By
From the * Department of Immunology and the Department of Virology, Medical Institute of
Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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Most T cells develop through the thymus, where they undergo positive and negative selection.
Some peripheral T cells are known to develop in the absence of thymus, but there is insufficient information about their selection. To analyze the selection of extrathymically developed
T cells, we reconstituted thymectomized male or female recipient mice with bone marrow
cells of mice transgenic for male H-Y antigen-specific T cell receptor (TCR). It was revealed that the T cells bearing self-antigen-specific TCR were not deleted in thymectomized male recipients. More importantly, the absence of H-Y antigen-specific T cells in thymectomized female recipients suggests positive selection of extrathymically developed T cells by the self-antigen. The extrathymically developed T cells in male mice expressed interleukin (IL)-2 receptor
chain (IL-2R
) and intermediate levels of CD3 (CD3int) but were natural killer cell
(NK)1.1
. They rapidly produced interferon
but not IL-4 after TCR cross-linking. Furthermore, a similar pattern of cytokine production was observed in CD3intIL-2R
+NK1.1
cells
in normal mice which have been shown to develop extrathymically. These results suggest that
extrathymically developed CD3intIL-2R
+NK1.1
cells in normal mice are also positively selected by self-antigens.
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Introduction |
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Most peripheral T cells require the thymus for their
development. Immature T cells undergo positive
and negative selection in the thymus, and consequently
only T cells whose antigen receptors are specific for foreign
antigen peptides in the context of self-MHC molecules can
mature and appear in the periphery. It is also known that
some peripheral T cells do not require the thymus for their development. These extrathymically developed T cells are
abundant in the gut (1) and liver (4). Among the intestinal intraepithelial lymphocytes (iIELs), those that express homodimeric /
CD8 chains have been shown to
develop extrathymically (2, 3). Recently, it was revealed
that all of the extrathymically developed T cells in both
spleen and liver express IL-2R
and intermediate levels of
CD3 (CD3int; reference 6). Many reports have demonstrated the incomplete negative selection of these extrathymically developed T cells by self-antigens as assessed by
anti-V
mAbs in conjunction with the endogenous superantigen Mls system (7). However, the conditions that
induce positive selection of extrathymically developed T cells are still unclear. Negative selection of extrathymically developed T cells by self-antigen peptides has not been
fully addressed.
In male mice expressing a transgenic TCR specific for
male H-Y antigen peptides in the context of H-2 Db molecules (H-Y transgenic mice), deletion of immature thymocytes was observed (12). This clearly demonstrates negative selection of intrathymically developed self-reactive T
cells. However, it has also been shown that there are many
male antigen-specific T cells with or without CD8 molecules in the periphery of male H-Y transgenic mice, although the origin of these T cells is still unclear (13, 14).
Rocha et al. reported that among the iIELs of male H-Y
transgenic mice, those that express heterodimeric /
CD8 chains are deleted, whereas those with CD8
/
are
not deleted. Moreover, iIELs expressing transgenic TCR
and CD8 are not detected in female mice (15). This suggests positive selection of extrathymically developed T
cells, especially iIELs, by self-antigens. Similar results were
also reported by Poussier et al. (16).
In this study, we reconstituted thymectomized, lethally irradiated male or female recipient mice with bone marrow cells from female H-Y transgenic mice to analyze the selection of extrathymically developed T cells in spleen and liver. We also examined the phenotypes and functions of the T cells developed in thymectomized recipients.
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Materials and Methods |
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Mice.
The mice transgenic for male H-Y antigen-specific TCR (H-Y transgenic mice) were provided by Dr. T.W. Mak, The Amgen Institute (Toronto, Ontario, Canada). C57BL/6 mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and maintained in specific pathogen-free conditions.Bone Marrow Transfer.
The recipient male or female C57BL/6 mice (H-2b) were thymectomized or sham operated. After 2 wk, they were lethally irradiated (11 Gy), then injected intravenously with 5 × 106 T cell-depleted bone marrow cells from female H-Y transgenic mice (H-2b) on the same day. 7 wk after bone marrow transfer, spleen and liver lymphocytes were collected and used for flow cytometric analysis or in vitro culture assays.Preparation of Liver Mononuclear Cells.
The liver mononuclear cells (MNC) were harvested as reported previously, with minor modifications (17). In brief, the livers were pressed through 100-gauge stainless steel mesh, then suspended in 5 ml 45% Percoll and layered on 5 ml 67.5% Percoll solution. The gradient was centrifuged at 2,500 rpm at 20°C for 25 min. The cells at the interface were harvested and washed with complete culture medium, then used as liver MNC.Flow Cytometric Analysis and Antibodies.
