presenting molecules or as genetic restricting molecules for cell interactions in the purified protein derivative (PPD)-specific human T-cell responses (5). Okubo et al. have reported that murine anti-I-Ek antibody cross-reacts with some types of the polymorphic determinants of human la-like molecules on APC in PPD-specific proliferative responses (6). Furthermore, Lindahl and Bach and others have shown that xenogeneic Class I and Class II antigens can stimulate both cytotoxic T cells and mixed lymphocyte reaction (MLR)-responding T cells (7-12). Specifically, human
’ To whom correspondence should be addressed. 2 Abbreviations used: APC, antigen-presenting cells; C, complement; FCS, fetal calf serum; [‘H]TdR,
[3H]thymidine; ILl, interleukin 1; IL-2, interleukin 2; MLR, mixed lymphocyte reactions; MMC, mitomycin C, PETLES, peritoneal exudate T-lymphocyte-enriched cells; PBL, peripheral blood leukocytes; PPD, purified protein derivative.
659
0008-8749184 $3.00 Copyright 0 1984 by Academic Press, Inc.
All rights of reproduction in any form reserved.
660 YAMAMOTO AND YANO
MLR-responding T cells (7) and cytotoxic T cells (8-10) recognize the polymorphic determinants of murine Class I or Class II antigens, and similarly murine cytotoxic T cells ( 12) and MLR-responding T cells ( 11) also recognize human Class I or Class II antigens. Thus, it may be reasonable to assume that certain human T cells recognize some polymorphic determinants of murine Ia antigens of APC as self-polymorphic determinants of human Ia antigens in antigen-specific T-cell proliferative responses. Therefore, it is of interest to test whether the serological cross-reactivity of the poly- morphic determinants of Ia antigens between mice and humans can permit xenogeneic APC and antigen-specific T-cell interaction. BlO.S(9R) mice may be the most potent and suitable strain, since the Ia molecules coded for by the I-A” and I-Ek subregions of H-2 cross-react with human Ia antigens (such as HLA-DR antigens) of APC as previously described (4, 5). Two volunteers were selected according to the serological cross-reactivity of their Ia determinants with murine allo-anti-Ia antibodies (anti-I- AS and anti-I-Ek antibodies). Ia antigens of Human A include a serologically cross- reactive determinant with murine I-Ek molecules but not with murine I-A” molecules. In contrast, Ia antigens of Human B include a serologically cross-reactive determinant with murine I-AS molecules but not with murine I-Ek molecules. By using the xeno- geneic combinations of Human A, B, and BlO.S(9R), in addition to other strains based on the serological cross-reactivity of anti-I-AS antibody, and anti-I-Ek antibody, the possibility of cooperative cell interaction between xenogeneic APC and T cells was examined.
The data presented here indicate that PPD-specific Human-A and -B T cells are not stimulated by PPD-pulsed B lO.S(9R) APC, just as in the case of allogeneic human APC. On the contrary, the data suggest that BlO.S(9R) T cells primed to PPD were stimulated by Human-A and -B APC as well as by syngeneic BlO.S(9R) APC. Thus, the antigen-presenting ability of human APC is dominant over that of murine APC in the stimulation of PPD-specific xenogeneic T cells. On the basis of the data, Ia antigen or HLA-DR antigen may be seen to have some role as antigen-presenting molecules rather than as restricting molecules in the xenogeneic APC-T-cell interaction.
MATERIALS AND METHODS
Animals. C57BL/6 CrSlc (B6), BlO.S/SgSn, and BlO.A/SgSn mice were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Japan). BlO.S(gR), A.TL, and A.TH mice were bred in our laboratory. Mice of both sexes of between 6 and 18 weeks of age were used in this study.
Antigens and immunization. Purified protein derivative of Mycobacterium tuber- culosis (PPD) was purchased from Japan BCG Company (Tokyo, Japan). PPD was emulsified in incomplete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.). Animals were immunized in the hind footpads with 25 pg of PPD in a total volume of 0.1 ml of emulsion.
Antisera and monoclonal antibody. Procedures for raising A.TH anti-A.TL and A.TL anti-A.TH antibodies are described in detail elsewhere (5,6). Monoclonal anti- HLA-DR antibody (clone L243) was purchased from Becton-Dickinson Company (Sunnyvale, Calif.) (13). This monoclonal antibody was dialized against phosphate- buffered saline (PBS, 0.005 M, pH 7.6) to eliminate sodium azide.
Preparation ofperitoneal exudate T-lymphocyte-enriched cells (PETLES). The orig- inal and slightly modified procedures for preparing PETLES are described in detail
INTERSPECIES CROSS-REACTION OF CLASS II ANTIGEN 661
elsewhere (14). Briefly, 2-3 weeks after immunization, thioglycolate-induced peritoneal exudate cells were harvested and PETLES were purified by passing over columns of nylon wool (Fenwall Laboratories, Morton Grove, Ill.).
Preparation of spleen cells. Nonimmunized animals were sacrificed by cervical dislocation and the spleens removed aseptically. The spleen cells were washed once with RPM1 1640 (GIBCO, Grand Island, N.Y.) and were then suspended in RPM1 1640, supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO), 50 pg/ml of kanamycin (Meiji Pharmaceutical Co., Tokyo, Japan) and 5 X lop5 2- mercaptoethanol (culture medium). The cells were counted and kept on ice un- til used.
