Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1999 Jan;96(1):57–65. doi: 10.1046/j.1365-2567.1999.00665.x

Extrathymic derivation of gut lymphocytes in parabiotic mice

S Sugahara *,†, T Shimizu *, Y Yoshida *,†, T Aiba *,†, S Yamagiwa *,†, H Asakura †, T Abo *
PMCID: PMC2326721  PMID: 10233678

Abstract

In adult mice, c-kit+ stem cells have recently been found in their liver, intestine and appendix, where extrathymic T cells are generated. A major population of such thymus-independent subsets among intraepithelial lymphocytes is T-cell receptor (TCR)γδ+ CD4− CD8αα+(β−) cells, but the origins of other lymphocyte subsets are still controversial. In this study, we examined what type of lymphocyte subsets were produced in situ by such stem cells in the small intestine, large intestine and appendix. To investigate this subject, we used parabiotic B6.Ly5.1 and B5.Ly5.2 mice which shared the same circulation by day 3. The origin of lymphocytes was identified by anti-Ly5.1 and anti-Ly5.2 monoclonal antibodies in conjunction with immunofluorescence tests. Lymphocytes in Peyer's patches and lamina propria lymphocytes (especially B cells and CD4+ T cells) in the small intestine became a half-and-half mixture of Ly5.1+ and Ly5.2+ cells in each individual of parabiotic pairs of mice by day 14. However, the mixture was low in CD8αα+, CD8αβ+ and γδ T cells in the small and large intestines and in CD3+ CD8+ B220+ cells in the appendix. These cells might be of the in situ origin. When one individual of a pair was irradiated before parabiosis, the mixture of partner cells was accelerated. However, a low-mixture group always continued to show a lower mixture pattern than did a high-mixture group. The present results suggest that extrathymic T cells in the digestive tract may arise from their own pre-existing precursor cells and remain longer at the corresponding sites.

INTRODUCTION

We, as well as other investigators, have recently revealed that some T-cell subsets in the digestive tract, including the liver,1–4 small intestine,5–11 and appendix,12 are generated extrathymically. Such T cell subsets include NK1.1+ T cells [i.e. natural killer (NK) cells] in the liver, intraepithelial lymphocytes (IEL) (especially CD8αα+γδ T cells and CD4+ CD8+αβ T cells) in the small intestine, and double-negative (DN) CD4− CD8− CD3+ B220+αβ T cells in the appendix. However, controversy still exists as to their extrathymic origin in some subsets of the digestive tract, e.g. lamina propria lymphocytes (LPL) and CD8αβ+αβ T cells among IEL in the small intestine.13–18 Although some subsets in the large intestine have already been characterized,19,20 other subsets in the large intestine and appendix have not. This matter was further investigated in this study by a different method (i.e. parabiosis) from that of the previous studies.

Parabiotic B6.Ly5.1 and B6.Ly5.2 mice21–23 were used in this study. These individuals share the same circulation by day 3 after parabiosis and T cells in the periphery (e.g. the spleen and lymph nodes) become a half-and-half mixture of a mouse's own cells and its partner's cells by 14 days after parabiosis. In contrast, we demonstrated in a preliminary study (manuscript submitted for publication), that extrathymic T cells, especially NK T cells, seen in the liver, showed a low mixture of partner cells (i.e. less than 10% entrance of partner cells into this cell fraction even by day 60 or more). It is conceivable that extrathymic T cells are generated by their own pre-existing stem cells. Recent studies have revealed that c-kit+Lin− (lineage marker) stem cells exist in the liver,24 small intestine25 and appendix.12 In contrast, conventional T cells originate in the thymus and therefore become a half-and-half mixture by 14 days. According to this concept, we investigated how lymphocytes at different sites and various lymphocyte subsets in the small intestine and appendix mixed with partner cells. Depending on lymphocytes at the sites and various lymphocyte subsets, unique mixture patterns were demonstrated. The combination of mice irradiated at various doses and non-irradiated mice was also used for parabiosis to determine how the mixture of partner cells was influenced.

MATERIALS AND METHODS

Mice

C57BL/6 (B6.Ly5.2, H-2b) and C57BL/6-Ly5.1 (B6.Ly5.1, H-2b) mice were used in this study. The latter were origin ally provided by Dr K. Kishihara (Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan) and were maintained in the animal facility of Niigata University. All mice were fed under specific pathogen-free conditions.

