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TCR-Independent Activation of Extrathymically Developed, Self Antigen-Specific T Cells by IL-2/IL-15¹

Hisakata Yamada^2,*, Takahiko Nakamura, Goro Matsuzaki, Yukihide Iwamoto^* and Kikuo Nomoto

^* Department of Orthopedic Surgery, Graduate School of Medical Sciences, and Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN- production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the thymus, immature T cells expressing self Ag-specific TCR are deleted during development. This is called negative selection and is a major mechanism to prevent autoimmunity. Consequently, mature T cells bearing foreign Ag-specific TCR are selectively exported to the periphery where they reside in a naive state until they encounter foreign Ags. Usually, naive T cells do not express IL-2R. After antigenic restimulation, they secrete IL-2 and express high affinity IL-2R, which is composed of IL-2R-chain, IL-2Rß-chain and common -chain, which enables IL-2 to be used as an autocrine growth factor (1). Such TCR-dependent, IL-2-mediated proliferation results in clonal expansion of invading foreign Ag-specific T cells, which is a hallmark of acquired immunity.

Some subsets of peripheral T cells exist even in the absence of the thymus and are therefore regarded as extrathymically developed T cells (2, 3, 4, 5). In our previous report, we analyzed the selections of extrathymically developed T cells in the spleen and the liver using male H-Y Ag-specific TCR transgenic mice (H-Y transgenic mice³; Ref. 6) (7). It was revealed that extrathymically developed T cells are not negatively selected but rather are positively selected by self Ags. The extrathymic development of self Ag-specific T cells has been shown in many systems (8, 9, 10, 11, 12, 13). Furthermore, through the analysis of intestinal intraepithelial lymphocytes (iIELs) in H-Y transgenic mice, the positive selection of extrathymically developed iIEL T cells by self Ags has been verified (14, 15). These findings suggest a possible occurrence of autoimmunity caused by extrathymically developed T cells. However, male Ag-specific T cells that developed extrathymically in male mice did not show any responses to male Ags in vitro. Furthermore, male H-Y transgenic mice, as well as normal mice in which self Ag-specific, extrathymically developed T cells should also exist, do not spontaneously develop any autoimmune diseases. Nevertheless, the extrathymically developed, self Ag-specific T cells could respond well to anti-TCR mAb, indicating that they are not in completely anergic state but can be activated under certain circumstances.

It has been shown that extrathymically developed T cells in naive normal mice selectively express IL-2Rß-chain but not -chain (4). Thus, they are supposed to spontaneously express low affinity receptors for IL-2. We showed that male Ag-specific T cells developed in athymic male mice were also IL-2Rß⁺ (7). Other than extrathymically developed T cells, NK cells express low affinity IL-2R and are known to respond to exogenous IL-2 (16). IL-15 is a novel cytokine that utilizes IL-2 receptors to exert biological activities (17). Similar to IL-2, IL-15 has been shown to stimulate NK cells via IL-2Rß-chain (18, 19). However, it has not been fully addressed whether extrathymically developed T cells, which also spontaneously express IL-2Rß-chain, respond to IL-2 or IL-15, although TCR iIEL T cells and NK1.1+ T cells, both of which could develop extrathymically, were shown to respond to IL-2 or IL-15 (20, 21). Furthermore, it is of interest to address whether IL-2 or IL-15 modulates the unresponsiveness of the self Ag-specific, extrathymically developed T cells to the self Ags.

In the present study, we examined the responses of IL-2Rß-chain-expressing, extrathymically developed T cells to IL-2 or IL-15. We further addressed the influences of TCR-mediated signaling on the responses to these cytokines using T cells in H-Y transgenic mice.

	Materials and Methods

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Mice

The mice bearing transgenic TCR specific for male H-Y Ag peptide on H-2D^b (H-Y transgenic mice) (6) were kindly provided by Dr. T. W. Mak (AMGEN Institute, Toronto, Canada). C57BL/6 mice and BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). BALB/c nu/nu mice were purchased from Japan Crea (Tokyo, Japan). These mice were maintained in specific pathogen-free conditions and used at 10–14 wk of age, with the exception of the BALB/c nu/nu mice, which were used at 20 wk of age.

