Title
[原著］Thy.1^- Bone Marrow Cells Passed through Nylon
Wool Can Augment the Generation of Cytotoxic T
Lymphocytes (CTL) with Preference for the
X-Chromosome-Linked Gene Product in Primary Cultures in vitro
Author(s)
Tanabe, Masao J
Citation
琉球医学会誌 = Ryukyu Medical Journal, 13(2): 133-145
Issue Date
1993
URL
http://hdl.handle.net/20.500.12001/3117
Ryukyu Med.J., 13 (2) 133-145, 1993
Thy.l Bone Marrow Cells Passed through Nylon Wool Can
Augment the Generation of Cytotoxic T Lymphocytes (CTL)
with Preference for the X-Chromosome-Linked Gene
Product in Primary Cultures in vitro
Masao J. Tanabe
Department of Bacteriology, Faculty of Medicine, University of the Ryukyus
(Received on November 2nd 1992, accepted on January 27th 1993)
Key words : X-chromosome, minor histocompatibility antigen, cytotoxic T lymphocytes, bone marrow cells
133
ABSTRACT
Thy.l bone marrow cells passed through nylon wool (NW-BM cell) helped to induce the cytotoxic
T lymphocytes (CTL) specific for allogeneic major histocompatibility complex (MHC) antigens in vitro
l)
as previously reported. In this report we studied whether they could also help to induce the CTL specific to minor histocompatibility (raH) antigens. When a limited number of responder lymph node (LN) cells and an excess of helper NW-BM cells from Balb/c (H-2 ) mice were cultured with stimula-tor spleen cells from BIOD2 mice (H-2 ), the CTL specific for mH antigens restricted to self MHC were easily induced in primary cultures in vitro. These CTL killed only BIOD2 target cells but not DBA/2 (H-2 ) which had some mH antigens in common with BIOD2. Genetic analysis showed that these CTL had a strict preference for X-chromosome linked gene products (Xir antigen). When re-sponder LN cells and NW-BM cells from Balb/c mice were replaced by those from DBA/2 mice, the same results were obtained. As the CTL specific to the Xir antigen was also induced either with (BIOBRxBalb/c) Fl male anti (Balb/CXBIOBR) Fl male or with a reciprocal combination, stimulation across the sex chromosomes was necessary and sufficient to induce these CTL. Stimulation across other mH antigens derived from autosomes was not required as the trans-activating bystander. This report suggests that the Xir antigen has a role in immune regulation, because non-self Xir anti-gen is the only stimulus in this culture system.
134 X-Chromosome-Linked Minor-H Antigen
INTRODUCTION
When stimulation across broad
non-H-2-coded genetic differences occurs, that is, across minor histocompatibihty (mH) antigen differ-ences, donor organ grafts are rejected 'and the graft versus host (GVH) reaction is inducedaf-3.6.7)
ter the transplantation of bone marrow cells The antigenicity of mH antigens was neither
8)
weak nor minor because the strength of the skin graft rejection by the incompatible mH antigens was to the same degree as that caused by incompatible major histocompatibility
com-9.10)
plex (MHC) antigens . Congenic strains of mice related to mH antigens have been
ll.12)
established . From the numbers of mrl anti-gens defined in these strains, it was speculated that several hundreds of genes on mH antigens
13I
were present in mice . These mH antigens
6,14)
were scattered over the entire chromosome The mH antigens are related to graft
trans-p一antation and contribute to the generation of
the repertoire of the T eel】 receptor by being
15- 17)
self antigens in the thymus or periphery Recently, it was suggested that mH antigens had relationships to some degree with retroviruses because the sites of mH antigen gene loci in chromosomes were present near the integration
14.18-21I
sites of retroviruses
In general, the cytotoxic T lymphocytes (CTL) specific to mH antigens can be induced by priming in vivo and boosting in vitro across broad non-H-2-coded genetic differences. These induced CTL recognized the mH antigens
22.23)
restricted to MHC . In these reports the CTL had specificity to many mH antigens, that is, they lysed not only the target cells from the stimulator strains but also the target cells which had the same MHC as the stimulator cells but a different genetic background. On the
24.25)
other hand, in a few reports the CTL in-duced were specific only to a few dominant mH antigens in spite of the stimulation across many
disparate mH antigens. The CTL can also be induced by a single mH anLIgen difference using
26- 28)
congenic mice strains . Stimu】ated across only two mH antigens, the CTL were specific only to one of them. Thus, there is a hierarchy between these two mH antigens, that is. one is
ご5-.'9サ
dominant
ll
In my previous paper , we found that bone
marrow cells passed through nylon wool (NW-BM cells) helped to induce the CTL specific to allogeneic MHC antigens in primary cultures in vitro. These helper NW-BM cells were Thy.lt CD4 CD8 cells and required MHC molecules identical with the CTL precursors to augment their induction. We show in this report that CTL can be easily induced against mH antigens in primary mixed lymphocytes eultures (MLC) in vitro, when NW-BM cells are used as the hel-per cells. Despite the stimulation across many mH antigens, these CTL had a strict preference
for X-chromosome 一inked gene products (Xir
antigen) and did not recognize any other mH antigens. Thus, the Xir antigen provided the only stimulation among, many mH antigens in our culture system, and behaved as the domi-nant type. We also discuss the Xir antigen which is objective for sejf-nonself discrimina-tion in our MLC system and′which plays a role
in the immune regulation.