The spleen cells and MNC were stained with FITC-conjugated T3.70 mAb (a gift of T.W. Mak), PE-conjugated anti-CD8 mAb (2.43; PharMingen, San Diego, CA), and allophycocyanin (APC)-conjugated anti-CD3 mAb (2C11; PharMingen), in combination with biotin-conjugated anti-IL-2RIn Vitro Proliferation and Cytokine Production Assay.
5 × 105 spleen cells of male thymectomized or female nonthymectomized mice reconstituted with bone marrow cells from female H-Y transgenic mice were cultured with 5 × 105 irradiated (30 Gy) spleen cells of female or male C57BL/6 mice in 96-well plates. 20 U/ml of recombinant human IL-2 were added to some cultures. The cultures were pulsed with [3H]thymidine on day 4, followed by harvesting 6 h later. To examine the proliferative response to TCR cross-linking, 5 × 105 responder cells were cultured in 96-well plates precoated with or without 5 µg/ml T3.70 mAb. After 2 d, the cultures were pulsed with [3H]thymidine, followed by harvesting 6 h later. To examine the cytokine production, each culture was also performed in 24-well plates at 2.5 × 106 responder cells/ml, and supernatants were collected 24 or 96 h later. IFN-Analysis of Intracellular Cytokine Synthesis.
C57BL/6 mice were injected intravenously with 4 µg of anti-CD3 mAb (2C11). After 90 min, the spleen cells were harvested, washed, and suspended at 106 cells/ml in complete culture medium, then incubated for 3 h at 37°C in the presence of 10 µg/ml Brefeldin A (Wako Pure Chemical Industries, Ltd., Osaka, Japan). These cells were harvested, washed, and incubated for 30 min at 4°C with PE-conjugated anti-TCR ![]() |
Results |
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The syngeneic male or female C57BL/6 mice with or without adult
thymectomy were lethally irradiated and transferred with T cell-depleted bone marrow cells from female H-Y transgenic mice. 7 wk later, spleen and liver lymphocytes were
analyzed using a flow cytometer (Fig. 1 A). In female nonthymectomized recipient mice, T cells bearing transgenic
TCR chain (T3.70+) appeared in both spleens and livers.
It has been observed in male H-Y transgenic mice that in
spite of negative selection in the thymus, many T3.70+
cells with or without CD8 were present in the periphery,
although the levels of CD8 expression are relatively low
compared with female mice (13). These two populations of
T cells developed in the male nonthymectomized recipients. T3.70+ cells were also observed in both spleens and
livers of male thymectomized recipients, but most of them
were CD8+. Thus, it is suggested that T3.70+CD8+ cells
in the periphery of male H-Y transgenic mice may develop extrathymically, whereas T3.70+CD8
cells may develop
through the thymus. Surprisingly, T3.70+ cells were scarcely
detected in either spleens or livers of the female thymectomized recipient mice. Therefore, T3.70+ cells in female H-Y
transgenic mice may be thymus derived, and none of them
can develop extrathymically. These results suggest not that
the extrathymically developed self-antigen-specific T cells
are not deleted, but rather that the extrathymic development of T cells requires expression of TCR specific for
self-antigens. Rocha et al. reported the positive selection of
T3.70+ cells in extrathymically developed iIELs in male H-Y
transgenic mice (15). They also showed that T3.70+ cells in
iIELs express higher levels of CD8 than T3.70+ cells in
lymph nodes. Similarly, we found that extrathymically developed T3.70+ cells in liver of male mice express higher
levels of CD8 than those in spleen (Fig. 1 B).
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Similar to unmanipulated male H-Y transgenic mice, the thymectomized male recipient mice reconstituted with bone marrow cells of H-Y transgenic mice showed no signs of autoimmunity. It has been reported that T3.70+ T cells in male H-Y transgenic mice are unresponsive to male antigen but can respond to anti-TCR mAbs in vitro (13). Therefore, we next examined the proliferative response of the T cells developed in male thymectomized mice to male antigens in vitro. T cells in the female nonthymectomized recipient mice proliferated in response to male stimulator cells, whereas those in male thymectomized mice showed no proliferative response (Fig. 2 A). The unresponsiveness of T cells in male thymectomized mice could not be reversed by the addition of IL-2 (Fig. 2 B). Nevertheless, T cells in male thymectomized mice showed significant proliferative response to anti-TCR antibodies (Fig. 2 C ). Therefore, self-antigen- specific T cells, which developed in the absence of thymus, seem to be unresponsive to the self-antigen but are not completely anergic to TCR stimulation. This discrepancy may be explained by their low avidity to H-Y antigen, possibly due to somewhat lower levels of CD8 than T3.70+ cells developed in female recipients (Fig. 1). However, the absence of T3.70+ T cells in female thymectomized mice strongly argued that the T3.70+ T cells in male thymectomized mice should functionally recognize H-Y antigens to develop.