Parabiosis

Parabiosis of B6.Ly5.1 and B6.Ly5.2 mice at the age of 8 weeks was produced as described previously.23 When a few individual mice of parabiotic pairs suffered from stress resulting in thymic atrophy, they were eliminated from the experiments.

Irradiation of one individual for parabiosis

Only B6.Ly5.2 mice were irradiated at various doses (3·5, 6·0 and 9·5 Gy) and then treated as parabiotic mice a few hours later. Parabiotic mice were killed at appropriate intervals and Ly5.1+ cells, which had immigrated from an individual partner mouse, were examined.

Cell preparations

IEL, LPL and Peyer's patches were collected from the intestine according to the method described in a previous report.26 Briefly, the small intestine, colon (large intestine) and appendix were removed and flushed with phosphate-buffered saline (PBS) to eliminate luminal contents. The mesentery and Peyer's patches, or appendiceal lymphoid follicles of the appendix were then resected. The intestine was opened longitudinally and cut into fragments 1–2 cm long. These fragments were incubated for 15 min in 20 ml Ca2+-free Dulbecco's PBS containing 5 mm ethylenediaminetetraacetic acid (EDTA), in a 37° shaking-water bath. The supernatant was then collected. The cell suspensions were collected and centrifuged in a discontinuous 40%/80% Percoll gradient at 900 g. for 25 min. Cells from the 40%/80% interface were collected.

LPL were prepared after the digestion of intestine with collagenase type II at a concentration of 90 U/ml in the medium. Samples were incubated for 45–90 min in a 37° shaking water bath. Digested intestine was then pressed through 200-gauge stainless steel mesh and suspended in medium. Cells were fractionated by a 35% Percoll solution.

Immunofluorescence tests

Phenotypes of lymphocytes were identified by two- or three-colour immunofluorescence tests using flow cytometry. For flow cytometric analysis, anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD8β (Ly-3), anti-TCRαβ (H57-597), anti-TCRγδ (GL3) and anti-B220 (RA3-6B2) monoclonal antibodies (mAb) were purchased from PharMingen (San Diego, CA).26 Mouse anti-Ly5.1 (A20.1.7) and anti-Ly5.2 (ALI-4A2) mAb were provided by Dr T. Kina (Kyoto University, Kyoto, Japan). All mAb were used with a fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or biotin-conjugated form. Biotinylated reagents were developed with TRI-COLOR-conjugated streptavidin (CALTAG Lab., San Francisco, CA). Cells were analysed by fluorescence-activated cell sorter (FACScan) using Lysis II software (Becton-Dickinson Co., Mountain View, CA). To prevent non-specific binding of mAb, CD32/16 (24G2) was added before staining with the labelled mAb. Dead cells were excluded by forward scatter or side scatter.

RESULTS

Retarded mixture of IEL in the digestive tract

As previously shown,22,23 lymphocytes in the spleen and lymph nodes become a half-and-half mixture of their own cells and their partner's cells in each individual of parabiotic mice by day 14 after parabiosis. This is because parabiotic mice share the same circulation and because such peripheral lymphocytes originate in the thymus and other organs. In this experiment, it was investigated whether or not IEL in the digestive tract became a half-and-half mixture after parabiosis (Fig. 1). IEL were isolated from the small intestine, large intestine and appendix. LPL in the small intestine and Peyer's patch lymphocytes were examined in parallel. IEL in the small intestine showed a low mixture of partner cells (i.e. less than 10% by day 60). A similar tendancy of low mixture was also seen in IEL of the large intestine and appendix (i.e. less than 20% on day 60). It is conceivable that the majority of IEL may give rise to their own stem cells in situ. In sharp contrast, Peyer's patch lymphocytes became a half-and-half mixture by day 14 after parabiosis. This pattern was quite similar to that of usual lymph node lymphocytes (data not shown). Interestingly, LPL in the small intestine showed an intermediate pattern between IEL of the digestive tract and Peyer's patch lymphocytes. It is speculated that some lymphocyte subsets of LPL gave rise to their own stem cells at this site but that the other subsets came from other sites.

Figure 1.