MATERIALS AND METHODS
Mice
We used 8- to 16-week-old male and female mice of inbred strains BIOBR 2k), BIOD2 (H-2), Balb/ (H-2d), and DBA/2 (H-(H-2), male
(BIOBRxBalb/c) Fl (H-2k′) and (Balb/CXBIO
BR) Fl (H-2 ) mice bred in the animal facility at the School of Medicine, University of the Ryukyus, Okinawa, Japan.
Mixed lymphocyte cultures (MLC)
Tanabe, M.J.
い
previously. In brief, cells from lymph nodes (LN) (inguinal, axillary, and brachial) or spleens were suspended into our standard culture medium (a-minimum essential medium [a MEMJ) (Flow Laboratories, Inc., Rockville, Md.) containing lO% fetal calf serum (FCS) (Lot No. 10006, General Scientific Laboratories, L.A., Calif.), 10 mM HEPES buffer, and 5xlO" M 2・ mercaptoethanol. Cell suspensions of bone marrow (BM) cells obtained from the femur were prepared by passing through syringe
nee-dies. Two milliliter (2.5xlO cells/ml) BM cell
suspensions were loaded on a nylon wool (Wako
Pure Chemical, Inc., Osaka, Japan) column.
Af-ter incubation for 45 minutes at 37℃,
nonadhe-rent cells were eluted, washed once by centri-fugation, resuspended in the culture medium as30)
described previously then referred to as
NW-BM ce一ls. Severa一 doses of LN cells (as respon-】)
der cells), NW-BM cells (as helper cells ), and
3xlO mitomycin-c treated spleen ce】Is (as sti-mulator cells) were co-cultured in microculture wells (Cat. No. 76-023-05, Flow Laboratories, Inc., McLean, Virginia) containing 0.2 ml of cul-ture medium supplemented with several doses of rat T cell growth factor (TCGF) (Cat. No. 40114, Collaborative Research, Inc., Lexington,い
Mass.) as described previously. Contro一 cu上 tures, from which were obtained the spon-taneous value, did not contain responder LN cells. These cultures were incubated for 5 days at 37℃ in an atmosphere of 5% C02 in air.
A ntisemm
The anti-Thy.1.2 monoclonal antibody was kindly provided by Dr. H. Ishikawa (Department of Microbiology, School of Medicine, Keio Uni-versity, Tokyo). Cells were incubated with antiserum for 30 minutes at 4℃, washed once in cytotoxicity medium (Cedarlane Laboratories, Inc., Hornby, Ontario, Canada), and incubated in a water bath with rabbit complement
(Low・Tox-135
M; Cedarlane Laboratories), at a fina一 di一ution
of 1:10, for 45 minutes at 37℃. ce】Is were
then washed three times in cu一ture medium.Cytotoxic assay
After five days in culture, MLC were assayed for cytotoxic activity by using Cr-labelled concanavalin A (Con A) blast target spleen cells, 1xlO cells/well, as described
31)
previously . Cells in each microculture wel】 were split into two wells. Target cells from stimulator strains were added into one well, and target cells from third party strains, which were different from either responder or stimula-tor strains, were added to the other. The pur-pose of these experiments was to test whether the CTL were specific either for only stimula-tor, or for both stimulator and third party strains. After four hours in culture, specific Cr release, p, was defined as observed counts minus spontaneous counts / total releasable counts minus spontaneous counts. Each ex-perimenta一 group contained 32 replicate cuト tures. Test wells were scored as positive if their counts were greater than the mean spon-taneous value by more than 2 standard devia-tions (pく0.05).