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Interestingly, after stimulation with anti-TCR antibodies,
IFN- was rapidly produced by T cells in male thymectomized mice, within 24 h, while T cells in female nonthymectomized mice produced IFN-
only 4 d, but not
until 24 h, after stimulation (Fig. 3). No IL-4 was detected
in the culture supernatants (data not shown). Neither IFN-
nor IL-4 were detected in the supernatants of the cultures
stimulated with male stimulator cells (data not shown).
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Rapid production of effector cytokines after
TCR stimulation has been shown to be a unique feature of
NK1.1+ T cells (NK-T cells; references 18 and 19). Although NK-T cells are found in the thymus, extrathymic
development of liver and spleen NK-T cells has also been
demonstrated (6, 20, 21). Therefore, the question remained
whether T cells developed in male thymectomized mice
have similar phenotypes of surface molecules as NK-T
cells. Interestingly, similar to NK-T cells in normal mice,
T3.70+ cells in male thymectomized mice expressed IL-2R, a high level of CD44, and an intermediate level of
CD3 (CD3int), but were NK1.1
(Fig. 4). Thus, T3.70+
cells in male thymectomized mice are phenotypically similar to CD3intIL-2R
+NK1.1
T cells in normal mice
which have also been shown to develop extrathymically
(22, 23). On the other hand, CD8+ T cells in female nonthymectomized mice were CD3highIL-2R
CD44low, similar to conventional intrathymically developed naive T cells.
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Although CD3intNK1.1+ T cells, most of which are
CD4CD8
or CD4+CD8
, have been shown to produce
IL-4 in addition to IFN-
rapidly after intravenous injection of anti-TCR antibodies (18), production of these cytokines by CD3intNK1.1
T cells, most of which are
CD4
CD8+, has not been fully addressed. Therefore, we
examined the cytokine production of CD3intNK1.1
CD8+
T cells in normal mice after in vivo administration of anti-TCR antibodies. As shown in Fig 5, IFN-
-producing
cells were observed in both CD8+ and CD8
populations
of CD3intIL-2R
+ cells, but not in those of CD3highIL-2R
cells. IL-4-producing cells were only detected in the
CD8
population of CD3int cells, which contains NK-T
cells. This implies that, like T3.70+CD8+ T cells in male
H-Y transgenic mice, CD3intIL-2R
+CD8+ cells in normal mice produce IFN-
but not IL-4 rapidly after TCR
stimulation. These results indicate that extrathymically developed T3.70+CD8+ T cells in the periphery of male H-Y
transgenic mice are phenotypically and functionally indistinguishable from CD3intNK1.1
CD8+ T cells in normal
mice. It can also be suggested that extrathymically developed CD3intNK1.1
CD8+ T cells in normal mice are positively selected by some self-antigens.
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Discussion |
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In this study, we have shown that extrathymically developed T cells expressing self-antigen-specific TCR are not
deleted, but rather cannot develop in the absence of the
self-antigen. This feature is in striking contrast to the positive and negative selection events in the thymus. There
have been many reports showing the incomplete negative
selection of extrathymically developed T cells (7).
Moreover, using H-Y transgenic mice, it was also reported
that among iIELs, those with male antigen-specific TCR and CD8 /
were positively selected independently of
the thymus only in male mice (15). These studies are consistent with our results showing positive selection of extrathymically developed splenic or liver T cells by self-antigens.