HLA typing and preparation of human peripheral blood lymphocyte (PBL). Cell donors were healthy volunteers whose HLA phenotypes were established by standard serologic techniques in Sakura National Hospital (Sakura, Chiba, Japan), and were tuberculin positive as judged by skin testing. Human PBL were prepared by Ficoll- Conray gradient centrifugation. After separation, the PBL were washed three times with RPM1 1640 and were then suspended in culture medium. Nylon-wool-adherent cells and nonadherent cells were separated using sterile nylon-wool columns as pre- viously described (6). The nonadherent cells were used as T cells, since the PPD- specific responding cells were already identified as humans T cells, namely, Leu l- positive cells ( 15).
Procedure for antigen pulsing. Murine spleen cells and unfractionated human PBL were used as APC in the assay system of the PPD-specific response as described elsewhere ( 16, 17).
Treatment of PPD-specific proliferative cells with murine allo-anti-la antibodies (anti-I-A” antibody and anti-I-Ek antibody) plus complement and cell cultures. One hundred microliters of culture medium, containing 1 X lo5 PBL treated with murine allo-anti-Ia antibodies (anti-I-AS antibody and anti-I-Ek antibody) plus complement (C), was placed in each well of a sterile, U-bottom microculture plate (Nunclon, Denmark). PPD (5 pg in culture medium) or medium alone as a control was added to each well. PBL (3 X 104) treated with mitomycin C (MMC) (Kyowa Hakko, Tokyo, Japan) were added as an APC source. The cells were incubated at 37°C in 3% COZ, 97% air in a humid atmosphere for 5 days and pulsed with 1 &i of [‘Hlthymidine ([3H]TdR) (New England Nuclear Corp., Boston, Mass.) per well 22 hr before har- vesting. The cells were harvested onto filter paper strips with a semiautomatic cell harvester (Abe Kagaku Co., Funabashi, Chiba). Incorporation of [3H]TdR into PBL was measured with a liquid scintillation counter (Packard, Downers Grove, Ill.). Each experimental group was assayed in triplicate and the results were expressed either as mean counts per minute f the standard error of the mean (SEM), or as the difference between antigen-stimulated and control responses (Acpm).
Analysis of the blocking eflects of murine allo-anti-la antibodies (anti-I-AS antibody and anti-I-Ek antibody) on PPD-spect$c human T-cell proliferative responses. The antiserum, either A.TL anti-A.TH antiserum (anti-I-A’) or A.TH anti-A.TL antiserum (anti-I-ES, was added at what would become a final concentration of 10 to 0.1% in the assay cultures. The blocking effect was measured by [3H]TdR incorporation. The blocking effect was calculated according to the following formula
% of blocking = 1 - PPD-specific responses with antibody (Acpm)
PPD-specific responses without antibody ( Acpm) x 100.
662 YAMAMOTO AND YANO
Assay of syngeneic, allogeneic, or xenogeneic APC-T-cell interactions in antigen- spectfic T-cell proltferative responses. In order to eliminate antigen-presenting cells from T-cell fractions, human nonadherent cells were pretreated with anti-HLA-DR antibody (1.0 pg) and C, while BlO.S(9R) PETLES were pretreated with A.TH anti- A.TL antibody (1: 10 diluted) and C. One hundred microliters of culture medium containing 5 X lo4 human nonadherent cells or 1 X lo5 B lO.S(9R) PETLES was placed in each well of a sterile, U-bottom microculture plate. A PPD-pulsed or non- pulsed suspension containing 1, 10, or 100 X lo4 APC was added in another 100 ~1 to give a total volume of 200 ~1. As a control, soluble PPD was added with a final concentration of 2 pg/ml. The APC-T-cell interaction was measured as antigen- specific T-cell proliferative responses, that is through [3H]TdR incorporation as already described. The data are expressed either as mean counts per minute + the standard error of the mean, or as the difference between antigen-pulsed and nonpulsed re- sponses (Acpm).
Interleukin 1 (IL-l) and interleukin 2 (IL-2). Highly purified human IL-1 and IL-2 were purchased from Genzyme Company (Boston, Mass.). IL1 (Catalog No. GuPI-1) and IL-2 (Catalog No. GuPI-2) were sterilized and stocked at -80°C until use. IL-l (final concentration 10 U/ml) or IL-2 (final concentration 10 U/ml) was added to the assay culture of antigen-specific T-cell proliferative responses.
RESULTS
Eflect of Cross-Reacting Murine A&Anti-la Antibodies on Human PPD-Spectfic Proliferative Responses
Effects of the treatment of human PBL with either cross-reacting A.TL anti-A.TH (anti-IaS) antibody or A.TH anti-A.TL (anti-Iak) antibody plus C was examined. As shown in Table 1, Experiment I, anti-Ias antibody plus C treatment eliminated PPD- specific proliferative responses of human B but not that of human A. On the other hand, anti-Iak antibody plus C aborted human A responses but not human B responses in PPD-specific T-cell responses (Table 1, Experiment II). Furthermore, blocking effects of these antibodies on PPD-specific responses of Human A and B were examined (Figs. 1A and B). Human B PPD-specific proliferative responses were blocked by anti-IaS antibody ( 100% blocking at 1: 10 dilution) but not by anti-Iak antibody, while human A PPD-specific proliferative responses were blocked by anti-Iak antibody (100% blocking at 1: 10 dilution) but not by anti-IaS antibody. These results suggest that murine allo-anti-IaS antibody and murine allo-anti-Iak antibody cross-react with some types of polymorphic determinants of la-like molecules on Human-B APC and Human-A APC, respectively. Furthermore, on the basis of the data from the blocking experiments, these result also indicate that the polymorphic determinants of Ia-like molecules on human APC detected by murine allo-anti-Ia antibody (anti-IaS and anti- Iak antibodies) function in the human APC-T-cell interaction.
