Functionally active CD8
ß+ TCR
intestinal intraepithelial lymphocytes in athymic nu/nu mice
Yoshiko Emoto1,
Masashi Emoto1,2,
Mamiko Miyamoto1,
Izumi Yoshizawa1 and
Stefan H. E. Kaufmann1
1 Department of Immunology, Max-Planck-Institute for Infection Biology, Schumannstrasse 21/22,10117 Berlin, Germany 2 Present address: Laboratory of Immunology, Department of Laboratory Sciences, Gunma University School of Health Sciences, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
The first two authors contributed equally to this work
Correspondence to: M. Emoto; E-mail: emoto{at}mpiib-berlin.mpg.de
Transmitting editor: H. R. MacDonald
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Abstract
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Murine intestinal intraepithelial lymphocytes (IEL) encompass a high proportion of TCR
cells. A vast majority of these TCR
IEL express CD8
, but not CD8ß (CD8
homodimer), and are considered to develop in intestinal epithelial layers independently of a functional thymus. Here we show that TCR
cells expressing both CD8
and CD8ß (CD8
ß heterodimer) appear in athymic nu/nu mice, although their appearance is random. The IEL comprising CD8
ß+ TCR
cells expressed pronounced cytolytic and IFN-
-producing activities after TCR
ligation, which were markedly stronger than activities of IEL lacking CD8
ß+ TCR
cells. Purified CD8
ß+ TCR
cells expressed strong cytolytic activities and produced large quantities of IFN-
after TCR engagement. CD8
ß+ TCR
cells were also identified among IEL from euthymic C57BL/6 mice, although their abundance varied among individual animals. However, cytolytic and IFN-
-producing activities in euthymic C57BL/6 mice were markedly lower than those in athymic nu/nu mice. Our findings suggest that CD8
ß+ TCR
cells can develop in the intestine independently of a functional thymus/thymic epithelial cells and that they perform biological functions in situ.
Keywords: cytotoxic T lymphocyte, IFN-
, intestine, nude mouse, thymus independent
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Introduction
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In the mouse, intestinal intraepithelial lymphocytes (IEL) comprise unique T cell populations which are distinct from mainstream T cells prevalent in central lymphoid organs. The IEL encompass roughly equal numbers of TCR
ß cells and TCR
cells, and most of them co-express CD8
(14). CD8
+ TCR
ß IEL segregate into two populations on the basis of CD8ß co-expressionone expressing CD8ß (CD8
ß heterodimer) and the other not (CD8
homodimer) (2). CD8
ß+ TCR
ß IEL are considered identical to conventional T cells in central lymphoid organs which develop in a thymus-dependent manner, although some CD8
ß+ TCR
ß IEL have been suggested to develop in a thymus-independent fashion (4,5). In contrast, CD8
+ TCR
ß cells are prominent in the gut and are considered to develop in intestinal epithelial layers independently of a functional thymus (1,2,6,7), although a recent report suggests that CD8
+ TCR
ß IEL in euthymic mice develop in a thymus-dependent manner (8). In contrast to CD8
+ TCR
ß IEL, virtually no CD8
+ TCR
IEL express CD8ß and the development of this subset is considered thymus independent (1,2,6,7).
T cells are hardly detectable in central lymphoid organs of nu/nu mice due to the lack of a functional thymus (9). However, a distinct population of T cells becomes detectable in peripheral lymphoid organs of these mice dependent on age, although they appear randomly (1017). In contrast to other organs, considerable numbers of T cells can be detected in the gut of nu/nu mice independent of age, and the vast majority express a TCR
and only a minority a TCR
ß (4,6,7). Consistent with the notion that the thymus is essential for the development of CD8
ß+ T cells, virtually all TCR
cells in the gut bear CD8
, but not CD8ß (4,6,7).
In the present study, we identified CD8
ß+ TCR
cells in athymic nu/nu mice, although their appearance was random. The CD8
ß+ TCR
cells expressed strong cytotoxic T lymphocyte (CTL) activities and produced large amounts of IFN-
after TCR ligation. Our results suggest that CD8
ß+ TCR
cells expressing functional activities can develop in the gut independently of a functional thymus/thymic epithelial cells.
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Methods
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Mice
Male nu/nu C57BL/6 mice were obtained from Bomholtgard Breeding and Research Centre (Ry, Denmark) and used at 28 months of age. C57BL/6 mice were bred and maintained under specific pathogen-free conditions at our animal facilities, and used at 28 months of age.