Flow cytometric analysis and Abs

mAbs used for flow cytometric analysis comprised FITC- or biotin-conjugated T3.70 mAb (specific for transgenic TCR -chain, a kind gift from Dr. Tak W. Mak), PE- or allophycocyanin-conjugated anti-CD8 mAb (2.43) (PharMingen, San Diego, CA), PE-conjugated anti-TCR ß mAb (H57-597) (PharMingen), PE- or allophycocyanin-conjugated anti-CD3 mAb (2C11) (PharMingen), biotin-conjugated anti-CD44 mAb (IM7) (PharMingen), FITC-conjugated anti-IL-2Rß (CD122) mAb (TM-ß1) (PharMingen), PE-conjugated streptavidin (Life Technologies, Gaithersburg, MD), and allophycocyanin-conjugated streptavidin (Becton Dickinson, Mountain View, CA). Freshly isolated or cultured cells were incubated with various combinations of mAbs before being analyzed by a FACScalibur flow cytometer with the CELLQuest program (Becton Dickinson). In some experiments, cell suspensions received an addition of 50 µg/ml of propidium iodide (PI) just before being run on the cytometer to detect and exclude dead cells.

Measuring surviving cell numbers after in vitro cultures

Spleen cells (5 x 10⁵) of male or female H-Y transgenic mice were cultured with various doses of recombinant human IL-2 (a kind gift from Takeda Pharmaceutical, Osaka, Japan) or recombinant human IL-15 (Genzyme, Cambridge, MA), as indicated in the figures, in 96-well plates. Some cultures were performed with 5 x 10⁵ irradiated spleen cells of male C57BL/6 mice (H-2^b) or anti-H-2D^b mAb (Meiji, Tokyo, Japan). After 4 days, cells were collected, and live cells were analyzed by a flow cytometer by gating out PI-positive cells. The numbers of surviving T3.70⁺CD8⁺ cells were calculated after FACS analysis. In the other experiments, 5 x 10⁵ spleen cells of C57BL/6 mice, BALB/c mice, or BALB/c nu/nu mice were cultured with various doses of IL-2 or IL-15 for 4 days. The numbers of several subsets of surviving cell numbers were calculated after FACS analysis.

Detection of apoptotic cells

Nylon wool nonadherent splenocytes from female or male H-Y transgenic mice were stained with biotin-conjugated anti-CD8 mAb (53-6.7, Life Technologies). CD8-positive cells were positively selected using streptavidin-conjugated MACS microbeads (Miltenyi Biotec, Auburn, CA) and were cultured with medium only, IL-2 (10 ng/ml), or IL-15 (100 ng/ml). After 18 h, cells were harvested and stained with FITC-conjugated T3.70 mAb, followed by fixation by 75% ethanol at 4°C. They were then incubated with RNase (Sigma, St. Louis, MO) at 37°C for 30 min before being stained with PI (50 µg/ml) (Sigma). The DNA content of FITC-positive cells was measured by a flow cytometer.

In vitro proliferation and cytokine production assay

Nylon wool nonadherent spleen cells of female or male H-Y transgenic mice were adjusted to contain 2 x 10⁴ T3.70⁺CD8⁺ cells per well in 96-well plates and were cultured for 4 days with IL-2 (10 ng/ml), IL-15 (100 ng/ml), or irradiated spleen cells of male C57BL/6 mice (5 x 10⁵/well). In some groups, CD8⁺ or CD44⁺ cells were depleted using Dynabeads M450 sheep anti-rat IgG (Dynal, Oslo, Norway) after staining with anti-CD8 (2.43, rat IgG) or anti CD44 (IM7, rat IgG) mAbs, respectively. The cultures were pulsed with 1 µCi/well of [³H]TdR for the final 18 h. To examine the cytokine production, culture supernatants were collected after 4 days. IFN- in the supernatants was measured by an ELISA assay using Mouse IFN- DuoSeT (Genzyme). In another experiment, CD8-positive cells in female or male H-Y transgenic mice were positively selected by using MACS microbeads. They were cultured at 2 x 10⁴ T3.70⁺CD8⁺ cells per well with irradiated spleen cells in female or male C57BL/6 mice in the presence of 0.1 ng/ml of IL-12 (Genzyme) with or without various dose of IL-2 or IL-15. After 4 days, culture supernatants were collected, and IFN- in the supernatants was measured.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
All of the male Ag-specific T cells in male H-Y transgenic mice have surface molecule phenotypes similar to those of extrathymically developed T cells in normal mice