Hierarchy ofAntigen-Presenting Ability of Human APC and Murine APC in Xenogeneic APC-T-Cell Interactions
In the next group of experiments, we investigated the antigen-presenting ability of human APC and B lO.S(9R) APC, which possess serologically cross-reacting I-AS and 1-E’ antigens, in xenogeneic APC-T-cell interactions. As shown in Table 2, the ex-
TAB
LE
I
Effe
ct o
f M
urin
e A
llo-A
nti-I
a A
ntib
ody
Plu
s C
Tre
atm
ent
on P
PD
-Spe
cific
PB
L R
espo
nses
Expt
A
ntib
ody
Res
pond
ing
PB
L”
PP
D-s
peci
fic p
rolif
erat
ive
resp
onse
s’
3 g A
nti-I
a’
or a
nti-l
a’
antib
ody
plus
C
%
C a
lone
8
Rec
onst
itute
d w
ith M
MC
- N
othi
ng
adde
d N
othi
ng a
dded
tre
ated
PB
LC
E
B
cpm
A
cpm
d
cm
Acp
md
cpm
A
cpm
d
0 F I
A.T
L an
ti-A
.TH
H
uman
A
Med
1,
740
f 38
0 4,
760
-+ 1
50
2,86
0 f
440
2 P
PD
16
,220
f 2,
200
14,4
80
19,7
40 *
300
14,9
80
15,2
00 +
2,0
80
12,3
40
t-5
c!
Hum
an
B
Med
3,
550
f 93
0 1,
510
+ 36
0 1,
160
+ 1,
100
2 P
PD
16
,700
+ 2
,350
13
,150
3,
180
+ 86
0 1,
670
11,6
00 f
3,52
0 10
,440
8
II A
.TH
an
ti-A
.TL
Hum
an A
M
ed
2,50
0 +
630
1,07
0 f
580
1,97
0 f
740
PP
D
13,7
00
@
k 2,
300
11,2
00
1,65
0 +
80
580
10,7
40 +
- 780
8,
770
g H
uman
B
Med
2,
800
+ 1,
520
2,75
0 f
200
1,50
0 f
40
v)
PP
D
28,4
30 f
2,
800
25,6
00
29,9
00 f
21
0 27
,150
27
,600
=:
f
2,65
0 26
,100
*
* PB
L (1
X
10s)
wer
e cu
lture
d w
ith o
r w
ithou
t P
PD
(5
&m
l) fo
r 5
days
. R
espo
ndin
g P
BL
wer
e tre
ated
with
m
urin
e al
lo-a
nti-I
a an
tibod
ies
(Exp
erim
ent
I: A
.TL
5
anti-
A.T
H
antib
ody;
Exp
erim
ent
II: A
.TH
an
ti-A
.TL
antib
ody)
plu
s C
or
with
C a
lone
, as
des
crib
ed u
nder
Mat
eria
ls a
nd M
etho
ds.
5
‘The
co
mpl
ete
HLA
typ
es o
f the
cel
l don
ors
wer
e: H
uman
A:
HLA
-AZ;
A
W24
; B
W52
; B
W55
; Cl;
DR
2; D
R9;
MTl
; M
T3;
Hum
an B
: HLA
-AI
1; A
W24
; B
35;
BW
5 1;
“z
CW
3; D
Rl;
DR
S; M
Tl;
MT2
. ‘A
ntig
en-p
rese
ntin
g ce
lls (3
X 1
04, m
itom
ycin
C
-trea
ted
PB
L) w
ere
adde
d to
the
tre
ated
pop
ulat
ion.
d
Acp
m =
PP
D-s
timul
ated
res
pons
e (c
pm)-
med
ium
al
one
resp
onse
(cpm
).
664 YAMAMOTO AND YANO
80
c” 60 .-
:: 40 0
n 20
z
8 O
-201 A
10 100 300 1000
reciprocal
80
60
I
10 20 40 80
dilution
FIG. 1. Blocking effect of murine allo-anti-Ia antibodies on PPDspecific responses of human PBL. Human-A (open circle) and Human-B (closed circle) PBL (I X 10’) were cultured with various concentrations of A.TL anti-A.TH antibody (A) or A.TH anti-A.TL antibody (B) in PPD-specific response assay culture for 5 days. The effect of the antibody on PPDspecific response. was expressed as percentage inhibition calculated by the following formula 96 of inhibition = [ 1 - (PPD-specific response with antibody (Acpm))/ (PPDspecific response without antibody (Acpm))] X 100.
periments were set up in a crisscross fashion so that the antigen-presenting ability of human and murine APC was tested against both syngeneic and xenogeneic T cells. According to the data from preliminary experiments set up to indicate the maximum responses, 1 X lo5 B lO.S(9R) PETLES or 5 X lo4 Human-A or -B T cells were cultured with 1 X lo5 B lO.S(9R) APC, 1 X lo5 Human-A APC, or 1 X lo5 Human- B APC. In order to eliminate APC, these responding cells were pretreated with either A.TH anti-A.TL antibody plus C for the murine T-cell preparation or anti-HLA-DR antibody plus C for the human T-cell preparations as described under Materials and Methods. To examine the effect of treatment on responding cells, soluble PPD was added to the treated cells and no significant PPD-specific responses were observed. PPD-pulsed B lOS(9R) APC stimulated the proliferation of BlOS(9R) PETLES (18,252 Acpm), while the same APC were ineffective in stimulating Human-A T cells (~0 Acpm) and Human-B T cells (810 Acpm). Human-A APC presented PPD well to autologous Human-A T cells (15,697 Acpm) but gave no effective stimulation for allogeneic Human-B T cells (3,806 Acpm). On the other hand, the same Human-A APC stimulated the proliferation of BlO.S(9R) PETLES (13,133 Acpm). Human-B APC presented PPD well to autologous Human-B T cells (9,641 Acpm) and provided significant stimulation for BlO.S(9R) PETLES (6,037 Acpm). But Human-B APC gave no effective stimulation for allogeneic Human-A T cells (473 Acpm). The data indicate that PPD-specific Human-A and -B T cells are not stimulated by PPD-pulsed BlO.S(9R) APC, and further suggest that BlO.S(9R) T cells primed to PPD are stim- ulated by PPD-pulsed Human-A and -B APC.