Figure 1

Open in a new tab

Entrance of partner cells into lymphocyte subsets at various sites of parabiotic pairs. Parabiotic B6.Ly5.1 and B6.Ly5.1 mice were examined at the indicated days after parabiosis. The percentages of Ly5.1+ cells in an Ly5.2 individual of a parabiotic pair are depicted. Peyer's patch lymphocytes showed a high mixture pattern (i.e. >40% mixture with partner cells by day 14). On the other hand, all IEL of the digestive tract showed a low mixture pattern. LPL in the small intestine had an intermediate mixture pattern.

Dissociation of the mixture pattern among lymphocyte subsets in the small intestine

When IEL of the small intestine were stained for CD4 and CD8, three subsets were identified, including single-positive CD4+ and CD8+ cells and double-positive (DP) CD4+ CD8+ cells. By gated analysis, the mixture patterns of these three subsets were directly compared (Fig. 2). In this experiment, three-colour staining for Ly5.1, CD4 and CD8 was conducted. In the left column of Fig. 2(a), the pattern of B6.Ly5.1 mice is represented as a positive control. In the right column of Fig. 2(a), the pattern in the B6.Ly5.2 individual of a parabiotic pair is represented. These immunofluorescence data were produced by using parabiotic mice on day 20 after parabiosis. As shown by the previous data (see Fig. 1), IEL of the small intestine contained only a small proportion (2·9%) of R2 (Ly5.1+) cells. On the other hand, LPL of the small intestine and Peyer's patch lymphocytes contained a large proportion (25·4% and 44·0%, respectively) of R2 (Ly5.1+) cells. When the staining pattern of CD4 and CD8 was observed in the small intestine, the R2 fraction was found to contain a larger proportion of CD4+ cells than that of the R1 fraction. In other words, CD4+ cells preferentially entered the other individuals as partner cells. This situation was further examined on various days after parabiosis and the data were plotted (Fig. 2b). It was clearly demonstrated that CD4+ cells of the small intestine mixed well in the other individual but that CD8+ and DP CD4+ CD8+ did not.

Figure 2.

Figure 2

Open in a new tab

Mixture pattern of lymphocytes with CD4+ or CD8+ phenotype in the small intestine. (a) Three-colour staining and gated analysis, (b) time-kinetics of the mixture with partner cells in IEL of the small intestine, and (c) time-kinetics of the mixture with partner cells in LPL of the small intestine. To determine the mixture rates in each lymphocyte subset, three-colour staining for Ly5.1, CD4 and CD8 was conducted at the indicated days. All experiments were repeated four times. A representative result (day 20 after parabiosis) is depicted for experiment (a), while the mean values at the indicated time-points are depicted for experiments (b) and (c). The axis of all FACS profiles in this study was logarithmic expression as usual.

Since the mixture ratio of LPL (25·4%) showed an intermediate pattern, among LPL, there might be lymphocytes of in situ origin as well as those originating at other sites. When the mixture ratio was compared between CD4+ and CD8+ cells of LPL, the CD4+ cells showed a greater mixture pattern than did the CD8+ cells (Fig. 2c). Similar to the case of IEL, CD4+ cells might contain a large population of lymphocytes originating at other sites.

Retarded mixture seen in both CD8αα+ and CD8αβ+ cells

IEL of the small intestine and appendix comprise both CD8αα+ and CD8αβ+ cells. It was then examined whether there were any differences of the mixture pattern between these subsets (Fig. 3). The data of parabiotic mice on day 20 are represented, in parallel with a positive control of B6.Ly5.1 mice. IEL of both the small intestine and appendix contained only a small proportion of partner cells (R2, Ly5.1+ cells). The gated analysis of R1 and R2 fractions for CD4 and CD8 showed that CD8αβ+ cells were more frequent than CD8αα+ cells. This situation was further examined in the small intestine (Fig. 3b), results showing the same tendency. However, the mixture was retarded in both subsets (i.e. less than 10%).

Figure 3.

Figure 3

Open in a new tab

A comparison of the mixture pattern between CD8αα+ and CD8αβ+ cells in IEL of the small intestine and appendix. (a) Three-colour staining and the gated analysis, (b) time-kinetics of the mixture pattern with partner cells. All experiments were repeated four times. A representative case (day 20 after parabiosis) is depicted for experiment (a), while the means at the indicated time-points are depicted in experiment (b).