In our previous report, we showed that H-Y Ag-specific T cells (T3.70⁺CD8⁺) developed in syngeneic male thymectomized recipient mice expressed IL-2Rß (CD122⁺), intermediate levels of CD3 (CD3^int), and high levels of CD44 (CD44^high) similar to the extrathymically developed T cells in normal mice (7). In this study, we first addressed whether all of the T3.70⁺CD8⁺ cells in unmanipulated male H-Y transgenic mice have such phenotypes of surface molecule expression. As shown in Fig. 1, T3.70⁺CD8⁺ cells in the spleens of male H-Y transgenic mice were uniformly CD3^intCD122⁺CD44^high. The expression levels of these molecules were very similar to CD122⁺CD8⁺ cells in normal mice and, furthermore, CD8⁺ cells in athymic nude mice. Among the T3.70⁺ cells in male mice, those without CD8 were CD3^highCD122^-CD44^low (data not shown) similar to the T3.70⁺CD8⁺ cells in female H-Y transgenic mice (Fig. 1), neither of which developed in the absence of the thymus, as we have reported previously (7).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Expression of surface molecules on H-Y Ag-specific T cells. Expression of various surface molecules on freshly isolated T3.70+CD8+ cells in female or male H-Y transgenic mice, on CD122 (IL-2Rß-chain)-positive or -negative cells among CD8 T cells in C57BL/6 mice and on CD8 T cells in athymic BALB/c nu/nu mice was analyzed by a flow cytometer by gating on each subset.

As shown in Fig. 1, all of the T3.70⁺CD8⁺ cells in male H-Y transgenic mice, as well as extrathymically developed T cells in naive normal mice, express IL-2Rß-chain, but none of them were IL-2R⁺ (data not shown; Ref. 4). Thus, they may spontaneously express low affinity receptors for IL-2. To examine their responses to exogenous IL-2, spleen cells of H-Y transgenic mice in male or female mice were cultured with IL-2. After 4 days, numbers of CD8⁺T3.70⁺ cells were analyzed (Fig. 2, A and B). CD8⁺T3.70⁺ cells in female transgenic mice showed only a slight increase in cell number with the presence of exogenous IL-2. On the other hand, CD8⁺T3.70⁺ cells in male mice showed a striking increase in cell number dose-dependently of IL-2. IL-2 directly acted on T3.70⁺CD8⁺ cells in male mice, since purified T3.70⁺CD8⁺ cells also increased after culture with IL-2 (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Numbers of male H-Y Ag-specific T cells in female or male mice after culture with IL-2 in the presence or absence of antigenic stimulation. Spleen cells (5 x 105) of female (A) or male (B) H-Y transgenic mice were cultured with or without various doses of IL-2. Some cultures were performed with anti-H-2Db mAb and/or 5 x 105 irradiated spleen cells of male C57BL/6 mice. After 4 days, cells were collected, and live cells were analyzed by a flow cytometer by gating out PI-positive cells. The numbers of surviving T3.70+CD8+ cells were calculated after FACS analysis. The cell numbers at day 0 are plotted along the y-axis. We obtained similar results in four independent experiments. Representative data are shown in the figure.