RESULTS
The induction of cytotoxic T lymphocytes (CTL) specific to minor histocompatibihty (mH) antigens
in Pγimary cultures in vitro
NW-BM cells helped to induce CTL from sponder LN cells specific for mH antigens re-stricted to self major histocompatibility complex (MHC) in primary cultures in vitro. The CTL were induced in three wells among 32 and kiト led only the target cells from BIOD2 mice (Fig. 1A). Although DBA/2 mice had some mH anti-gens in common with BIOD2 mice, CTL with
specificity to these common mH antigens were not induced at all. This result suggested that
136 ( N ＼ V m Q ) sisXi oiアioadの % 0 0 [ t v ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ l
A
Ⅹ-Chromosome-Linked Minor-H Antigen
( U ＼ q ￨ B 皿 ) s i s A 一 o i j . i o a d s % 20 0 -1 蝣--● 20 40 60 (BIOD2)
% specific lysis
A-1寸 ォ-- -i J 10 20 30 (B-1 0D2)% specific lysis
Fig. 1. The mH specific CTL were induced by the helper effect of NW-BM cells in primary
cul-tures in vitro. A) Three thousands cells/well of Balb/c LN cells, 3xlO cells/well of
mitomycin-c treated BIOD2 spleen cells, and 3xlO4 cells/well of Balb/c Thy.l" NW-BM
cells were c0-cultured in 32 replicate culture wells supplemented with 0.25 units/ml of rat TCGF. Duplicate sets of these cultures were prepared. In one set, each well was split into two wells after 5 days in culture, then BIOD2 target cells were added to one well and DBA/2 target cells were added to the other. Only the positive wells were plotted accord-ing to MATERIALS AND METHODS. The broken lines show two standard deviations (p
く0.05) of the spontaneous lysis value. In the other set of cultures (parallel cu一tures)
BIOBR target cells were added, but no lysis was detected in any wells. B) The experi-ment was carried out as for Fig. 1A. One thousand cells/well of DBA/2 LN cells, 3xlO5
cells/wel一 of BIOD2 spleen cells, and lxlO ce一ls/well of DBA/2 Thy.l" NW-BM cells
were c0-cultured. Wells were supplemented with 0.4 units/ml of rat TCGF. In one set of cultures* BIOD2 and Balb/c target cells were used, and positive wells alone were plot-ted. In the other set (parallel cultures), BIOBR target cells were used but no lysis was observed.
these CTL had a narrow spectrum of specificity, that is, against one or a few mH antigens.
We also obtained the same results when LN cells, 1,000 cells/well, from DBA/2 mice as the responders were cultured with spleen cells from
BIOD2 mice as the stimulators, then NW-BM, 1xlO cells/well, from DBA/2 mice were added to these cultures. The CTL were induced in 4
among 32 wells and they were specific against BIOD2 but not Balb/c (Fig. IB).
The CTL induced in parallel cultures (32 wells) with the same protocol did not kill the target cells from BIOBR mice (H-2, BIO back-ground), thus they did not recognize the mH antigen alone but did recognize the mH antigen restricted to self-MHC.
Tanabe, M. I. ( e ￨ e w ∝ 皿 O L g X u ＼ q i e g )
s
j
s
X
￨
o
n
l
o
a
d
s
%
10 20 30(BIOBRXBalb/c male)
% specific lysis
(B¥e∈∝皿OlaXu＼q一eg)s
j
s
A
￨
o
j
j
j
o
e
d
s
%
137 I l l [ l -+-1 0 20 30(BIOBRXBalb/c male)
% specific lysis
Fig. 2. The CTL had a strict preference for the X-chromosome linked gene products (Xir antigen).A) One thousand cells/well of Ba】b/c LN cells, 3xlO cells/we日of mitomycin-c treated
BIOD2 spleen eells, and 3xlO cells/well of Balb/c Thy.l" NW-BM cells were c0-cu】tured
in 32 replicate culture wells supp】emented with 0.33 units/ml of rat TCGF. Each well was
split into two wells after 5 days in culture, then (BIOBRxBalb/c) Fl ma一e target cells
were added to one well and (Balb/CXBIOBR) Fl male target cells were added to the other. Only the positive wells were plotted according to MATERIALS AND METHODS. The
broken lines show two standard deviations (pく0.05) of spontaneous lysis value. B) The
ex-periment was carried out as for Fig. 2A. One thousand cells/well of DBA/2 LN cells, 3xlO5 cells/well of BIOD2 spleen cells, and lxlO4 cells/well of DBA/2 Thy.l" NW-BM cells were c0-cultured. Wells were supplemented with 0.33 units/ml of rat TCGF. Target cells were from (BIOBRxBalb/c) Fl male and (Balb/CXBIOBR) Fl male mice.