Retarded mixture seen in both αβ TCR+ and γδ TCR+ cells of IEL in the small intestine

Characterization was then extended to αβ TCR+ and γδ TCR+ cells in the small intestine. IEL, LPL and Peyer's patch lymphocytes were examined (Fig. 4). In the control B6.Ly5.1 mice, the composition pattern of αβ TCR+ and γδ TCR+ cells was unique, the proportion of γδ TCR+ cells being greater in IEL than in LPL. The γδ TCR+ cells were almost completely absent in Peyer's patch lymphocytes. All sites in the small intestine contained a large proportion of αβ TCR+ cells.

Figure 4.

Figure 4

Open in a new tab

A comparison of the mixture pattern with partner cells between αβ T and γδ T cells at various sites of the small intestine. (a) Three-colour staining and gated analysis, (b) time-kinetics of the mixture pattern with partner cells. All experiments were repeated four times. A representative result (day 20) is depicted for experiment (a) while the means of the indicated time-points are depicted for experiment (b).

When lymphocytes in the small intestine of parabiotic mice were examined on day 20 after parabiosis, it was confirmed that IEL did not mix well with partner cells (R2, Ly5.1+ cells). Peyer's patch lymphocytes mixed well, while LPL showed a low mixture pattern. Attention was then focused on the subsets of αβ TCR+ and γδ TCR+ cells. The mixture of γδ TCR+ cells was found to be more retarded than that of αβ TCR+ cells in the R2 fraction. This was true in both IEL and LPL. This result for the small intestine was confirmed by further experiments (Fig. 4b). However, both αβ TCR+ and γδ TCR+ cells showed primarily a low mixture pattern (≤10%).

Characterization of DN CD4− CD8− cells and other subsets in the appendix

It is well established that some T cells, including γδ T cells in the small intestine, have the DN CD4− CD8− phenotype.5–11 However, we recently demonstrated the presence of a large proportion of αβ T cells with DN CD4− CD8− phenotype in IEL of the appendix.12 This situation is represented by the data of Fig. 5(a), in which IEL of the appendix were examined in comparison with lymphocytes at various sites in the small intestine. The proportion of CD3+ CD4− CD8− cells (40·9%) was extremely high in IEL of the appendix.

Figure 5.

Figure 5

Open in a new tab

A comparison of the mixture pattern among lymphocyte subsets with DN CD4− CD8− or single-positive CD4+ and CD8+ phenotype in the appendix. (a) Three-colour staining for Ly5.1, CD3 and a mixture of CD4 and CD8 (day 20), (b) time-kinetics of the mixture with partner cells. Since DN CD4− CD8− cells were abundant in IEL of the appendix, the mixture pattern of such subsets was compared with others (various sites in the small intestine). Experiments were repeated four times at the indicated time-points.

The mixture patterns of partner cells among all these subsets were then examined (Fig. 5a, centre columns). The level of partner cells which entered IEL of the appendix was very small (7·2%) on day 20 after parabiosis. More interestingly, the entrance of partner cells into the DN cells of the R2 fraction was low. This situation was confirmed by repeated experiments (Fig. 5b). In this experiment, the results of single-positive CD4+ and CD8+ cells in the appendix are represented in parallel. CD8+ cells and DN CD4− CD8− cells showed a lower mixture than did CD4+ cells in the appendix.

Retarded mixture of B220+CD3+ cells, but not B220+CD3− cells, in IEL of the appendix

Experiments thus far revealed that IEL of the appendix contained a large proportion of DN CD4− CD8− cells. As shown previously,12 the majority of such DN cells expressed B220 antigens, as did conventional B cells. In this experiment, it was examined how these B220+ subsets mixed with partner cells in the appendix of parabiotic mice (Fig. 6). The high expression of B220 antigens in CD3+ cells was unique in IEL of the appendix when compared with lymphocytes at various sites in the small intestine (Fig. 6a). It was also demonstrated that the mixture of partner cells in B220+ CD3+ cells of the R2 fraction (Ly5.1+ cells) was retarded on day 20 after parabiosis (Fig. 6a, centre column). This situation was confirmed in the appendix by repeated experiments (Fig. 6b). Both total CD3+ cells and the subset of CD3+ B220+ cells showed a low mixture, while B220+ B cells mixed well in the appendix.

Figure 6.

Figure 6

Open in a new tab

A comparison of the mixture pattern between DN CD3+ B220+ cells in IEL of the appendix and others and B220+ B cells in LPL and Peyer's patches of the small intestine. (a) Three-colour staining for Ly5.1, CD3 and B220 and the gated analysis (day 20), (b) time-kinetics of the mixture pattern in IEL of the appendix, and (c) time-kinetics of the mixture pattern in LPL of the small intestine. Experiments were repeated four times at the indicated time-points.