Dose Effects of Murine APC on PPD-Specific Proliferative Human T-Cell Responses and Kinetics of Xenogeneic APC-T-Cell Interaction
Because the cell dose of murine APC may account for the failure of antigen pre- sentation by murine APC to PPD-specific human T cells, the proliferative responses
TAB
LE
2
Hie
rarc
hy o
f A
ntig
en-P
rese
ntin
g A
bilit
y of
Hum
an
AP
C a
nd M
urin
e A
PC
in
Xen
ogen
eic
T-C
ell-A
PC
In
tera
ctio
n
PP
D-s
peci
fic p
rolif
erat
ive
resp
onse
s”
BlO
S(9
R)
PE
TLE
S
Hum
an
A n
onad
here
nt c
ells
H
uman
B
non
adhe
rent
cel
ls
AP
Cb
cpm
+ S
EM
A
cpm
’ cp
m f
S
EM
A
cpm
’ cp
m k
SE
M
Acp
m’
Non
e M
edd
3,16
5 k
1,26
2 1,
472
k 31
0 51
0 +
64
PP
D
3,66
1 k
692
496
991
f 11
5 <o
18
0 +
16
to
BlO
S(9
R)
sple
en c
ells
N
onpu
lsed
37
8 f
21
4,08
2 k
1,09
6 17
,716
+
1,49
1
PP
D-p
ulse
d 18
,630
k 2
,733
18
,252
3,
628
_t 1
,925
to
18
,526
f 1,
424
810
Hum
an-A
P
BL’
N
onpu
lsed
8,
256
+ 51
5 4,
041
f 1,
923
22,9
60 +
972
PP
D-p
ulse
d 21
,398
k
359
13,1
33
19,7
38 k
7,0
63
15,6
97
26,7
66 +
3,2
60
3806
Hum
an-B
PB
L’
Non
puls
ed
8,40
1 k
1,23
9 5,
619
+ 1,
890
4,70
1 -c
249
PP
D-p
ulse
d 14
,438
-+ 7
02
6,03
7 6,
092
k 1,
561
473
14,3
42 +
1,4
52
9641
’ BlO
.S(9
R)
PE
TLE
S (
1 X
105
) pre
treat
ed w
ith A
.TH
an
ti-A
.TL
antib
ody
(1: 1
0 di
lute
d) a
nd c
ompl
emen
t, or
Hum
an-A
or
-B
non
adhe
rent
cel
ls (
5 X
104
) pre
treat
ed
with
ant
i-HLA
-DR
an
tibod
y (1
.0 rg
) an
d co
mpl
emen
t w
ere
cultu
red
with
eith
er 1
X 1
0’ B
lO.S
(9R
) A
PC
, 1
X 1
0’ H
uman
-A
AP
C,
or 1
X l
o5 H
uman
-B
AP
C.
b Mur
ine
and
hum
an A
PC
wer
e tre
ated
with
mito
myc
in
C (
50 &
ml)
and
puls
ed w
ith o
r w
ithou
t P
PD
(25
&m
l) as
des
crib
ed u
nder
Mat
eria
ls a
nd M
etho
ds.
’ See
foot
note
d in
Tab
le
1.
d BlO
S(9
R)
PE
TLE
S,
Hum
an-A
, an
d -B
non
adhe
rent
cel
ls w
ere
cultu
red
with
PP
D (
2 &
ml)
or i
n m
ediu
m a
lone
(M
ed)
for
5 da
ys.
‘See
foo
tnot
e b
in T
able
I.
666 YAMAMOTO AND YANO
of human T cells to varying numbers of murine APC were examined. The responses of 5 X lo4 Human-A and -B T cells to PPD associated with varying numbers of autologous and BlO.S(9R) APC are shown in Table 3. The maximum proliferative response by autologous human APC was achieved with 1 X 1 O5 APC, while 1, 10, and 100 X lo4 B lO.S(9R) APC could not stimulate Human-A and -B T cells at all, although 10 X lo4 APC stimulated syngeneic PPD-specific PETLES. Thus, the cell dose of BlOS(9R) APC cannot account for the failure of antigen presentation by BlO.S(9R) APC to PPD-specific human T cells. Next, 5 X lo4 Human-A and -B T cells were cultured with either autologous or BlO.S(9R) APC for 3-6 days. As shown in Fig. 2, the responses by antigen-pulsed Human-A and Human-B APC peaked after a 5-day in vitro incubation, while significant responses by xenogeneic BlO.S(9R) APC were not observed through a 6-day in vitro incubation. Thus, the failure of antigen presentation by BlO.S(9R) APC to Human-A and -B T cells was not caused by differences in the time course of the proliferative responses.
The Influence of Xenogeneic Mixed Lymphocyte Reaction (MLR) in Antigen Presen- tation of Xenogeneic Murine APC to Human PPD-SpeciJic T Cells
One might explain the failure of antigen presentation of xenogeneic murine APC to human T cells seen in this assay by postulating a suppressive effect in cultures with an ongoing xenogeneic MLR. Human-A T cells (5 X 104) were cultured with mixtures of equal numbers of autologous Human-A APC and xenogeneic BlO.S(9R) APC and the ability of these mixtures to stimulate Human-A T cells was compared when antigen was bound to one or the other type of APC. As shown in Fig. 3, Human- A APC presented PPD well to autologous Human-A T cells despite the presence of BlO.S(9R) but BlO.S(9R) APC could not present PPD to Human-A T cells at all. The same patterns of stimulation were observed between Human-B and BlO.S(9R). Therefore, we conclude that ongoing xenogeneic MLR suppression cannot account for the failure of antigen presentation by murine APC to human PPD-specific T cells.