Antibodies
mAb against TCR
(GL3), CD8
(YTS 169.4), CD8ß (H35-17.2), TCR
ß (H57-597), IFN-
(R4-6A2, AN-18.17.24) and Fc
receptor (2.4G2) were purified from hybridoma culture supernatants. mAb against TCR
, CD8
, CD8ß, TCR
ß and IFN-
(AN-18.17.24) were biotinylated, and mAb against CD8
and CD8ß were conjugated with FITC by standard methods. Phycoerythrin (PE)-conjugated anti-CD4 mAb (H129.19) and hamster IgG were purchased from Life Technologies (Gaithersburg, MD) and Dianova (Hamburg, Germany) respectively.
Cell preparation, flow cytometry and cell sorting
The IEL and hepatic leukocytes were prepared as described previously (18). Splenocytes were prepared by conventional methods. After blocking with anti-Fc
receptor mAb, cells were stained with conjugated mAb at 4°C for 30 min and biotinylated mAb was visualized by streptavidin (SA)-conjugated Red 670 (Life Technologies). Stained cells were washed with PBS containing 2.5% normal human serum and 0.1% sodium azide (Merck, Darmstadt, Germany), fixed with 1% paraformaldehyde (Serva Feinbiochemica, Heidelberg, Germany), acquired by FACScan (BD Biosciences, Mountain View, CA) and lymphoid cells were analyzed with Lysis II or CellQuest software. To obtain CD8
ß+ cells, IEL were stained with FITC-conjugated anti-CD8ß mAb and PE-conjugated anti-CD4 mAb, and CD4CD8ß+ cells as well as CD4CD8ß cells were positively sorted by FACS Vantage (BD Biosciences). The purity was >98%. Since virtually all CD8ß+ TCR
cells expressed CD8
, but not CD4, the CD4CD8ß+ cells were regarded as CD8
ß+ cells.
Redirected CTL assay
CTL activities of TCR
IEL were measured in a standard redirected 51Cr-release assay using P815 as target cells as described previously (19). Briefly, various numbers of IEL were incubated with 1 x 103 51Cr-labeled P815 cells at 37°C in 7% CO2 at various E:T ratios for 4 h. Assays were performed in the presence or absence of 2 µg/ml anti-TCR
mAb or hamster IgG in 96-well V-bottom plates (Nunc, Wiesbaden-Biebrich, Germany) in a total volume of 200 µl/well. After centrifugation, supernatants (100 µl) were harvested from each well and radioactivity was determined using a
-counter (Cobra, Canberra Packard, Germany). Percent specific lysis was calculated as follows: (experimental 51Cr release spontaneous 51Cr release)/(maximum 51Cr release spontaneous 51Cr release) x 100. Spontaneous release was always <15% of total incorporated radioactivity into target cells.
ELISPOT
Frequencies of IFN-
producers among TCR
IEL were estimated by ELISPOT as described previously (20). Briefly, various numbers of IEL were incubated with 1 x 104 P815 cells in the presence of 2 µg/ml anti-TCR
mAb or hamster IgG in ELISPOT plates (Millipore, Eschborn, Germany) coated with 1 µg/ml anti-IFN-
mAb (R4-6A2) at 37°C for 18 h in 5% CO2. After washing, plates were incubated with 0.25 µg/ml biotinylated anti-IFN-
mAb (AN-18.17.24) at 37°C for 2 h. Plates were subsequently washed and incubated with SA-conjugated alkaline phosphatase in PBS containing 0.1% BSA (Serva) at 37°C for 1 h. After washing, nitroblue tetrazolium chloride (Fluka, Basel, Switzerland) and 5-bromo-4-chloro-3-indolylphosphate (Fluka) were added, and plates were incubated at 37°C in the dark for 15 min. Plates were then washed, dried and spots were counted using a dissecting microscope.