In the case of IEL of the small intestine, there are extremely few B cells (i.e. B220+ CD3−). On the other hand, some B cells are present in LPL of the small intestine. In this regard, the origin of such B220+ CD3− B cells in LPL was examined (Fig. 6c). It was demonstrated that such B cells in LPL showed a high mixture pattern (i.e. they originated at other sites).

Acceleration in the mixture of partner cells by irradiation

To investigate how total body irradiation changed the degree of mixture of partner cells in various subsets in the digestive tract of parabiotic mice, B6.Ly5.2 mice were first irradiated at the indicated doses and then parabiosis was conducted with non-irradiated B6.Ly5.1 mice. On day 20 after parabiosis, the percentages of partner cells (Ly5.1+ cells) in various subsets in the digestive tract of B6.Ly5.2 individuals were examined (Fig. 7). All stainings and the gated analysis were the same as in the previous experiments in this study. It was demonstrated that the irradiation accelerated the mixture of partner cells in all tested subsets and at all tested sites in a dose-dependent manner. Usually, the lymphocyte subsets which showed a high mixture rate under non-irradiated conditions also showed a much more accelerated mixture than those which showed a low mixture rate. It is noteworthy that the lymphocyte subsets which showed a low mixture rate eventually showed a high mixture rate under a lethal dose of 9·5 Gy. However, the mixture differences became more remarkable in lymphocytes which had originated at sites other than those of in situ origin.

Figure 7.

Figure 7

Open in a new tab

Modulation of the mixture pattern with partner cells into various lymphocyte subsets by irradiation. (a) Various sites of the digestive tract, (b) IEL with CD4+, CD8+, and DP phenotype in the small intestine, (c) CD8αα+ and CD8αβ+ cells in IEL of the small intestine, (d) αβ T and γδ T cells in IEL of the small intestine, (e) IEL with CD4+, CD8+, and DN phenotype in the appendix, (f) CD3+ B220+ cells, CD3− B220+ cells, and CD3+ B220− cells in IEL of the appendix. B6.Ly5.2 mice were irradiated at the indicated doses and then parabiosis was produced with non-irradiated B6.Ly5.1 mice.

In addition to this general rule, the mixture pattern of CD8αα+ cells and αβ T cells IEL of the small intestine was unique. CD8αβ+ cells showed a much more accelerated mixture pattern than did CD8αα+ cells. Similarly, αβ T cells showed a much more accelerated mixture pattern than did γδ T cells. In some situations, these CD8αβ+ cells and αβ T cells were preferentially supplied from other sites. In Fig. 7, 0 Gy indicates no irradiation. By gated analysis, we determined the percentages of partner cells. In this regard, non-specific infiltration by cells from the partner mouse was not estimated in this figure. However, in the FACS profile, we learned that even 9·5-Gy irradiation did not increase such non-specific infiltration (data not shown). In other words, the infiltration occurred specifically, even in the gut of 9·5-Gy-irradiated mice.

DISCUSSION

In the present study, we investigated how lymphocytes at different sites and various lymphocyte subsets in the digestive tract mixed with partner cells after parabiosis. The most rapid mixture was seen for Peyer's patch lymphocytes in the small intestine where these lymphocytes became a half-and-half mixture by 14 days after parabiosis. This mixture pattern was quite similar to those of lymphocytes in the spleen and lymph nodes.5–11 It is estimated that almost all of the lymphocytes in the Peyer's patches, as well as those in the spleen and lymph nodes, originated at another site (i.e. the thymus). On the other hand, all IEL of the small intestine, large intestine and appendix showed a retarded mixture (less than 20% on day 60). It is speculated that these IEL originated from their own pre-existing stem cells. This concept arose from recent evidence that c-kit+ Lin− cells with stem cell ability are present in the small intestine (i.e. cryptopatches)25 and appendix.12 In other words, IEL of the large intestine originate from their own stem cells as do those of the small intestine and appendix. In the case of LPL in the small intestine, they showed an intermediate mixture pattern. It is conceivable that some subsets of LPL originated in situ in the lamina propria while some other subsets came from other sites (e.g. the thymus).