Efect of Human Lymphokines on PPD-SpeciJic Proliferative Responses
Because the lack of human lymphokines, such as IL-1 or IL-2, might account for the failure of antigen presentation by murine APC to PPD-specific human T cells, the effects of human IL-1 and IL-2 on the murine APC-human T-cell interaction were examined. As shown in Table 4, PPD-pulsed BlO.S(9R) APC could not present PPD to Human-A and -B T cells at all, irrespective of the addition of human IL1 and IL-2. The effects of interleukins on the human APC-T-cell interaction were also observed. Human-A and -B T cells (5 X 104) were stimulated well by PPD-pulsed autologous APC without the addition of human IL- 1 or IL-2 ( 18,222 Acpm in Human A; 21,272 Acpm in Human B). When IL-l (final concentration 10 U/ml) was added to culture medium, slight augmentations of PPD-specific proliferative responses were observed in Human A and B (21,045 Acpm in Human A; 23,344 Acpm in Human B). On the other hand, in the case of IL-2, a decrease of PPD-specific proliferative responses of Human A and B was observed, because of the increase of background responses by IL-2. Therefore, the failure of antigen presentation by murine APC to human T cells is attributed to the xenogeneic APC-T-cell interaction level and not to the lack of lymphokines, such as IL-l or B-2.
TAB
LE
3
Pro
lifer
ativ
e R
espo
nse
of H
uman
N
onad
here
nt C
ells
to V
aryi
ng N
umbe
rs o
f B
lO.S
(9R
) Spl
een
Cel
ls
Res
pond
ing
hum
an
nona
dher
ent
cells
A
PC
b
1 X
lo4
AP
C
cpm
+ S
EM
A
cpm
d
PP
D-s
peci
fic p
rolif
erat
ive
resp
onse
s”
1 X
10’ A
PC
1
X l
o6 A
PC
cpm
+ S
EM
A
cpm
d cp
m +
SE
M
Acp
md
Hum
an-A
no
nadh
eren
t ce
lls’
Hum
an-A
N
onpu
lsed
14
01 f
952
13,4
47 -t
1,3
74
3,60
0 f
270
PB
L P
PD
-pul
sed
1445
k 4
93
44
64,4
20 +
7,6
86
50,9
73
19,5
53 +
2,1
09
15,9
53
BIO
S(9
R)
Non
puls
ed
1679
k 2
80
5,44
3 +
1,96
3 91
6 +
229
sple
en c
ells
P
PD
-pul
sed
3220
-t
149
1541
7,
973
+ 49
2 2,
530
369
-+ 2
7 to
Hum
an-B
H
uman
-B
Non
puls
ed
266
f 93
1,
389
k 37
8 74
3 +
192
nona
dher
ent
PB
L P
PD
-pul
sed
343
+ 10
3 79
27
,709
+ 2
,738
26
,320
2,
976
f 1,
772
2,23
3 P
cells
’ B
lO.S
(9R
) N
onpu
lsed
67
3 k
263
1,41
0 +
528
197
+ 16
h
sple
en c
ells
P
PD
-pul
sed
1079
+ 8
30
406
3,01
5 +
288
1,60
5 33
0 f
60
133
z
’ H
uman
-A a
nd -
B n
onad
here
nt c
ells
(5 X
IO
“) w
ere.
cultu
red
with
var
ying
num
bers
of
auto
logo
us A
PC
or
BIO
.S(9
R)
AP
C f
or 5
day
s.
b See
foot
note
b in
Tab
le 2
. ‘S
ee f
ootn
ote
b in
Tab
le
1.
d See
foot
note
d in
Tab
le
1.
668
Acpmx10-3
A
6
YAMAMOTO AND YANO
3 4 5 6
days in culture
FIG. 2. Kinetics of antigen presentation by autologous (A) or xenogeneic BIOS(9R) (0) APC to 5 X lo4 Human-A nonadherent cells (A) or 5 X IO4 Human-B nonadherent cells (B). The results are expressed as Acpm.
cplnx 10-4
human A 1 10 100
BlO.S(9R) 1 10 100
CPm
B 3
x 10-4
human B 1 10 100
BlO.S(gR) 1 10 100
number of APC (x10-4
1
PIG. 3. (A) Human-A nonadherent cells (5 X 10’) were cultured with mixtures of equal numbers of PPD-pulsed Human-A APC and nonpulsed BlOS(9R) APC (O), nonpulsed Human-A APC and PPD- pulsed BlOS(9R) APC (A), or nonpulsed Human-A APC and nonpulsed BlOS(9R) APC (m). Stimulation was assessed 5 days later by measuring the incorporation of a 204~ pulse of [3H]TdR. The results are expressed as cpm. (B) Human-B nonadherent cells (5 X 104) were cultured with mixtures of equal numbers of PPD-pulsed Human-B APC and nonpulsed BlOS(9R) APC (O), nonpulsed Human-B APC and PPD pulsed BlOS(9R) APC (A), or nonpulsed Human-B APC and nonpulsed BIO.S(9R) APC (m). Stimulation was assessed 5 days later by measuring the incorporation of a 20-hr pulse of [‘H]TdR. The results are expressed as cpm.