It has been shown that many T cells expressing male antigen-specific TCR with or without CD8 are present in
the periphery of male H-Y transgenic mice, despite the absence of mature CD8 single positive thymocytes (13). Our
results suggest that those with CD8 may develop extrathymically and those without CD8 may develop intrathymically. This was confirmed by analyzing the phenotypes of T
cells in unmanipulated male H-Y transgenic mice. Of the
T cells in male transgenic mice, those with and without
CD8 show phenotypes of extrathymically and intrathymically developed T cells, respectively (our unpublished observations). It is possible that because of the absence of
CD8 molecules and the resulting low avidity to H-Y antigen, CD8 cells may escape the negative selection in the
thymus and appear in the periphery of male H-Y transgenic mice. A previous report showing the absence of T
cells in male H-Y transgenic mice crossed with nude mice
suggests thymic origin of all peripheral T cells in male H-Y
transgenic mice (14). We have no precise explanation for
the discrepancy between those results and ours. We could
not detect any remnant thymus in our male thymectomized recipients macroscopically. The striking difference in
the proportion of CD8+ to CD8
cells between nonthymectomized and thymectomized recipients also argues
against remanence of thymus. Moreover, studies showing extrathymic development of iIELs in H-Y transgenic mice
support our results (15, 16). Unfortunately, liver and iIELs
of H-Y transgenic mice crossed with nude mice have not
been studied. Because extrathymically developed T cells
are abundant in the gut (1) and liver (4), T3.70+ cells
may be detected only in these locations. It is well known that the number of extrathymically developed T cells in
adult thymectomized radiation chimera is close to that in
euthymic mice and is much larger than that in nude mice
(24, 25). This could also account for the discrepancy. Another possibility is that T3.70+CD8+ T cells found in male
thymectomized mice were the progeny of a few T cell
contaminants in the donor bone marrow cells from female
H-Y transgenic mice and expanded only in male recipients. However, it has been shown that T3.70+ cells from female
H-Y transgenic mice became anergic to TCR stimulation and downmodulated CD8 expression after transfer into
male recipient mice (14), in contrast to T3.70+ cells developed in our male recipient mice, which showed proliferative response to T3.70+ mAbs (Fig. 2 C) and expressed
high levels of CD8, especially those in the liver (Fig. 1 B).
It has been shown that CD3intIL-2R+ cells are the only
splenic or liver T cells that develop extrathymically (6, 22).
Among CD3intIL-2R
+ cells, those with NK1.1 (NK-T
cells) have been shown to produce IL-4 and IFN-
rapidly
after TCR stimulation without prior priming (18, 19). Interestingly, Yoshimoto and Paul observed the expression of
IFN-
mRNA in splenic CD8 T cells after intravenous injection of anti-CD3 mAb, although it was not addressed
whether they were also CD3intIL-2R
+ cells or CD3high
conventional T cells (18). In addition, rapid IFN-
production after administration of anti-TCR antibodies was
also observed in CD1 knockout mice, which selectively
lack NK-T cells (26). We have shown in this study that
the CD8+ T cells that produce IFN-
rapidly after TCR
stimulation are CD3intIL-2R
+ cells (Fig. 4). We also
showed that extrathymically developed male antigen-specific T cells in male recipient mice rapidly produce IFN-
but not IL-4 after TCR cross-linking. In addition, they are
CD3intIL-2R
+ but NK1.1
. Therefore, extrathymically
developed CD3intIL-2R
+ T cells in normal mice, especially those of the CD8+NK1.1
population, are phenotypically and functionally indistinguishable from extrathymically developed T3.70+CD8+ T cells in male transgenic
mice. These results imply that extrathymically developed T
cells in normal mice may be also positively selected by self-antigens.
We could not detect any response of the extrathymically developed, potentially self-reactive T cells to the self-antigens in vitro. However, a previous study showing slow expansion of T3.70+CD8+ T cells in male transgenic mice upon transfer into syngeneic male recipients (14) suggests responsiveness of these T cells to male antigen in vivo which may be too weak to be detected in vitro. Expansion of extrathymically developed T cells with age has also been observed in normal mice (4, 29, 30). Similar to male antigen-specific T cells in male H-Y transgenic mice, extrathymically developed, potentially self-reactive T cells in normal mice can respond to TCR cross-linking by antibodies (6). Nevertheless, neither male H-Y transgenic mice nor athymic nontransgenic mice develop autoimmune disease. It will be important to further clarify the in vivo function of extrathymically developed self-antigen-specific T cells.
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Footnotes |
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Address correspondence to Hisakata Yamada, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6822; Fax: 81-92-642-6776; E-mail: hisako{at}bioreg.kyushu-u.ac.jp
Received for publication 30 March 1998 and in revised form 12 May 1998.
We are grateful to T.W. Mak for providing us with H-Y transgenic mice and T3.70 hybridomas.
This work was supported in part by a grant from the Ministry of Education, Science, and Culture of Japan.
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