The CTL had a strict preference for X-chγ抑0-so桝e linked gene products (Xir antigen) over other mtt antigens
We analyzed the genetics of antigens recog-nized by the CTL, which were induced as shown in Figs. 1A and IB. Firstly, we used Fl male mouse target cells to examine whether the genes of these antigens were linked to the
sex chromosomes. We set up the same experi-merit as described in the legend to Fig. 1A. A
limited number of LN ce一ls (1,000 cells/well)
from Balb/c mice as responder ce】Is were
stimulated with mitomycin-c treated spleen cells
from BIOD2 mice, and T cell depleted NW-BM cells, 3xlO cells/well, from Balb/c mice were added to these cultures as helper cells.
The CTL induced killed the target cells from (BIOBRxBalb/c) Fl male mice but not those from (Balb/CXBIOBR) Fl male mice (Fig. 2A). As both (BIOBRxBalb/c) Fl and (Balb/c XBIOBR) Fl male mice had the same autosomes but reciprocally different sex chromosomes de-rived from either parental strains, the antigens recognized by the CTL were sex chromosome
138 ( a ¥ e ∈ ∝ 田 o i e X o / q ￨ B 血 )
s
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0 0 ' i l lA
X-Chromosome-Linked Minor-H Antigen
g 8 ( a ￨ e i u ∝ a o i a X u ＼ q i ^ a )
s
i
s
A
i
o
i
j
j
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a
d
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%
「 l ● 1 0 20 30 40 (BIOBRXBalb/c ma一e)% specific lysis
JI T∴⊥∵∴∴∴‥∵:「∵ 20 40 60 (BIOBRXBalb/c ma一e)% specific lysis
Fig. 3. The stimulation across sex chromosome difference was necessary and sufficient to induce the CTL. A) The experiment was carried out as described in the legend to Fig. 2A. Three thousands cells/well of (Balb/CXBIOBR) Fl male LN cells, 3xlO cells/well of
(BIOBRxBalb/c) Fl male spleen cells, and 3xlO4 eel】s/well of (Balb/CXBIOBR) Fl ma一e
Thy.l NW-BM cells were c0-cultured and supplemented with 0.5 units/ml of rat TCGF. Target cells were from (BIOBRxBalb/c) Fl male and (Balb/CXBIOBR) Fl male mice. B) The experiment was carried out as described in the legend to Fig. 2A. One thousand cells/well of (BIOBRxBalb/c) Fl male LN ceHs, 3xlO cells/well of (Balb/CXBIOBR) Fl male spleen cells, and 3xlO cells/well of (BIOBRxBalb/c) Fl male Thy.l NW-BM cells were c0-cultured and supplemented with 0.5 units/ml of rat TCGF. Target cells were from (BIOBRxBalb/c) Fl male and (Balb/CXBIOBR) Fl male mice.
linked gene products. These CTL also killed the target cells from 】〕10D2 mice as shown in Fig. 1A. Both BIOD2 and (BIOBRxBalb/c) Fl male mice had the X-chromosome in common but had a different Y-chromosome. Thus, these CTL had a strict preference for the X-chromo-some linked gene product (Xir antigen) to other mH antigens. The Xir antigen was the absolute dominant antigen because it behaved as the ma-jor antigen like MHC, since it was the only one which elicited the generation of CTL in this cul-ture system despite stimulation across broad non-H-2-coded genetic differences.