TAB
LE
4
Effe
ct o
f H
uman
IL
-l an
d IL
-2 o
n H
uman
PP
D-S
peci
fic P
rolif
erat
ive
Res
pons
es
2
PP
D-s
peci
fic p
rolif
erat
ive
resp
onse
s”
2 R
espo
ndin
g Fi
hu
man
N
othi
ng a
dded
IL
- 1
adde
d ’
IL-2
add
edd
8 no
nadh
eren
t w
ce
lls
AP
Cb
cpm
A
cpm
’ cp
m
Acp
m’
cm
Acp
m ’
0
Hum
an-A
’ H
uman
-A
Non
puls
ed
3,27
0 +
21
5,58
9 +-
21
12,8
64 +
994
8
nona
dher
ent
PB
L P
PD
-pul
sed
21,4
92 +
1,
026
18,2
22
26,6
34 f
. 3,
191
21,0
45
25,1
08 f
1,
987
12,2
44
F ce
lls
BIO
S(9
R)
Non
puls
ed
2,39
3 -+
18
2,01
1 +
406
8,41
3 +
456
F
sple
en c
ells
P
PD
-pul
sed
2,57
4 +
245
181
2,08
8 +-
866
77
7,
741
?z 1
,111
<o
E
; 2
Hum
an-B
’ H
uman
-B
z N
onpu
lsed
4,
585
f 82
3 2,
437
2~ 46
0 23
,100
f
3,34
1 no
nadh
eren
t P
BL
PP
D-p
ulse
d 25
,875
+ 1
,419
21
,272
25
,781
-t
12,2
37
23,3
44
27,5
36 k
1,
378
4,43
6 e
cells
B
IO.S
(I)R
) N
onpu
lsed
1,
335
+ 69
0 2,
200
t- 21
3 11
,879
+ 3
03
$ sp
leen
cel
ls
PP
D-p
ulse
d 98
7 +
63
to
1,87
7 E
!Z 26
8 co
14
,365
_t
1,03
2 2,
486
g
’ H
uman
-A a
nd -
B n
onad
here
nt c
ells
(5 X
104
) wer
e cu
lture
d w
ith e
ither
aut
olog
ous
AP
C o
r B
lOS
(9R
) A
PC
for
5 d
ays.
=
‘See
foo
tnot
e b
in T
able
2.
> 5 ’ H
uman
IL-
l (fi
nal
conc
entra
tion
10 U
/ml)
was
add
ed in
PP
D-s
peci
fic p
rolif
erat
ive
assa
y cul
ture
. 5
d Hum
an I
L-2
(fina
l co
ncen
tratio
n 10
U/m
l) w
as a
dded
in P
PD
-spe
cific
pro
lifer
ativ
e as
say c
ultu
re.
“z
’ See
foot
note
d in
Tab
le
I. /S
ee f
ootn
ote
b in
Tab
le
I.
670 YAMAMOTO AND YANO
Failure of Antigen Presentation by Various H-2 Congenic Murine APC to Human PPD-SpeciJic T Cells The next group of experiments was performed in order to examine various com-
binations of both mouse and human cells in terms of biological cross-reactivity of Class II antigen in the xenogeneic APC-T-cell interaction (Table 5). By using murine APC from various H-2 congenic strains, the antigen-presenting ability of murine APC to various human T cells was examined. All strains used in these experiments have been shown to possess serological cross-reactive determinant(s) with human Ia antigens. Humans were chosen to overlap various serotypes of the HLA-DR antigens. The full HLA types of the cell donors were: Human A: HLA-A2; AW24; BW52; BW55; Cl; DR2; DR9; MT1 ; MT3.
Human B: HLA-All; AW24; B35; BW51; CW3; DRl; DR5; MTl; MT2. Human C: HLA-A 11; AW24; B 15; BW6 1; CW3; CW4; DR4; MT3. Human D: HLA-A2; AW24; B46; BW54; CWl; CW3; DR4; DR8; MT3. Human E: HLA-A33; B12; DRl; DR6; MTl; MT2.
Human-A, -B, -C, -D, and -E T cells were stimulated well by autologous human APC (40,885 Acpm in Human A; 23,408 Acpm in Human B, 19,575 Acpm in Human C; 57,541 Acpm in Human D; 15,398 Acpm in Human E). In contrast, antigen- pulsed BlO.S(9R), BlO.A, BlO.S, and B6 APC could not stimulate human T cells from any of the experimental subjects. Thus, the serological cross-reactivity of murine and human Ia antigen does not permit cooperative cell interaction between murine APC and human T cells.
DISCUSSION Many studies have already been reported indicating the biological cross-reactivity
of Class I and Class II antigens in xenogeneic MLR (7, 11) or xenogeneic-antigen- specific cytotoxic T cells (8, 12). Swain et al. (9, 10) have recently reported that the same human T-cell population is responsible for cytotoxic T-cell responses against both allogeneic and xenogeneic antigens. However, one of the important biological functions of a Class II antigen, antigen presentation for T cells, has not been examined in xenogeneic cell interactions. In this paper, we examine the biological cross-reactivity of the polymorphism of Class II antigen in the xenogeneic murine APC-human T- cell interaction. A hierarchy between the antigen-presenting ability of human APC and murine APC in the xenogeneic APC-T-cell interaction is shown to exist (Table 2). The data indicate that PPD-specific Human-A and -B T cells were not stimulated by PPD-pulsed BlO.S(9R) APC, and further suggest that BlO.S(9R) T cells primed to PPD were stimulated by PPD-pulsed Human-A and -B APC. In order to analyze the failure of antigen presentation by murine APC to PPD-specific human T cells, we examined various experimental conditions such as cell dose responses and kinetics of response. As shown in Table 3 and Fig. 2, these various experimental conditions cannot account for the failure of antigen presentation by BlO.S(9R) APC to PPD- specific human T cells. Furthermore, xenogeneic MLR did not affect the autologous human APC-T-cell interaction (Fig. 3). Thus, the failure of the murine APC-human T-cell interaction is not caused by the suppressive effect in cultures with ongoing xenogeneic MLR.