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Results
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CD8
ß+ TCR
IEL develop in the absence of a functional thymus
Cell-surface phenotypes of TCR
IEL in athymic nu/nu mice of different ages were analyzed by flow cytometry. Six staining patterns from 30 mice analyzed are shown in Fig. 1 and the results are summarized in Table 1. High proportions of TCR
cells were identified among IEL in nu/nu mice, although the abundance of this subset markedly varied among individual animals. High fractions of CD4CD8
+ß cells were detected among TCR
IEL in all nu/nu mice examined, although the proportion of this subset also varied among individual animals. Interestingly, CD4CD8
+ß+ cells were also identified among TCR
IEL in some nu/nu mice, although their appearance was random and rare. Note that the appearance of CD4CD8
+ß+ TCR
cells was independent of age. CD4CD8
+ß+ TCR
cells were restricted to the intestine, and virtually absent in other organs such as liver and spleen (data not shown). These results suggest that not only CD8
+ TCR
IEL, but also CD8
ß+ TCR
IEL, develop in the absence of a functional thymus.

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Fig. 1. Cell-surface expression of CD4, CD8 and CD8ß on TCR IEL from nu/nu mice of different ages. The IEL were stained with FITC-conjugated anti-CD8 mAb or FITC-conjugated anti-CD8ß mAb respectively, PE-conjugated anti-CD4 mAb and biotinylated anti-TCR mAb followed by SA-conjugated Red 670. Profiles of TCR are displayed as histograms after gating on lymphoid cells. Numbers in histograms represent percentages of TCR cells. Data of CD4/CD8 or CD4/CD8ß are displayed as dot-plots after gating on TCR cells. Numbers in dot-plots represent percentages of each cell population among TCR cells. Data from six individual nu/nu mice are shown. Recovery numbers of IEL: no. 1, 0.4 x 107; no. 2, 0.6 x 107; no. 3, 0.7 x 107; no. 4, 1.7 x 107; no. 5, 1.4 x 107; no. 6, 1.3 x 107 Age: no. 1, 3 months old; no. 2, 3 months old; no. 3, 8 months old; no. 4, 8 months old; no. 5, 8 months old; no. 6, 3 months old.
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CD8
ß+ TCR
IEL in athymic nu/nu mice express strong CTL activities
We raised the question of whether CD8
ß+ TCR
IEL in nu/nu mice express functional activities. To address this issue, mAb-redirected CTL activities were examined using P815 cells as target cells. CTL activities after TCR
ligation were virtually undetectable among IEL from the nu/nu mouse comprising a small proportion of CD4CD8
+ß TCR
cells (Fig. 2A, no. 1; see also Fig. 1). The IEL with a large fraction of CD4CD8
+ß TCR
cells showed weak CTL activities after TCR
engagement (Fig. 2A, nos 2 and 3; see also Fig. 1). Importantly, TCR
ligation induced pronounced CTL activities in IEL from nu/nu mice encompassing a small, but distinct, population of CD4CD8
+ß+ TCR
cells (Fig. 2A, nos 46; see also Fig. 1). These results suggest that CD8
ß+ TCR
IEL express CTL activities.
To directly assess whether CD8
ß+ TCR
IEL in nu/nu mice express CTL activities, CD4CD8ß+ IEL were purified from eight adult (3 months old) nu/nu mice and their CTL activities were determined by 51Cr-release assays as described above. Because the appearance of CD4CD8ß+ cells was random and rare in nu/nu mice, results from the experiment in which sufficient numbers of CD4CD8ß+ cells were obtained are shown in Fig. 2B. Purified CD4CD8ß+ and CD4CD8ß IEL populations comprised considerable numbers of TCR
cells (data not shown), whereas TCR
ß cells were scarce among these populations (Fig. 3). Purified CD4CD8ß+ cells showed strong CTL activities against P815 cells in the presence of anti-TCR
mAb, but not isotype-matched antibody, hamster IgG. In contrast, CTL activities of CD4CD8ß cells were low even after TCR
engagement. Thus, CD8
ß+ TCR
IEL in nu/nu mice express potent CTL activities.
CD8
ß+ TCR
IEL in athymic nu/nu mice are potent IFN-
producers
We examined IFN-
production by CD8
ß+ TCR
IEL in nu/nu mice using the ELISPOT assay. TCR
engagement failed to induce noteworthy IFN-
production by IEL from the nu/nu mouse comprising a small proportion of CD4CD8
+ß TCR
cells (Fig. 4A, no. 1; see also Fig. 1). Measurable frequencies of IFN-
-producing cells, however, were found among IEL from nu/nu mice with a high proportion of CD4CD8
+ß TCR
cells (Fig. 4A, nos 2 and 3; see also Fig. 1). Importantly, high frequencies of IFN-
producers were identified among IEL encompassing a small, but distinct, population of CD4CD8
+ß+ TCR
cells (Fig. 4A, nos 46; see also Fig. 1). Note that CTL activities and IFN-
production were correlated (see Figs 2A and 4A). These results suggest that CD8
ß+ TCR
IEL are more potent IFN-
producers than CD8
+ TCR
IEL.
To directly assess whether CD8
ß+ TCR
IEL in nu/nu mice produce IFN-
, frequencies of IFN-
producers among CD4CD8ß+ IEL purified from eight adult (3 months old) nu/nu mice were analyzed by ELISPOT assay as described above. Remaining CD4CD8ß+ and CD4CD8ß IEL used for CTL assay (Fig. 2B) were employed for this assay. High frequencies of IFN-
-producing cells were detected among purified CD4CD8ß+ cells after TCR
engagement (Fig. 4B). In contrast, numbers of IFN-
producers were low among CD4CD8ß cells even after TCR
engagement. These results indicate that CD8
/ß+ TCR
IEL in nu/nu mice are potent IFN-
producers.
CD8
ß+ TCR
IEL in euthymic C57BL/6 mice
We wondered whether CD8
ß+ TCR
IEL are also present in euthymic C57BL/6 mice. Results from 30 C57BL/6 mice of different ages analyzed are summarized in Table 1. Consistent with previous findings (14), high proportions of TCR
cells were identified among IEL in all C57BL/6 mice. High proportions of CD4CD8
+/ß cells were detected among TCR
IEL in all C57BL/6 mice examined, although the proportion of this subset varied among individual animals. In contrast to nu/nu mice, in all C57BL/6 mice CD4CD8
+ß+ cells were identified among TCR
IEL, although their abundance varied among individual animals. Similar to nu/nu mice, the appearance of CD4CD8
+ß+ TCR
cells was independent of age. CD4CD8
+ß+ TCR
cells were restricted to the intestine, and virtually absent from the liver and spleen (data not shown).
CTL and IFN-
-producing activities of CD8
ß+ TCR
IEL in euthymic C57BL/6 mice
We examined whether CD8
ß+ TCR
cells in euthymic C57BL/6 mice were also functionally active. CD4CD8ß+ IEL were purified from eight adult (3 months old) C57BL/6 mice, and CTL and IFN-
-producing activities were analyzed by 51Cr-release assay or ELISPOT assay respectively, as described above. Purified CD4CD8ß+ cells expressed CTL activity after TCR
ligation (Fig. 5A). CTL activities of CD4CD8ß+ cells from euthymic mice were markedly lower than those of cells from athymic mice (see Fig. 2B). In contrast to nu/nu mice, IFN-
-producing cells were virtually undetectable in C57BL/6 mice even after TCR
ligation (Fig. 5B). These results suggest that CD8
ß+ TCR
IEL in euthymic mice are functionally different from those in athymic mice.
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Discussion
|
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In the mouse, intestinal epithelial layers constitute a rich provenance of TCR
cells (14). In contrast to the rat in which TCR
IEL comprise large numbers of cells expressing both CD8
and CD8ß (21), the vast majority of TCR
IEL in the mouse surface express CD8
, but lack CD8ß (CD8
homodimer) (14). In the present study, we identified a novel TCR
IEL population expressing CD8
ß in some nu/nu mice. This suggests that not only CD8
+ TCR
cells, but also some CD8
ß+ TCR
cells, develop in the absence of a functional thymus. Since thymic rudiments are present in nu/nu mice (22), we cannot formally exclude that CD8
ß+ TCR
IEL in nu/nu mice are derived from thymic rudiments. However, we assume that CD8
ß+ TCR
cells in nu/nu mice develop independently of a functional thymus/thymic epithelial cells for the following reasons: (i) thymic epithelial cells are undetectable in nu/nu mice (23), (ii) whn, forkhead winged-helix transcription factor, has been identified as a nude gene and whn gene-deletion mutants show identical phenotypes as nu/nu mice (24,25), and (iii) T cells are hardly detectable in whn gene-deletion mutants due to the lack of thymic epithelial cells (24,25). CD8
ß+ TCR
cells were restricted to the intestine only, and virtually undetectable in other organs such as spleen and liver. Evidence has been presented that precursors of CD8
+ TCR
IEL are present in cryptopatches and they develop independently of thymus (26,27). It is therefore possible that, at least in nu/nu mice, CD8
ß+ TCR
cells develop in a similar manner.
The appearance of CD8
ß+ TCR
IEL in nu/nu mice markedly varied among individual animals. This suggests that the development of CD8
ß+ TCR
IEL is influenced by microenvironmental stimuli. Because the CD8
+ IEL have been suggested to represent an intermediate stage in the development of CD8
ß+ IEL (6), we cannot exclude that CD8
ß+ TCR
IEL were derived from CD8
+ TCR
IEL under the influence of environmental stimuli. Also, we cannot exclude formally that CD8ß is adsorbed onto these cells from other CD8ß+ cells. Because modulation of TCR
ß IEL by bacterial colonization has been described (28), it can be assumed that CD8
ß+ TCR
IEL are influenced by microbial colonization. Because frequencies in appearance of CD8
ß+ TCR
IEL were comparable in mice maintained under germ-free, specific pathogen-free and conventional conditions (Emoto, unpublished observation), we consider it unlikely that the appearance of CD8
ß+ TCR
IEL is directly influenced by bacterial colonization.
Pronounced CTL and IFN-
-producing activities were detected among IEL in nu/nu mice by TCR
engagement, implying that TCR
IEL in nu/nu mice were functionally active. Similarly, profound CTL activities and IFN-
production were determined in purified CD4CD8ß+ IEL after TCR
engagement, suggesting that CD8
ß+ TCR
IEL are functionally active. The functional activities of CD4CD8ß+ TCR
cells were higher than those of CD4CD8ß TCR
cells, although both populations expressed activation markers such as CD69 and IL-2Rß at comparable levels (Emoto, unpublished observation). It is possible that TCR
ß cells influenced and/or modified the functional activities of CD8
+ and CD8
ß+ TCR
IEL in nu/nu mice. Yet, we consider this possibility unlikely because TCR
ß cells were scarce among IEL in adult nu/nu mice (5). Although CD8ß is not involved in effector functions of CD8+ T cells in certain experimental systems (29,30), the CD8
ß heterodimer is generally more effective than the CD8
homodimer in promoting T cell activation (3134).
CD8
ß+ TCR
cells were detectable in all euthymic C57BL/6 mice, albeit in small numbers, whereas the frequencies in appearance of CD8
ß+ TCR
cells were rare in athymic mice. Our findings suggest that the thymus plays a major role in the development of CD8
ß+ TCR
cells. The maturation of thymus-independent IEL has been suggested to be influenced by thymic factors which are induced by thyrotropin-releasing hormone, although this seems to be restricted to TCR
ß IEL (3537). It is possible that the development of CD8
ß+ TCR
IEL is regulated by these thymic factors. A recent study demonstrates that the development of extrathymic T cells is repressed in the presence of thymus (8). Hence, the vast majority of CD8
ß+ TCR
IEL in euthymic mice appear thymus dependent, whereas CD8
ß+ TCR
IEL in nu/nu mice could develop independently of a functional thymus/thymic epithelial cells.
CTL activities and IFN-
-producing activities of purified CD8
ß+ TCR
IEL were markedly lower in euthymic mice as compared to athymic mice. It remains to be established whether the differences of CD8
ß+ TCR
IEL in athymic and euthymic mice are qualitative or quantitative. In either case, the CD8
ß+ TCR
cells in athymic mice differ from their counterparts in euthymic mice. We assume that the thymic organ per se or thymic factors, at least in part, participate in the regulation of functional activities of CD8
ß+ TCR
IEL. However, we cannot formally exclude additional mechanisms, which control functional activities of thymus-independent CD8
ß+ TCR
IEL, e.g. influence of TCR
ß IEL on the functional activities of TCR
IEL in euthymic mice.
In summary, we conclude that not only CD8
+ TCR
IEL, but also some CD8
ß+ TCR
IEL, develop in the absence of a functional thymus/thymic epithelial cells. Since CD8
ß+ TCR
IEL in athymic mice expressed potent CTL and IFN-
activities, we assume that this subset participates in regional immune responses. It is possible that the thymus not only serves as a source of CD8
ß+ TCR
IEL, but also as a crucial regulatory organ for functional activities of CD8
ß+ TCR
IEL.
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Acknowledgements
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We thank Dr Hisakata Yamada for critical reading this manuscript. We are grateful to Manuela Stäber for mAb purification. This work was supported by a grant from the German Science Foundation (SFB421).
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Abbreviations
|
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CTLcytotoxic T lymphocyte
IELintestinal intraepithelial lymphocytes
PEphycoerythrin
SAstreptavidin
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