When responder LN cells, 1,000 cells/well,
and NW-BM cells, 1xlO cells/well, from DBA/2 mice were stimulated with BIOD2 spleen cells in Fig. 2B, the results were the same as shown in Fig. 2A. As the CTL not only from Balb/c but also from DBA/2 mice, despite the different genetic background in both strains, recognized the common mH antigen, namely, the Xir antigen of BIOD2 mice, the dominancy of the Xir antigen was dependent on the intrinsic nature of this antigen on BIOD2 stimulator cells, but was not dependent on the relation between the responder and stimulator strain of mice.
Tanabe, M. I. 40 10 ( o ＼ q i e g )
s
i
s
A
i
o
i
i
i
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e
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s
%
l l l l l l J l 】 J l l l l l -U---一一ll l l ● ● 10 20 30 (BIOD2) specific lysis ( U ＼ q i e e )s
j
s
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￨
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p
e
d
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0 0 4 3 -11 - -1 1 0 20 30 (BIOD2) specific lysis 139Fig. 4. The CTL induced by the stimulation across sex chromosome difference were specific for the Xir antigen. A) The experiment was carried out as described in the legend to Fig. 2A. One thousand cells/well of (Balb/CXBIOBR) Fl male LN cells, 3xlO cells/well of (BIOBRxBalb/c) Fl male spleen cells, and 3xlO* cells/well of (Balb/CXBIOBR) Fl male Thy.l NW-BM cells were co-cultured. Wells were supplemented with 0.5 units/ml of rat TCGF. Target cells were from BIOD2 and Balb/c mice. B) The experiment was carried out as described in the legend to Fig. 2A. One thousand cells/well of (BIOBRxBalb/c) Fl
male LN cells, 3xlO cells/well of (Balb/c耳BIOBR) Fl male spleen cells, and 3xlO'
cells/well of (BIOBRxBalb/c) Fl male Thy.l NW-BM cells were c0-cultured. Wells were supplemented with 0.33 units/ml of rat TCGF. Target cells were from BIOD2 and Balb/c mice.
Stimulation across seズ chromosome difference
was necessary and sufficient to induce CTL
We examined nextly whether the CTL could
be induced by sLimulation across only the sex chromosome difference. A limited number ofLN ce一ls from (Balb/CXBIOBR) Fl male mice as
responder cells were cultured with mitomycin-c treated spleen cells from (BIOBRxBalb/c) Fl male mice, and NW-BM cells from the same strain as the responder cells were added to these cultures.
As the CTL induced killed target eel】s both from (BIOBRxBa】b/c) Fl male mice (Fig. 3A) and BIOD2 mice (Fig. 4A) but did not kin
target cells from (Balb/CXBIOBR) Fl male mice as self (Fig. 3A) and from Balb/c mice (Fig. 4A), it was found that the Xir antigen alone was recognized by these CTL. These results sug-gested that stimulation across only a sex chromosome difference was necessary and suHi-cient for the induction of the CTL and that other mH antigens derived from autosomes were not necessary as trans-activating bystander sti-mull (Figs. 3A and 4A).
When the reciprocal combination of respond-er and stimu】ator cells was cultured, CTL wrespond-ere a一so induced which k川ed both target cells from (BaJb/CXBIOBR) Fl male mice (Fig. 3B) and
MO X-Chromosome-Linked Minor-H Antigen
those from Balb/c mice (Fig. 4B) but never the reciprocal set of target cells. This showed that Balb/c mice (Figs. 3B and 4B), as well as BIOD2 mice (Figs. 1, 2, 3A and 4A), had the Xir antigen, that the Xir antigen derived from Balb/c mice also elicited the induction of CTL (Figs. 3B and 4B), and that the Xir antigen was polymorphic or ol,igomorphic because both BIOD2 and Balb/c mice had different Xir anti-gens (Figs. 3 and 4).
DISCUSSION
In general, cytotoxic T lymphocytes (CTL) specific to the minor histocompatibility (mH) antigens can be induced by in vivo priming fol-lowed by in vitro boosting across broad non-H-2-coded genetic differences ・23) This report showed that the CTL specific to mH antigens were induced easily in primary mixed 】ympho-cyte cultures (MLC) in vitro (Figs. 1A and IB).