Recently, several research groups have examined the participation and functional relationship of HLA-DR antigens and interleukins in the process of activating T cells (18, 19). Palacios postulated that the interaction of HLA-DR molecules plus a foreign
APC
b
TAB
LE
5
Failu
re o
f Ant
igen
Pre
sent
atio
n of
Var
ious
H-2
Con
geni
c M
urin
e A
PC
for
Hum
an P
PD
-Spe
cific
T C
ells
PPD
-spe
cific
pr
olife
rativ
e re
spon
ses”
Hum
an
A H
uman
B
Hum
an C
H
uman
D
cpm
+
SEM
Ac
pm
d cp
m
+ SE
M
Acpm
d cp
m
-e S
EM
Acpm
d cp
m
f SE
M
Acpm
d
Hum
an
E
cpm
&
SEM
Ac
pmd
Non
e M
ed’
PPD
Auto
logo
us
APC
BIO
.S(9
R)
APC
BI0.
A AP
C
Non
puls
ed
PPD
-pul
sed
Non
puls
ed
PPD
-pul
sed
Non
puls
ed
PPD
-puk
ed
BI0.
S AP
C
Non
puls
ed
PPD
-pul
sed
B6 A
PC
Non
puls
ed
PPD
-puk
ed
1,84
3 f
388
1,97
1 i
371
13.5
36
i 3,
104
54,4
21
+ 2,
372
920
+ 32
798
+ I8
3
1,11
3 *
7
806
? 22
778
+ 14
9
1,28
3 2
703
1,52
1 k
720
1,67
2 +
632
824
f 52
I28
1,02
2 +
26
4,18
3 k
850
40,8
85
27,5
91
k 4,
067
2,28
7 f
1,12
2
10
1,85
2 f
1,09
3
764
f 37
<o
726
+ 84
771+
5
505
748
+ 48
1,08
2 +
216
151
797
rt 91
1,13
2 +
240
198
835
+ 81
<o
4,74
2 -c
797
23,4
08
24,3
17
+ 10
,144
19
,575
933
5 9
10
1,41
4 f
140
481
944
zi 3
3
to
867
f 72
<o
1,27
5 zt
447
<o
1,16
6 &
364
<o
1,60
7 -c
885
-CO
1,
080
+ 21
7 9
958
+ 41
3,83
0 f
540
10,9
62
k 2,
175
68,5
03
+ 11
,643
764
f 1 I
852
+ 16
0
1,49
8 f
350
1,72
0 f
247
2,45
1 f
1,05
2
2,28
3 f
147
1,07
1 +
231
1,08
0 -t
217
814
2 92
2,87
2 1,
114
+ 13
2 30
0
1,42
7 k
31 I
57,5
41
16,8
25
+ 91
3 15
,398
1,48
4 +
179
88
1,00
9 3z
94
<o
1,26
8 f
436
22
1,27
4 rk
261
6
825
+ 23
6
to
1,66
0 +
BOB
835
884
f 23
6
9 90
6 f
125
22
’ H
uman
no
nadh
eren
t ce
lls (
5 X
10’)
wer
e cu
lture
d w
ith
eith
er
auto
logo
us
APC
or
m
urin
e AP
C
from
va
rious
st
rain
s fo
r 5
days
in
mic
rotit
er
plat
es.
The
full
HLA
ty
pes
of t
he c
ell
dono
rs
wer
e de
scrib
ed
unde
r R
esul
ts.
‘See
fo
otno
te
b in
Tab
le
2.
‘Hum
an
nona
dher
ent
cells
wer
e cu
lture
d w
ith
PPD
(2
&m
l) or
in
med
ium
al
one
(Med
) fo
r 5
days
. d
See
foot
note
d
in T
able
1.
672 YAMAMOTO AND YANO
antigen from APC with the corresponding “receptors” on T cells results in the expres- sion of receptors for IL-2 (20). If so, the failure of antigen presentation by murine APC to human T cells may be caused by the lack of human lymphokines, such as IL1 or IL-2. It has been shown convincingly that IL-l, a product of DR/Ia+ mac- rophage, is an essential signal required by IL2-producing T cells and that human IL-l can substitute for murine IL-1 (2 1). Even if murine APC participate in the activation of human T cells through their Ia molecule (for example, the expression of IL-2 receptor on T cells), it is possible that human T cells cannot be stimulated on account of the lack of human lymphokines, such as human IL-1 or IL-2, in the xenogeneic cell interaction. In order to test this possibility, human IL-1 or IL-2 was added to this assay system. As shown in Table 4, the addition of human IL-l or IL- 2 had no significant effect on the murine APC-human T-cell interaction. Therefore, the failure of antigen presentation by murine APC to human T cells is to be attributed to the xenogeneic APC-T-cell interaction level and is not caused by the lack of nonspecific stimulatory factors, such as human IL-1 and IL-2.
In order to examine the interspecies biological cross-reactivity of Class II antigens, we used murine APC from various H-2 congenic strains and human T cells from many donors. Humans A, B, C, D, and E, who do not share individual HLA-DR specificities with each other, were chosen to provide responder T cells. As shown in Table 5, human T cells from donors possessing various HLA-DR phenotypes could not be stimulated by murine APC from BlO.S(9R) (serologically cross-reacting I-A” and I-Ek positive), B 10.A (serologically cross-reacting I-Ak positive), BIOS (serologically cross-reacting I-A” positive), and B6 (serologically cross-reacting I-Ab positive) (3). Thus, the sharing of polymorphic determinants of Class II antigens between mice and humans did not permit the successful xenogeneic murine APC-human T-cell interaction, although the reverse combination, the human APC-murine T-cell in- teraction, could be achieved (Table 2).