These experimental designs and the concept for interpreting the results were then extended to various lymphocyte subsets in the small intestine and other sites. In IEL of the small intestine, there are three subsets with CD4+, CD8+ and DP CD4+ CD8+ phenotypes. The mixture ratios of both CD8+ cells and DP CD4+ CD8+ cells were extremely low (less than 5% even on day 60 after parabiosis). In other words, it is possible that these subsets of IEL were highly dependent on in situ generation from their own pre-existing stem cells. On the other hand, the majority of CD4+ cells in IEL might originate from other sites (e.g. the thymus). The mixture ratio of these CD4+ cells rose only 30% or less. Therefore, some CD4+ cells at this site were found to be generated in situ from their own pre-existing stem cells. Another important finding in this experiment was that the composition pattern of the R2 fraction (Ly5.1+ cells) was quite similar to that of the R1 fraction at various sites. This result was interpreted to mean that only selected partner cells which should home in on the corresponding sites migrate to specific sites of another partner individual.

The similarity of the low mixture of CD8αα+ and CD8αβ+ cells in the small intestine was very interesting. This was also true in the appendix. CD8αα+ cells are presumed to be phylogenetically more primitive than CD8αβ+ cells.5–11,26 However, with regard to their having the same low mixture pattern, both subsets of CD8+ cells might originate from their own pre-existing stem cells in situ. This was also the case for αβ T cells and γδ T cells in the small intestine.

The existence of γδ T cells and DP CD4+ CD8+αβ T cells in IEL was unique in the small intestine, whereas that of DN CD4− CD8− and CD3+ B220+ cells in IEL was unique in the appendix. As shown previously, DN CD4− CD8− cells and CD3+ B220+ cells are mostly overlapping populations and they mimic abnormal DN CD4− CD8− B220+ cells in the spleen and lymph nodes of MRL-lpr/lpr mice which have an abnormal Fas gene (i.e. lpr gene).12,27 In this study, attention was also focused on this population in the appendix of the parabiotic mice. Similar to the case of IEL in the small intestine, CD4+ cells showed an intermediate mixture pattern. On the other hand, both DN CD4− CD8− cells and CD8+ cells showed a low mixture pattern. It is therefore concluded that the majority of DN CD4− CD8− cells, as well as the majority of CD8+ cells, are generated in situ in the appendix. As expected, CD3+ B220+ cells behaved in the same manner as DN CD4− CD8− cells in the appendix.

We used mice irradiated at various doses as one individual of a parabiotic pair. In a dose-dependent manner, partner cells (i.e. non-irradiated mice) replaced those of irradiated mice. Especially at the lethal dose (9·5 Gy), the majority of irradiated cells of the individual were replaced by partner cells even if they had been generated in situ or had originated at other sites. However, a difference in mixture was still present between lymphocytes originating at other sites and those of in situ origin.

In this study, we used parabiotic pairs of B6.Ly5.1 and B6.Ly5.2 mice. According to the mixture pattern with partner cells, we speculated that lymphocyte subsets existing in the digestive tract were of extrathymic origin or had originated at other sites. It was estimated that extrathymic T cells generated in situ in the digestive tract were CD8+ (both CD8αα+ and CD8αβ+ cells), DP CD4+ CD8+, and CD4+ (partial) cells, including both αβ and γδ T cells, in the small and large intestines and were DN CD4− CD8− (B220+), CD8+, and CD4+ (partial) cells in the appendix. Some results just confirmed those of previous reports using different methods.16,23 However, this means that the method applied herein was efficient. Since some other new results were produced (e.g. lymphocyte subsets in the appendix and large intestine), this study yielded new information about mucosal immunology.