1)
My previous paper reported that bone mar-row cells passed through nylon wool (NW-BM cells) then treated with anti-Thy.l monoclonal antibody and complement had helper activity which was involved in the induction of CTL specific for the allogeneic major histocompatibiト Ity complex (MHC). In these cultures, respon-der lymph node (LN) cells were present in such limited numbers that helper T cells were di-luted out from the responder cells. Thus, alia MHC specific CTL were generated from their precursors by receiving helper effects from
NW-BM cells. NW-BM cells also had helper activity in inducing the mH specific CTL as shown here. There has been only one previous report stating that mH specific CTL were
in-32.33)
duced in primary cultures in vitro . In that study, leukemia cells were used as the target, which may be critical because leukemia cells are more susceptible to cytolysis by CTL than
normal concanavalin A (Con A) blast cells
In our system, mH specific CTL, which killed
normal Con A blast target cells, were easily in-duced in primary cultures in vitro when NW-BM cells were used as helper cells (Figs. 1A and lB),
When Balb/c (H-2 ) responder LN cells were cultured with BIOD2 (H・2) stimulator spleen cells in our MLC system, the CTL killed BIOD2 target cells but did not affect the third party DBA/2 (H-2 ) target cells, which were different from either responder or stimulator cells (Fig. 1A). Despite BIOD2 and DBA/2 having many mH antigens in common, the CTL killed only BIOD2 target cells. In parallel cultures the CTL, which killed BIOD2, did not kill BIOBR (H-2 ) target ce】】s. Thus, these CTL recognized the mH antigens with MHC in a restricted man-ner. These results were contradictory to many
22.23)
other reports except a few from one
21.251 22.23)
group . Most of these reports showed cross reactive specificity of the CTL, namely, they killed both BIOD2 and DBA/2 target cells as in the casein Fig. 1A.
The CTL which kil一ed BIOD2 target eel】S
(Fig. 1A), also killed (BIOBRxBalb/c) Fl male target cells (Fig. 2A), but did not kill (Balb/CXBIOBR) Fl male cells (Fig. 2A). These results showed that the CTL recognized the X-chromosome linked gene products called Xir antigen in BIOD2 and (BIOBRxBalb/c) Fl male mice (Figs. 1A and 2A). When LN and NW-BM cells derived from DBA/2 mice were
used as the responder cells, the same results were obtained (Figs. IB and 2B).
In this culture system, the Xir antigen was the only stimulatory antigen, and the other mH antigens had no stimulating activity. Thus, the Xir antigen was dominant. Similar phenomena
24.25)
have been reported in C57BL/6 (H-2b) mice primed m vivo and then boosted in vitro with BALB.B (H-2) mice; the induced CTL recog-nized only a few, not single, mH antigens, which were called dominant antigens. These CTL might be selected during the m vivo and in vitro
Tanabe. M.J.
incubation period. On the other hand, the Xir antigen was the absolutely dominant antigen and codominant antigens never appeared although the responder cells received less pressure of selection during the short period inour primary cultures. These resu一ts showed
that the Air antigen behaved as MHC-like major antigens against many other mH antigens, since MHC antigens completely dominated the other mH antigens, stimulating across both H-2 and non-H-2 coded genetic differences.
At least a single precursor of CTL was con-tained in 3 to5out of32 wells,when 3xlO re-sponder LN cells were added to each well (Figs. 1 and 2). From these results we could esti-mate a single precursor of CTL per approx-imately 2xlO responder LN cells. In another
<B
study , in which NW-BM cells were treated with a lower dose of anti-Thy.l antibody than in this report, we have demonstrated that the cytolysis activity was positive in 17 out of 32 wells when each well contained 3xlO respon-der LN cells. The specificity of those CTL had strict preference for mH antigens of stimulator cells, as shown here, but occasionally anoma-lous killer cells were induced in that condition. The frequency of CTL precursors in that inves-tigation was about 1 per 4,000 responder cells. The frequency of allo-MHC-specific CTL precur-sors was about 1 per 5,000 responder cells
36
according to a previous report . Despite the
re一atively high frequency of anti-mH CTL
pre-cursors in our culture syste町 none of the in-duced CTL specific for the Xir antigen had cross reactivity to the other mH antigens. These results confirmed the conclusion de-scribed above, that the Air antigen was
abso-一utely dominant. Thus, regardless of the other
mH antigens, immune responses did not occur when the Xir antigen was self, but did when it was nonself. In our culture system, the Xir antigen was the key substance for self-nonself discrimination and played a role in the immune
lEI
regulation.