Another interpretation of the failure of antigen presentation by murine APC to human T cells is possible. In addition to the HLA-DR antigens, other human Class II antigens, such as DC or SB antigen, have recently been identified (22-24). On the basis of limited N-terminal amino acid sequence data and the base sequence of DNA, it has been suggested that the HLA-DR region is the human counterpart of the I-E subregion of the murine H-2 gene complex (25). The human analogs of the I-A subregion antigens have been shown to be DC antigens (26). Genetic mapping studies in mice have demonstrated that the I-A molecule has a more dominant function than the I-E molecule and that identity at the I-A subregion of the H-2 gene is essential for the APC-T-cell interaction in antigen-specific proliferative response such as ova- lubumin or PPD (17). Data from human studies have shown that the HLA-DR molecules of APC have a more dominant function than other Class II molecules in antigen-specific proliferative responses and that HLA-DR identity between priming and restimulating APC is essential for optimal proliferative responses to soluble antigens such as PPD (27, 28). Therefore, the failure of antigen presentation by murine APC to human T cells may be caused by the inconsistency of the molecules used in the xenogeneic APC-T-cell interaction, that is, that the I-A molecule (analog of human DC molecule) of murine APC, rather than the I-E molecule (analog of HLA-DR molecule), plays a dominant role in PPD-specific murine T-cell proliferative responses. This may imply that Class II molecules do not simply function for T-cell triggering through the association of their unique polymorphic structures and nominal antigens.
INTERSPECIES CROSS-REACTION OF CLASS II ANTIGEN 673
Some research groups have already reported that human T cells recognize the polymorphic determinants of murine Ia antigens in the human anti-mouse xenogeneic MLR (7, 9, 10). Nevertheless, the question remains to be answered whether the murine Ia antigen recognition performed by human PPD-specific proliferative T cells is the same as that performed by the xenogeneic-antigen (mouse)-specific human T cells. Thus, another possible explanation of the failure of antigen presentation by murine APC to human T cells is that the murine Ia antigen lacks polymorphic determinants for triggering human PPD-specific T cells. A further possible explanation is that serological interspecies cross-reactive Ia determinants (for example, detected by murine allo-anti-Ia” antibody and anti-Iak antibody) cannot function as either restricting molecules or presenting molecules in the murine APC-human T-cell in- teraction.
The mechanism of successful antigen-presentation by human APC to murine T cells is now under active study. These studies using xenogeneic cell interaction may shed some light on the immunobiological function of polymorphism of Class II antigens in antigen presentation.
ACKNOWLEDGMENT The authors thank Ms. Rieko Okae for her excellent secretarial help.
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1983. 2. Brickell, P. M., McConnel, I., Milstein, C., and Wright, B., Immunology 43, 493, 1981. 3. Aosai, F., Yui, K., Yamamoto, M., and Yano, A., Fed. Proc. 42, 1229, 1983. 4. Lunney, J. K., Mann, D. L., and Sachs, D. H., &and. J. Immunol. 10,403, 1979. 5. Uemura, K., and Yano, A., Immunol. Lett. 5, 293, 1982. 6. Okubo, Y., Kusama, S., Yamashita, Y., Kitazawa, K., Nagasaka, M., and Yano, A., Cell. Immunol.
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10. Swain, S. L., Immunol. Rev. 74, 129, 1983. 11. Russo, C., Quaranta, V., Indiveri, F., Pellegrino, M. A., and Ferrone, S., Immunogenetics 11, 4 13,
1980. 12. Engelhard, V. H., and Benjamin, C., Immunogenetics 18, 461, 1983. 13. Lampson, L. A., and Levy, R., J. Immunol. 125,293, 1980. 14. Schwartz, R. H., Jackson, L., and Paul, W. E., J. Immunol. 115, 1330, 1976. 15. Okubo, Y., Kusama, S., and Yano, A., Microbial. Immunol. 26, 5 11, 1982. 16. Yano, A., Schwartz, R. H., and Paul, W. E., J. Exp. Med. 146, 828, 1977. 17. Schwartz, R. H., Yano, A., and Paul, W. E., Immunol. Rev. 40, 153, 1978. 18. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151, 155 1, 1980. 19. Watson, J., and Mochizuki, D., Immunol. Rev. 51, 257, 1980. 20. Palacios, R., Immunol. Rev. 63, 73, 1983. 21. Oppenheim, J. J., and Gery, I., Immunol. Today 3, 113, 1982. 22. Shaw, S., Kavathas, P., Pollack, M. S., Charmot, D., and Mawas, C., Nature (London) 293,745, 1981. 23. Shaw, S., Johnson, A. H., and Shearer, G. M., J. Exp. Med. 152, 565, 1980. 24. Tanigaki, N., and Tosi, R., Immunol. Rev. 66, 5, 1982. 25. Allison, J. P., Walker, L. E., Russell, W. A,, Pellergrino, M. A., Ferrone, S., Reisfeld, R. A., Frelinger,
J. A., and Silver, J., Proc. Natl. Acad. Sci. USA 75, 3953, 1978. 26. Bono, R., and Strominger, J. L., Immunogenetics 18, 453, 1983. 27. Bergholtz, B. O., and Thorsby, E., Stand. J. Immunol. 8,63, 1978. 28. Bergholtz, B. O., and Thorsby, E., Stand. J. Immunol. 6, 779, 1977.