If stem cells and subsequent precursors of T cells in the digestive tract are rapidly supplied from other sites (e.g. the bone marrow), the low mixture pattern seen in T cells of the digestive tract (e.g. αβ T and γδ T cells in IEL of the small intestine) would not have occurred. However, we also have to be concerned with their renewal rates and lifespans. In a previous study, Penney et al.28 reported that the 50% population renewal time for αβ T cells was 10 days and that for γδ T cells was ≈50 days in IEL of the small intestine in mice. Since the sizeable pool of these T-cell subsets in IEL of the small intestine is usually stable, it is conceivable that γδ T cells have a lifespan which is five times longer than that of αβ T cells in IEL of the small intestine. In the data from this study, both αβ T cells and γδ T cells belonged to the low mixture group. It is therefore presumed that the renewal rates and lifespans of lymphocyte subsets did not significantly affect the interpretation of data from parabiosis. In other words, the fact that lymphocyte subsets undergo self-renewal from their own pre-existing stem cells or precursors (without the supply of mature cells from the circulation) is important for the low mixture pattern. However, we cannot deny the following possibility. Interesting results were forthcoming from the parabiosis experiments, including first, the mixture rate of partner cells remained 55:45 in the spleen and lymph nodes of the recipient individual, and second the mixture rate of γδ T cells was lower than that of αβ T cells in IEL of the small intestine. These two results may be due to the following reasons: first, some of the T cells which homed to the spleen and lymph nodes from the recipient thymus had a significant high renewal capability, and second γδ T cells have a longer lifespan than αβ T cells in IEL of the small intestine, respectively.

Finally, if one lymphocyte subset in the corresponding area has a long lifespan but its precursor cells are always supplied from other sites, the mixture rate of partner cells should finally have increased. However, such a pattern was not observed in extrathymic T-cell subsets in the digestive tract. In other words, after long intervals of parabiosis, the mixture rate of such populations did not increase and became stationary. Only when one individual was irradiated before parabiosis did the mixture rate increase. Results using the present experimental protocol mainly indicate that the low-mixture group might be generated from their own pre-existing precursor cells.

Acknowledgments

We wish to thank Mrs Masako Watanabe for preparation of the manuscript and Mr Tetsuo Hashimoto for animal maintenance.