When the CTL were induced by stimulation
across a sex chromosome difference, they had specificity for the Xir antigen derived from stimu】ator strains (Figs. 3A, 3B, 4A and 4B). Even if both responder and stimulator cells had the same autosome, the CTL specific for the Xir antigen were induced. Thus, mH antigens from the autosome had no effect as trans-activating bystander stimuli in our culture system. There was, however, the possibility that X-chromo-some linked gene products, except the Xir anti-gen, or Y-chromosome linked gene products had bysLander stimuli activity. To test this possi-bility, we should have to set up MLC between the congenic mice related to the Xir antigen.From these results (Figs. 3B and 4B), we suggest that Balb/c mice also had the Air anti-gen which differed from that of BIO conanti-genic mice and that it also acted as a unique stimula-tory antigen. Thus, the Xir antigen might have polymorphic or oligomorphic alleles.
37)
Berryman and Silvers reported that the H-X antigen, which is a product of an H- X-chromo-some linked gene, was detected by skin graft re-jection. They used a combination of mice which differed only in the X-chromosome be-tween donor and recipient. There has been no report, however, as to whether the H-X antigen is dominant or not. At present it is not clear whether the Xir and H-X antigens are the same.
There are many important genes invo】ved in immune responses on the X-chromosome. They inc】ude X-chromosome linked severe combined
38)
immunodeficiency , X linked
agammaglobulin-39) 40)
emia , Wiscott-Aldrich syndrome and agam一
JHI
maglobulinemia after E.B. virus infection in
42)
humans, and the Aid gene in mice . It might be important to define the relationship between the Xir antigen and these X-chromosome linked genes. In mice, the Xid gene has been
inten-sive一y investigated, but the Xir and Aid
142 X-Chromosome-Linked Minor-H Antigen
42)
generated against the Xid gene product
In all experiments in this report the positive responses were obtained in 3 to 5 wells out of
32 wells (Fig. 1 to 4). We had already dis-cussed above that maximum anti-mH responses were obtained in these culture systems as the frequency of CTL precursors (CTLp) against mH antigens was relatively high, that is, about 1 per 2xlO responder cells by the estimation of these results. There were two ways to prove the significance of these weak anti-mH CTL responses. One was the large scale cul-ture method inducing greater responses in cuト
ture dishes or bigger wells. When a large
number of responder LN cells and NW-BM cells
from Balb/c mice were stimulated with a large number of BIOD2 spleen cells in the large scale culture, the induced CTL had, however, the cross reactive specificity, that is, they killed equa】ly both BIOD2 and third party DBA/2target ce一ls (data not shown). This cross
reac-tive specificity of CTL was the same as the
re-22.231
su】ts in many reports published previously
Thus, the limiting dilution method was essentia一
to induce the CTL specific for the Xir antigen. Another one was the repetition of the small scale culture experiments as carried in this re-port. Fortunate】y, the induced CTL had the
specificity for the Xir antigen. Thus, we cou一d
set up the MLC between very similar responder and stimulator cells (Figs. 3 and 4) and set up very similar target cells (Figs. 2 and 3), such as (BIOBRxBalb/c) Fl male and (Balb/CXBIOBR) Fl male mice. Responder LN cells (Figs. 3 and 4) and the induced CTL (Figs. 2 and 3) could recognize the difference between the Fl male cells, that is, Xir antigen. As the same results could be obtained in these six repetitions (Figs. 2A, 2B, 3A, 3B, 4A and 4B), the significance of the responses in this report might be proved.
In this report we used the NW-BM cells as
the helper cells. We had the data in advance
that Thy.l spleen cells also had the same
abil-lty (data not shown). It is very interesting to
study whether normal helper T ce一ls have the
same ability as NW-BM cells to augment the generation of CTL specific for the Xir antigen, and so we are now studying this issue.
In this system, the minimum required dose of TCGF was used to maintain the culture condi-tions. It would be of interest to study whether
recombinant IL2 0r other interleukins can re-place TCGF, since this would shed some light upon the kinds of mediators which helper NW-BM cells can produce.
ACKNOWLEDGEMENT
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The author would like to thank Mr. T. Koga, Mr. Y. Kowan and Mr. H. Higa, who belonged to the animal facility at the Sclool of Medicine, Uni-versity of Lhe Ryukyus, for breeding the mice.REFERENCES
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