It seems possible that male Ags expressed on spleen cells, including T cells themselves, in male H-Y transgenic mice may stimulate the male Ag-specific TCR to affect the response to exogenous IL-2. In other words, IL-2 may abrogate the unresponsive state of T3.70⁺CD8⁺ cells in male mice to the self male Ag, since IL-2 has been shown to be able to reverse T cell anergy (22). To examine the influences of TCR-mediated signaling on the responses observed above, the cultures were added with male stimulator cells or anti-H-2D^b mAbs (Fig. 2, A and B). The numbers of T3.70⁺CD8⁺ cells in female mice strikingly increased when they were cultured with syngeneic male spleen cells. An addition of IL-2 further increased the numbers of the Ag-stimulated T cells. These clonal expansions of H-Y Ag-specific T cells in female mice were effectively inhibited by an addition of anti-H-2D^b mAbs, indicating that the anti-H-2D^b mAb used in this experiment was suitable to block the interaction between transgenic TCRs and male H-Y Ag peptides on H-2D^b molecules. Interestingly, the dose-dependent increase of T3.70⁺CD8⁺ cells in male mice by IL-2 was scarcely affected by the presence of male stimulator cells or anti-H-2D^b mAbs. This clearly showed that self Ag-specific T cells, which developed extrathymically, respond to IL-2 independently of TCR stimulation.

Ag-independent increase of male Ag-specific T cells in male H-Y transgenic mice after culture with IL-15

In addition to IL-2, IL-15 also binds to IL-2Rß-chain to exert similar biological activities on activated T cells and NK cells (16, 18, 19). We next examined the responsiveness of IL-2Rß-chain-expressing extrathymically developed, self Ag-specific T cells to IL-15 (Fig. 3, A and B). Similar to IL-2, IL-15 induced a dose dependent increase of T3.70⁺CD8⁺ cells in male H-Y transgenic mice, independent of TCR-stimulation. T3.70⁺CD8⁺ cells in female mice were hardly affected by an addition of IL-15, unless they were stimulated by male Ags. From these findings, it was revealed that extrathymically developed T cells respond to any stimulus via IL-2Rß-chain, which is spontaneously expressed on them. However, there seem to be slight differences in the activities of IL-2 and IL-15. A high dose of IL-2 elicited a greater increase of cell number than similar dose of IL-15, whereas a low dose of IL-15 was more efficient than a similar dose of IL-2.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Numbers of male H-Y Ag-specific T cells in female or male mice after culture with IL-15 in the presence or absence of antigenic stimulation. Spleen cells of female (A) or male (B) H-Y transgenic mice were cultured with various doses of IL-15 similar to Fig. 2. The numbers of surviving T3.70+CD8+ cells were calculated after FACS analysis. Data are representative of four independent experiments.

We next examined the responses of extrathymically developed T cells in normal mice, which also express IL-2Rß-chain spontaneously (4), to exogenous IL-2 or IL-15 (Fig. 4). Whole spleen cells of naive C57BL/6 mice were cultured with IL-2 or IL-15 for 4 days. The numbers of various subsets of viable T cells were analyzed by flow cytometry. CD122^-CD3⁺ cells, which are regarded as intrathymically developed T cells, showed only a slight increase of cell number in the presence of exogenous IL-2 or IL-15. On the other hand, extrathymically developed CD122⁺ T cells including CD8⁺ cells showed an apparent increase in cell numbers dose-dependently of IL-2 or IL-15 similar to NK cells. They did not seem to be generated from CD122^- cells since, after removal of CD122⁺ cells from the spleen cells, CD122⁺ cells were scarcely generated after the culture (data not shown). CD8⁺ T cells in athymic BALB/c nude mice also increased after culture with IL-2 or IL-15 (Fig. 5). These results further confirm the extrathymic origin of IL-2/15-responding CD8⁺ T cells and indicate that the differences in the response to IL-2 or IL-15 between extrathymically and intrathymically developed CD8⁺ T cells are general properties of these cells, and are not specific for those in H-Y transgenic mice. There seemed some differences in the responses to IL-2, but not in the response to IL-15, between CD8⁺ T cells in BALB/c nude mice and CD8⁺CD122⁺ cells in normal BALB/c mice. Although we do not know the precise reason for this discrepancy, it is possible that this difference may have resulted from the presence of additional IL-2-responsive, IL-15-nonresponsive cell populations, other than extrathymically developed T cells, in CD8⁺CD122⁺ cells in euthymic BALB/c mice.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Numbers of various subset of T cells in C57BL/6 mice after culture with IL-2 or IL-15. Spleen cells (5 x 105) of C57BL/6 mice were cultured with various doses of IL-2 (A) or IL-15 (B) for 4 days. The numbers of various subsets of surviving cells were calculated after FACS analysis. Data are representative of five independent experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Numbers of CD8 T cells in athymic nude mice after culture with IL-2 or IL-15. Spleen cells (5 x 105) of BALB/c mice or BALB/c nu/nu mice were cultured with various doses of IL-2 (A) or IL-15 (B) for 4 days. The numbers of various subsets of surviving cell numbers were calculated after FACS analysis. Data are representative of three independent experiments.

Both IL-2 and IL-15 have been shown to have antiapoptotic effects on T cells, especially on activated T cells (23, 24, 25). It seems possible that the increases in cell number of T3.70⁺CD8⁺ cells of male mice after culture with IL-2 or IL-15 were attributable to their antiapoptotic effect. Actually, the numbers of T3.70⁺CD8⁺ cells in male mice decreased strikingly from the beginning of the culture in the absence of the cytokines (Figs. 2 and 3). This striking decrease of cell number was also observed in CD122⁺ T cells in normal mice (Figs. 4 and 5), whereas the numbers of CD122^- T cells were only moderately decreased similar to T3.70⁺CD8⁺ cells in female mice. We directly addressed this issue by measuring the percentage of apoptotic cells at 18 h after the beginning of the culture (Fig. 6). In the absence of any cytokines, a large portion of T3.70⁺CD8⁺ cells in male mice are apoptotic. An addition of IL-2 or IL-15 greatly diminished the percentage of apoptotic cells. Therefore, it is indicated that an inhibition of apoptosis contributed to the increase of extrathymically developed T cells cultured with IL-2 or IL-15. IL-2 or IL-15 also showed some antiapoptotic effects on T3.70⁺CD8⁺ cells in female mice, although the percentage of apoptotic cells among T3.70⁺CD8⁺ cells in female mice cultured without any cytokines was fewer than that in male mice, consistent with the data of surviving cell numbers. It is also suggested from these findings that extrathymically developed T cells have a greater tendency to be apoptotic than intrathymically developed T cells in vitro.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Apoptosis of male H-Y Ag-specific T cells in female or male mice after culture with IL-2 or IL-15. CD8-positive cells in spleen cells of female or male H-Y transgenic mice, positively sorted using magnetic beads, were cultured with medium only, IL-2 (10 ng/ml), or IL-15 (100 ng/ml) for 18 h at 37°C. As negative controls, cells were incubated at 4°C without cytokines for 18 h. Apoptosis was determined by measuring DNA content after ethanol fixation.

There still remains a possibility that IL-2 or IL-15 also selectively activate and induce proliferation of T3.70⁺CD8⁺ cells in male mice, in addition to inhibiting their death. So, we directly measured the proliferation of spleen cells in male or female H-Y transgenic mice cultured with IL-2 or IL-15. In this experiment, spleen cells of female mice were depleted of CD44^high cells, since an increase of the T3.70^-CD8⁺CD44^high cells in female mice was observed after culture with IL-2 or IL-15 (data not shown) and none of the T3.70⁺CD8⁺ cells in female mice were CD44^high (Fig. 1). As shown in Fig. 7, spleen T cells in male H-Y transgenic mice proliferated vigorously in response to 10 ng/ml of IL-2, and mildly in response to 100 ng/ml of IL-15, which induce equal levels of cell increase as 10 ng/ml of IL-2 as shown in Figs. 2 and 3. These proliferative responses were not detected when CD8⁺ cells were depleted. In contrast, female spleen cells did not show any proliferative responses to IL-2 or IL-15, but did proliferate in response to male Ags. These results indicated that IL-2 or IL-15 actually has a proliferative effect, in addition to an antiapoptotic effect, selectively on T3.70⁺CD8⁺ cells in male but not in female H-Y transgenic mice.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Proliferation of male H-Y Ag-specific T cells in female or male mice after culture with IL-2 or IL-15. Nylon wool nonadherent spleen cells of female or male H-Y transgenic mice, depleted or not depleted of CD8+ cells, were adjusted to contain 2 x 104 T3.70+CD8+ cells or equivalent numbers of cells in each well, before being cultured for 4 days with IL-2 (10 ng/ml) (A), IL-15 (100 ng/ml) (B), or irradiated spleen cells of male C57BL/6 mice (5 x 105/well) (C). Cell proliferation was measured by counting the incorporation of [3H]TdR pulsed for the final 18 h.

View larger version (17K): [in this window] [in a new window]   FIGURE 8. IFN- production of male H-Y Ag-specific T cells in female or male mice after culture with IL-2 or IL-15. A, T cell-enriched spleen cells of female or male H-Y transgenic mice, containing 2 x 104 T3.70+CD8+ cells in each well, were cultured for 4 days with IL-2 (10 ng/ml), IL-15 (100 ng/ml), or irradiated spleen cells of male C57BL/6 mice (5 x 105/well). B, Magnetically selected CD8-positive cells in female or male H-Y transgenic mice were cultured at 2 x 104 cells per well with irradiated spleen cells of female or male C57BL/6 mice in the presence of 0.1 ng/ml of IL-12 (Genzyme) with or without various dose of IL-2 or IL-15. After 4 days, culture supernatants were collected, and IFN- in the supernatants was measured by an ELISA assay. Dotted lines in Fig. 8 (A) show the detection limit (<10 pg/ml).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several subsets of T cells have been shown to respond to IL-2 without TCR stimulation. These include iIEL T cells expressing CD8 and NK-T cells, both of which develop extrathymically (20, 21). iIEL contains self Ag-specific T cells (10, 11, 14, 15). NK-T cells express a limited repertoire of TCR specific for self CD1 molecules (26). Other than male Ag-specific TCR ß T cells in male H-Y transgenic mice, self TL Ag-specific TCR transgenic T cells were also shown to respond to IL-2 without antigenic stimulation (27). From these findings, it can be hypothesized that, in addition to the expression of IL-2Rß-chain, the responsiveness to IL-2 is a common feature of extrathymically developed, self Ag-specific T cells. It has been well known that NK cells are also activated by IL-2 (Ref. 16 ; Fig. 4). NK cells develop extrathymically and express IL-2Rß-chain but not IL-2R-chain, similar to extrathymically developed T cells. It seems possible that NK cells and extrathymically developed T cells are evolutionally of closely related lineage and exert similar functions in vivo. Since IL-2 is produced by conventional activated T cells, extrathymically developed T cells can be activated in vivo at the site where conventional, intrathymically developed T cells proliferate and secrete IL-2 in response to antigenic stimulation. Once activated, extrathymically developed T cells can influence the immune responses by secreting cytokines such as IFN-. Importantly, activation of extrathymically developed T cells is independent of TCR-mediated signaling. Therefore, they may hardly cause autoimmune disease in such situations, although they express self Ag-specific TCR.

It has been well known that anergic T cells restore responsiveness to TCR stimulation after culture with IL-2 (22). However, they can be activated only in the presence of the Ags, which is very different from IL-2-induced TCR-independent activation of extrathymically developed, self Ag-specific T cells. This implies that extrathymically developed T cells are not merely the self Ag-specific anergic T cells escaping clonal deletion.

We also showed that extrathymically developed T cells are activated by IL-15, which also binds IL-2Rß-chain to transduce signals (17). Similar to IL-2, IL-15 stimulates iIEL T cells and NK-T cells (20, 21). Recently, selective stimulation of memory phenotype CD44⁺CD8⁺ T cells by IL-15 was reported (28). It was shown that CD44⁺CD8⁺ T cells in naive mice express IL-2Rß. We and others also showed that IL-2Rß⁺ T cells, including CD8⁺ cells, in naive mice express CD44, conversely (Fig. 1; Ref. 4). Therefore, IL-15-responsive CD44⁺ T cells in naive mice should contain extrathymically developed T cells that correspond to T3.70⁺CD8⁺ cells in male mice. Interestingly, as we showed in our previous report (7), extrathymically developed T cells produced effector cytokines immediately after stimulation with anti-TCR Abs, which is the most important characteristic of memory T cells.

Although IL-2 and IL-15 share many biological activities, one important difference between IL-2 and IL-15 is in their producers: IL-2 is produced by activated T cells, whereas IL-15 is produced by many kinds of cells including professional APCs and even non-bone marrow-derived cells (17). Generally, the stimuli that induce IL-15 production consist of inflammation such as infection (28, 29). It seems possible that IL-15-activated extrathymically developed, self Ag-specific T cells play some protective roles at the site of infection by secreting IFN-, especially in the presence of IL-12, which greatly enhanced IFN- production from them (Fig. 8B). Actually, IL-15-mediated protective roles of iIELs during bacterial infection have been reported (30, 31). CD8⁺IL-2Rß⁺ T cells selectively expanded in vivo after poly(I:C) administration, which mimics viral infection and induces IL-15 production (28). An increase of self-reactive T cells was observed after bacterial infection, although participation of IL-15 was not clear in this case (32). IL-15 was also shown to be involved in the pathogenesis of rheumatoid arthritis or murine models of arthritis (33, 34, 35). It is possible that extrathymically developed T cells play some roles locally in such chronic inflammatory disease.

Recently, several kinds of gene-disrupted mice were generated that commonly showed strikingly decreased numbers of NK cells and extrathymically developed T cells, including CD8 IELs and NK-T cells. The disrupted molecules targeted in these mice were IL-2Rß-chain (20, 36), IFN regulatory factor (IRF)-1 (37, 38), and IL-15R-chain, which constitutes a high affinity receptor for IL-15 together with IL-2Rß-chain and common -chain (39). All of these molecules participate in IL-15-mediated signaling, and, thus, the IL-15/IL-15R pathway plays an important role in the normal development or maintenance of these cells. This is supported from our finding that IL-15 has an antiapoptotic effect and proliferation-inducing effect on extrathymically developed T cells, which can participate in in vivo maintenance of these cells. We also found that extrathymically developed, self Ag-specific T cells selectively express IL-15R-chain mRNA, whereas intrathymically developed T cells never express it (our unpublished data). Expression of IL-15-chain mRNA in TCR iIEL cells was also reported (19). However, since we could not obtain appropriate mAbs to perform flow cytometer analysis of IL-15R expression, it is still unclear whether all of these cells or only a part of them express high affinity IL-15R, with the rest simply expressing low-affinity IL-2R.

Until now, we could not detect Ag-specific functions of extrathymically developed T cells that express self-specific TCR, even in the presence of IL-2R-mediated signaling. Their self reactivity and potentials to cause autoimmune disease are still in question. It is important to further address the conditions in which the extrathymically developed T cells would acquire self reactivity. More detailed studies to clarify the roles of TCR-mediated signaling in the functions of extrathymically developed T cells are also required.

	Acknowledgments

 
We thank T. W. Mak for providing us with H-Y transgenic mice and hybridoma T3.70. The English used in this manuscript was revised by Miss K. Miller (Royal English Language Centre, Fukuoka, Japan).

	Footnotes

 
¹ This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.

² Address correspondence and reprint requests to Dr. Hisakata Yamada, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address:

³ Abbreviations used in this paper: H-Y transgenic mice, mice transgenic for male H-Y Ag-specific TCR; CD3^int, intermediate levels of CD3; iIEL, intestinal intraepithelial lymphocytes; PI, propidium iodide.

Received for publication August 9, 1999. Accepted for publication December 1, 1999.

	References

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