Abbreviations

DN

double-negative

DP

double-positive

IEL

intraepithelial lymphocytes

Lin

lineage

LPL

lamina propria lymphocytes

REFERENCES

  • 1.Abo T, Ohteki T, Seki S, et al. The appearance of T cells bearing self-reactive T cell receptor in the livers of mice injected with bacteria. J Exp Med. 1991;174:417. doi: 10.1084/jem.174.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Seki S, Abo T, Ohteki T, Sugiura K, Kumagai K. Unusual αβ-T cells expanded in autoimmune lpr mice are probably a counterpart of normal T cells in the liver. J Immunol. 1991;147:1214. [PubMed] [Google Scholar]
  • 3.Sato K, Ohtsuka K, Hasegawa K, et al. Evidence for extrathymic generation of intermediate TCR cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation. J Exp Med. 1995;182:759. doi: 10.1084/jem.182.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Watanabe H, Miyaji C, Kawachi Y, et al. Relationships between intermediate TCR cells and NK1.1+T cells are present within a population of intermediate TCR cells. J Immunol. 1995;155:2972. [PubMed] [Google Scholar]
  • 5.Ferguson A, Parrott DM. The effect of antigen deprivation on thymus-dependent and thymus-independent lymphocytes in the small intestine of the mouse. Clin Exp Immunol. 1972;12:477. [PMC free article] [PubMed] [Google Scholar]
  • 6.Mosley RL, Styre D, Klein JR. Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras. J Immunol. 1990;145:1369. [PubMed] [Google Scholar]
  • 7.Bandeira A, Itohara S, Bonneville M, et al. Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor gamma delta. Proc Natl Acad Sci USA. 1991;88:43. doi: 10.1073/pnas.88.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Rocha B, Vassalli P, Guy-Grand D. The Vβ repertoire of mouse gut homodimeric α CD8+ intraepithelial T cell receptor α/ β+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J Exp Med. 1991;173:483. doi: 10.1084/jem.173.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Mosley RL, Styre D, Klein JR. CD4+CD8+ murine intestinal intraepithelial lymphocytes. Int Immunol. 1990;2:361. doi: 10.1093/intimm/2.4.361. [DOI] [PubMed] [Google Scholar]
  • 10.Taguchi T, Aicher WK, Fujihashi K, et al. Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, γ/δ TCR+ T cells produced IFN-γ and IL-5. J Immunol. 1991;147:3736. [PubMed] [Google Scholar]
  • 11.Fangmann J, Schwinzer R, Wonigeit K. Unusual phenotype of intestinal intraepithelial lymphocytes in the rat: predominance of T cell receptor α/Gb+/CD2− cells and high expression of the RT6 alloantigen. Eur J Immunol. 1991;21:753. doi: 10.1002/eji.1830210331. [DOI] [PubMed] [Google Scholar]
  • 12.Yamagiwa S, Sugahara S, Shimizu T, et al. The primary site of CD4−B220+αβT cells in lpr mice – the appendix in normal mice. J Immunol. 1998;160:2665. [PubMed] [Google Scholar]
  • 13.Dunon D, Cooper MD, Imhof BA. Thymic origin of embryonic intestinal γ/δ T cells. J Exp Med. 1993;177:257. doi: 10.1084/jem.177.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lynch S, Kelleher D, McManus R, O'Farrelly C. RAG1 and RAG2 expression in human intestinal epithelium: evidence of extrathymic T cell differentiation. Eur J Immunol. 1995;25:1143. doi: 10.1002/eji.1830250502. [DOI] [PubMed] [Google Scholar]
  • 15.Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med. 1991;173:471. doi: 10.1084/jem.173.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Poussier P, Edouard P, Lee C, Binnie M, Julius M. Thymus-independent development and negative selection of T cells expressing T cell receptor α/β in the intestinal epithelium: evidence for distinct circulation patterns of gut-and thymus-derived T lymphocytes. J Exp Med. 1992;176:187. doi: 10.1084/jem.176.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.De Geus B, Van den Enden M, Coolen C, Nagelkerken L, Van der Heijden P, Rozing J. Phenotype of intraepithelial lymphocytes in euthymic and athymic mice: implications for differentiation of cells bearing a CD3-associated γδT cell receptor. Eur J Immunol. 1990;20:291. doi: 10.1002/eji.1830200210. [DOI] [PubMed] [Google Scholar]
  • 18.Guy-Grand D, Malassis-Seris M, Briottet C, Vassalli P. Cytotoxic differentiation of mouse gut thymodependent and independent intraepithelial T lymphocytes is induced locally. Correlation between functional assays, presence of perforin and granzyme transcripts, and cytoplasmic granules. J Exp Med. 1991;173:1549. doi: 10.1084/jem.173.6.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Camerini V, Panwala C, Kronenberg M. Regional specialization of the mucosal immune system. Intraepithelial lymphocytes of the large intestine have a different phenotype and function than those of the small intestine. J Immunol. 1993;151:1765. [PubMed] [Google Scholar]
  • 20.Beagley KW, Fujihashi K, Lagoo AS, et al. Differences in intraepithelial lymphocyte T cell subsets isolated from murine small versus large intestine. J Immunol. 1995;154:5611. [PubMed] [Google Scholar]
  • 21.Scheid MP, Triglia D. Further description of the Ly-5 system. Immunogenetics. 1979;9:423. [Google Scholar]
  • 22.Donskoy E, Goldschneider I. Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life: a study in parabiotic mice. J Immunol. 1992;148:1604. [PubMed] [Google Scholar]
  • 23.Hirokawa K, Utusyama M, Sado T. Immuno-histological analysis of immigration of thymocyte-precursors into the thymus: evidence for immigration of peripheral T cells into the thymic medulla. Cell Immunol. 1989;119:160. doi: 10.1016/0008-8749(89)90232-3. [DOI] [PubMed] [Google Scholar]
  • 24.Watanabe H, Miyaji C, Seki S, Abo T. C-kit+ stem cells and thymocyte precursors in the livers of adult mice. J Exp Med. 1996;184:687. doi: 10.1084/jem.184.2.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kanamori Y, Ishimaru K, Nanno M, et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+IL-7R+Thy1+ lympho-hemopoietic progenitors develop. J Exp Med. 1996;184:1449. doi: 10.1084/jem.184.4.1449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ohtsuka K, Iiai T, Watanabe H, et al. Similarities and differences between extrathymic T cells residing in mouse liver and intestine. Cell Immunol. 1994;153:52. doi: 10.1006/cimm.1994.1005. [DOI] [PubMed] [Google Scholar]
  • 27.Yamagiwa S, Kuwano Y, Hasegawa K, et al. Existence of a small population of IL-2Rβhi TCRint cells in SCG and MRL-lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene. Eur J Immunol. 1996;26:1409. doi: 10.1002/eji.1830260702. [DOI] [PubMed] [Google Scholar]
  • 28.Penney L, Kilshaw PJ, MacDonald TT. Regional variation in the proliferative rate and lifespan of αβ TCR+ and γδ TCR+ intraepithelial lymphocytes in the murine small intestine. Immunology. 1995;86:212. [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES