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Double-Negative T Regulatory Cells Can Develop Outside the Thymus and Do Not Mature from CD8⁺ T Cell Precursors¹

Megan S. Ford^2,*, Zhu-Xu Zhang^2,3,*, Wenhao Chen^* and Li Zhang^4,*,

^* Multiorgan Transplantation Program, Toronto General Research Institute, University Health Network, and Departments of Laboratory Medicine and Pathobiology, and Immunology, University of Toronto, Toronto, Ontario, Canada

	Abstract

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Recent studies have demonstrated that activated peripheral TCR⁺CD3⁺CD4^–CD8^–NK1.1^– (double-negative, DN) regulatory T cells (Tregs) from both mice and humans are able to down-regulate immune responses in vitro and in vivo. However, the origin and developmental requirements of functional DN Tregs remain unclear. In this study, we investigated the requirement for CD8 expression as well as the presence of a thymus for the development of functional DN Tregs. We demonstrate that DN Tregs exist in CD8-deficient mice and that stimulation of CD8⁺ T cells in vivo with TCR-specific Ag does not convert CD8⁺ T cells into DN Tregs. In addition, we found that DN T cells are present in the spleens and lymph nodes of thymectomized mice that are irradiated and reconstituted with T cell-depleted bone marrow cells. Interestingly, DN Tregs that develop in thymectomized mice can suppress syngeneic CD8⁺ T cells more effectively than those that develop in sham-thymectomized mice. Taken together, our data suggest that DN Tregs are not derived from CD8⁺ T cell precursors and that functional DN Tregs may preferentially develop outside of the thymus. These data suggest that DN Tregs may represent a developmentally and functionally unique cell population.

	Introduction

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Regulatory T cells (Tregs)⁵ have been demonstrated to play a very important role in controlling the development of autoimmune diseases, transplant rejection, malignancy, and infectious diseases (1, 2, 3). Several different regulatory cell populations have been identified, including the naturally occurring and induced populations of CD4⁺CD25⁺ Tregs, Tr1 cells, Th3 cells, NK T cells, CD8⁺ T cells, TCR⁺ cells, and TCR⁺CD4^–CD8^–NK1.1^– Tregs (1, 2, 3, 4, 5, 6, 7). Tregs are considered a natural and essential feature for the development of normal self-tolerance and acquired immunity (1). Therefore, investigating the origins and developmental pathways of Tregs is important because it may lead to the identification of novel therapies for the induction, activation, or inhibition of Treg function.

The majority of mature TCR⁺-T cells in normal mice and humans express either the CD4 or CD8 coreceptor molecules. However, 1–5% of the peripheral TCR⁺ T cell population expresses CD3, but neither the CD4 nor the CD8 coreceptor. Thus, they are termed CD4 and CD8 double-negative (DN) T cells. Based on the expression of NK cell markers, DN T cells can be divided further into two subpopulations: NK⁺-DN T cells referred to as NKT cells (8, 9) and NK^–-DN T cells referred to as DN Tregs (3, 10, 11). DN Tregs, first characterized in mice in 2000 (10) and in humans in 2005 (11), express a unique cell surface and cytokine profile and are able to suppress syngeneic antidonor CD4⁺ and CD8⁺ T cells in an Ag-specific fashion both in vitro and in vivo (10, 11, 12, 13). Murine DN Tregs have been shown to expand in the spleen and lymph nodes upon encountering Ags. Activated DN Tregs can specifically inhibit immune responses, at least partially through killing syngeneic CD8⁺ and CD4⁺ T cells that are activated by the same Ags as those used to activate the DN Tregs (11, 12, 14). Furthermore, when adoptively transferred into syngeneic mice, DN Tregs can prolong both allo- and xenograft survival compared with untreated controls (10, 12, 15, 16). In addition, DN Tregs have been shown to play a role in the regulation of autoimmune diseases (12, 17), inhibition of graft-vs-host disease (18), and the control of infectious diseases (19, 20). Human DN Tregs have also been shown to become activated upon encountering Ags and suppress the proliferation of syngeneic CD8⁺ T cells (11). These studies clearly demonstrate the immune regulatory function of DN Tregs and suggest that they may be used therapeutically to control human immune disorders.

It is known that NKT cells mature through the thymus and are positively selected by interactions with the MHC-like molecule CD1 (9). However, the location and the requirements for the maturation of DN Tregs remain unclear. Although a significant portion of T cells in the thymus possess the DN T cell phenotype, whether any of them go on to become Tregs in the lymphatic periphery is not known. Because DN Tregs are defined by their lack of TCR coreceptor expression, this has led to the hypothesis that they may develop from either CD4⁺ or CD8⁺ precursors and thus may also require the thymus at least for their initial development (21, 22, 23, 24, 25, 26, 27). Encountering Ags in vitro (28, 29) or in vivo (30, 31) has been shown to decrease CD8⁺ and increase DN T cell numbers, suggesting that DN T cells may develop from CD8⁺ T cell precursors. However, DN T cell numbers may also increase due to direct activation and expansion of a pre-existing population of DN T cells. Furthermore, studies have also suggested that some DN T cell populations may develop extrathymically in the appendix (32), female genital tract (20), nasal-associated lymphoid tissue (NALT) (33), or the liver (34, 35). Given the significant roles that DN Tregs play in various disease models, further characterization of their developmental pathways may facilitate expansion or reduction of DN Tregs and therefore better control of pathological immune responses.

In this study, we have addressed the question of whether DN Tregs mature from CD8⁺ T cell precursors or represent a separate T cell lineage. We also examined the role of CD8-molecule expression in the development of DN Tregs. Lastly, we investigated the requirement of the thymus in the maturation of functional DN Tregs. Our results indicate that DN Tregs do not develop from CD8⁺ T cell precursors, nor is CD8 expression required for their development in vivo. Furthermore, we found that functional DN Tregs develop outside the thymus. These results suggest that DN Tregs may mature from a unique and previously uncharacterized lineage of T cells.

	Materials and Methods

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Mice

C57BL/6 (B6, H-2^b), BALB/c (H-2^d), BALB/c-H-2-dm2 (dm2, a BALB/c L^d loss mutant), B6.C-H-2^bm1 (MHC class I-D^b, K^bm1), B6 CD8^–/– and BALB/c, and CD8^–/– and C57BL/6-Tg(UBC-GFP)30Scha/J mice were purchased from The Jackson Laboratory. B62C transgenic (Tg) mice breeders were provided by Dr. D. Y. Loh (Nippon Roche Research Center, Kamakura, Japan). B62C mice express a Tg TCR reactive against MHC-class I-L^d molecules (10), which can be detected by the clonotypic mAb, 1B2. B62C Tg mice were bred with dm2 (D^d, K^d, and L⁰) mice to produce the (2C x dm2)F₁ mice (H-2^b/d, L⁰, and anti-L^d TCR⁺) that were used in this study. All mice were maintained in the animal facilities at the University Health Network.

Cell surface marker staining

1B2 mAb was produced in our laboratory from the 1B2 hybridoma that was provided by Dr. H. Eilson (Massachusetts Institute of Technology, Cambridge, MA). Primary and activated T cells were stained with fluorescence-conjugated mAbs specifically recognizing the CD3, CD4, CD8, TCR-, NK1.1, and the pan-NK marker DX5 (eBioscience). Data were acquired and analyzed on an EPICS XL-MCL flow cytometer (Coulter).

Isolation of CD8⁺ and DN T cells

Spleen and lymph node cells were obtained from naive (2C x dm2)F₁, B6, or B6 CD8^–/– mice. DN T cells were isolated by depletion of RBC, followed by passage through nylon wool columns to enrich the T cell population. The cells were then treated with depleting mAbs specific for murine CD4 (RL172-4, rat IgM) and CD8 (3.168.8, rat IgM) at 4°C for 45 min, followed by the addition of rabbit complement (Cedarlane Laboratories) at 37°C for 45 min. CD8⁺ T cells were purified by staining with anti-CD8-biotin on ice for 30 min, followed by incubation with streptavidin-MACS microbeads (Miltenyi Biotec). Cells were then eluted from the positive fraction using an MS magnetic column (Miltenyi Biotec). The viability and purity of T cells was monitored by flow cytometry, and was >96%.

In vivo and in vitro activation of CD8⁺ and DN T cells

To assess whether DN Tregs are generated from the in vivo activation of CD8⁺ T cells, 2 x 10⁶ 1B2⁺CD8⁺ T cells were purified from (2C x dm2)F₁ mice and were i.v. injected to sublethally irradiated (700 rad) (B6 x BALB/c)F₁ CD8^–/– mice. The presence of CD8⁺ or DN T cells was monitored at different time points in various tissues by staining with mAbs specific for the 1B2-TCR and CD8, followed by flow cytometry analysis.

B6-CD8^+/+ or CD8^–/– mice were either infused with 4 x 10⁷ viable spleen cells collected from B6^bm1 mice or left untreated as controls. One week later, lymphocytes from different tissues were stained with mAbs. DN T cells from the spleen and lymph nodes were cultured in vitro with irradiated alloantigen (B6^bm1), 50 U/ml IL-2, and 30 U/ml IL-4, before use as effector cells in cytotoxicity assays.

Cytotoxicity assays

Target cell death resulting from coculture with DN Tregs was measured as reported previously (10, 12). Briefly, DN T cells were stimulated with irradiated (2000 rad) allogeneic splenocytes for 4–8 days in the presence of IL-2 (50 U/ml) and IL-4 (30 U/ml). Viable DN T cells were used as effector cells. Syngeneic CD8⁺ T cells were stimulated with the same type of irradiated allogeneic splenocytes in vitro for 4 days in the presence of IL-2 (50 U/ml), labeled with 10 µCi/ml [³H]TdR at 37°C overnight, washed three times with -MEM, and used as targets. After coculture with the effector cells at 37°C for 18 h in the presence of fresh irradiated allogeneic splenocytes, the cells were harvested and counted in a TopCount beta scintillation counter (Packard). Specific cell lysis was calculated using the following equation: percentage of specific killing = (S – E)/S x 100, where E (experimental) is cpm of retained DNA in the presence of effector cells, and S (spontaneous) is cpm of retained DNA in the absence of effector cells.

Suppression assays

Spleen and lymph node tissues were isolated from irradiated/reconstituted thymectomized and sham-thymectomized mice and passed through wire-mesh screens to create single-cell suspensions in -MEM plus 10% FBS plus 0.2% 2-ME. Flow cytometry was performed to confirm that >98% of the lymphocytes expressed the GFP transgene, indicating that the bone marrow reconstitution was complete. DN Tregs were isolated as described above and used as putative effector cells by plating in serial dilutions in 96-well, round-bottom plates either alone, or in the presence of 10⁴ naive syngeneic T cell responders. Cultures with suppressors or responders alone were used as controls. Each well was given 10⁵ irradiated (2000 rad) allogeneic splenocytes (H-2^b/d) as stimulators, as well as 50 U/ml IL-2 and 30 U/ml IL-4. Cells were incubated at 37°C in 5% CO₂ for 3.5 days. Each well was then given 1 µCi of [³H]TdR. Eighteen hours later, cells were harvested and counted in a TopCount beta scintillation counter (Packard).

Bone marrow transplantation

Eight-week-old B6 mice were surgically thymectomized or sham thymectomized 3 wk before bone marrow transplantation. No lymphocytes were found in any of the residual tissues around the thymic area in the thymectomized mice at the time of sacrifice. Bone marrow cells were obtained by flushing the femurs and tibia of sex-matched B62C or B6GFP mice with cold (4°C) 1% FBS/PBS. T cells were depleted by staining with depleting anti-Thy1.2 mAb (Cedarlane Laboratories), along with anti-CD4 (RL172-4, rat IgM) and anti-CD8 (3.168, rat IgM), followed by incubation with low-tox rabbit complement as before. The absence of T cells was confirmed before the transfer of bone marrow cells by staining with anti-CD3 and anti-Thy1.2 mAb and flow cytometry analysis. A total of 5 x 10⁶ T cell-depleted bone marrow cells was i.v. injected into sublethally irradiated (700 rad) thymectomized or sham-thymectomized B6 mice. The complete reconstitution of recipient mice was confirmed at the time of sacrifice by flow cytometry analysis for the presence of the 2C or GFP transgene on the lymphocytes and was consistently >98%.

	Results

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Encountering Ag in vivo does not convert CD8⁺ T cells into DN T cells

Previous data has shown that CD4⁺CD25^– T cells can convert into CD4⁺CD25⁺ Tregs after encountering Ag in the periphery (36). Similarly, stimulation with alloantigen has been shown to induce clonal expansion, followed by contraction of CD8⁺ T cells that is sometimes concomitant with an increase in DN T cells in the peripheral lymphoid organs (12, 15, 18, 30, 37). In this study, we attempted to determine whether the origin of the increased DN T cell population was from CD8⁺ T cells that had decreased their expression of CD8 molecules or whether they proliferated from a precursor DN T cell population. Naive MHC class I-L^d-specific 1B2⁺CD8⁺ T cells were purified from (B62C x dm2)F₁ (H-2^b/d, L^d–) mice and injected into sublethally irradiated L^d+ CD8^–/– (B6 x BALB/c)F₁ (H-2^b/d, L^d+) mice. In this setting, recipients lack CD8⁺ T cells and do not express the 1B2⁺ TCR; therefore, any 1B2⁺DN T cells found in the recipient mice must arise from the infused allogeneic 1B2⁺CD8⁺ T cells. Mice were sacrificed at various time points, and the presence and phenotype of the infused 1B2⁺ cells in different lymphoid organs were monitored by flow cytometry. As seen previously (13, 37), the number of 1B2⁺CD8⁺ cells increased in the lymph nodes 4 days after adoptive transfer and reduced thereafter (Fig. 1). 1B2⁺CD8⁺ T cells were barely detectable at 14 days following the infusion. Importantly, the number of 1B2⁺ DN T cells did not increase above background levels at any of the time points studied (Fig. 1). In addition, no 1B2⁺DN T cells were found in the blood, spleen, or liver of mice that had been infused with 1B2⁺CD8⁺ T cells. These data indicate that it is unlikely that CD8⁺ T cells can develop into DN T cells following alloantigen stimulation in vivo.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Ag-specific CD8+ T cells do not down-regulate CD8 expression and become DN T cells after encountering Ag in vivo. Purified 1B2+CD8+ cells from (B62C x dm2)F1 mice (left panel) were injected into sublethally (700 rad) irradiated CD8–/– (B6 x BALB/c)F1 (H-2b/d, Ld+) mice. Mice were sacrificed, and cells were collected from the lymph nodes at days 0, 4, 8, 14, and 60 and stained with 1B2 and CD8 Abs. The percentage of 1B2+CD8+ cells (CD8, box P) and 1B2+CD8– (DN, box Q) cells at each time point is shown. Spleen cells from each mouse were also stained and similar results were obtained (data not shown). The data represent staining from three mice sacrificed at each time point.

We next addressed the question of whether CD8 expression is required for the development of functional DN Tregs. First, we studied whether DN T cells exist in the peripheral lymphoid organs of CD8 gene-deficient mice. The lymph nodes and spleens of naive wild-type as well as CD8 gene-deficient B6 and BALB/c mice were stained with mAbs against CD3, CD4, and CD8, and the percentage of DN T cells was compared. As shown in Fig. 2a, both B6 and BALB/c mouse strains showed a relatively equivalent proportion of DN T cells in the spleens of CD8^–/– and CD8^+/+ mice. However, in the lymph nodes, the percentage of DN T cells is higher in B6 CD8^–/– mice compared with wild-type B6 mice (Fig. 2a).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. DN T cells are present in CD8–/– mice. a, Lymphocytes from the lymph nodes and spleens of naive wild-type () as well as CD8–/– () B6 and BALB/c mice were stained with anti-mouse TCR-, CD4, and CD8 mAbs and analyzed by flow cytometry. Cells were gated on the TCR-+ population, and the percentage of DN cells within the TCR-+ population from the spleens and lymph nodes of B6CD8+/+ (n = 9) and CD8–/– mice is shown (n = 9). b, Spleen and lymph node cells from B6 CD8+/+ (top panels) and B6 CD8–/– (bottom panels) cells were triple stained with anti-mouse CD3, CD4, CD8, and NK1.1 mAbs. Data shown are gated on the CD3+ T cell population and represent the results of nine mice.

DN Tregs from both CD8^–/– and wild-type mice can be activated and expanded in vivo

Donor lymphocyte infusion (DLI) has been shown to expand DN Tregs in CD8 wild-type animals. DLI-activated DN Tregs can inhibit T cell proliferation in vitro and can inhibit graft rejection when adoptively transferred into recipient mice in an Ag-specific fashion in vivo (10, 12, 38). To determine whether DN Tregs can also expand in response to DLI in the absence of CD8⁺ cells, allogeneic lymphocytes were prepared from the spleens of one MHC class I-mismatched B6^bm1 mice and infused into B6 CD8^–/– or B6 CD8^+/+ control mice. Seven days later, lymphocytes from the thymus, spleen, lymph nodes, and blood of DLI-treated and untreated control mice were stained using mAbs specific for TCR-, CD4, CD8, and NK1.1. At this time point, donor bm1 cells had been eliminated from the periphery because CFSE-labeled donor cells could not be found in the spleen or lymph nodes of recipient mice (data not shown). However, we found that the percentage of TCR⁺NK^– DN T cells significantly increased following DLI in both CD8^+/+ and CD8^–/– mice (Fig. 3a, p < 0.002 and 0.005, respectively).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. DN T cells that have developed in CD8–/– mice have regulatory function. a, Spleen cells from B6bm1 mice were infused into B6 CD8–/– mice (n = 5) and wild-type littermates (n = 9). Seven days later, lymphocytes from the spleens were stained using mAbs that recognize TCR-, CD4, CD8, and NK1.1. The percentage of NK– DN T cells was compared between CD8+/+ and CD8–/– mice treated with B6bm1 cells () and untreated naive mice (). Similar patterns were observed when the thymi, lymph nodes, and blood samples were analyzed (data not shown). b, DN T cells were purified and cultured for 4 days as described in Materials and Methods, then stained with mAbs specific for the TCR, CD4, CD8, and NK1.1. Representative staining is shown. c, DN T cells from either B6bm1 DLI-treated B6 CD8+/+ () or B6 CD8–/– () mice were cultured in vitro for 4 days, then used as effector cells in cytotoxicity assays. The result represents two independent experiments each with five replicates.

DN T cells can develop extrathymically

The thymus has been shown to be important for the development of both CD4⁺CD25⁺ (39) and NK Tregs (9). To determine whether the thymus plays a role in the development of DN Tregs, we conducted reconstitution studies. Groups of normal B6 and thymectomized B6 mice were sublethally irradiated and reconstituted with T cell-depleted bone marrow cells from B62C mice, which express the anti-L^d Tg (1B2⁺) TCR. Fifty days following bone marrow reconstitution, the donor-derived 1B2⁺CD8⁺ and 1B2⁺DN T cells in the spleen and lymph nodes of the recipients were monitored by staining lymphocytes with the 1B2 mAb in combination with CD4 and CD8 mAbs. The percentage of 1B2⁺DN and 1B2⁺CD8 T cells that had accumulated in the spleens of nonthymectomized mice was similar to that found previously in bone marrow donor B62C mice (40), indicating that reconstitution had occurred (Fig. 4a). As expected, CD8⁺ T cells were reduced in the spleen from 36.7% in nonthymectomized mice to 3.1% of the T cell population in thymectomized mice, demonstrating the successful removal of the thymus (Fig. 4a). Likewise, the total number of CD8⁺ T cells in the spleen was also reduced in the thymectomized compared with nonthymectomized mice (Fig. 4b, left panel). Interestingly, DN T cells comprised over 90% of the splenic 1B2⁺ T cells in thymectomized mice and over 60% of the splenic 1B2⁺ T cells in nonthymectomized mice (Fig. 4a). Despite the higher percentage of DN T cells in thymectomized mice, the total number of DN T cells was similar in the spleens of thymectomized and nonthymectomized mice (Fig. 4b, right panel).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. DN T cells develop in the absence of a thymus. T cell-depleted B62C (a and b) or B6GFP (c) bone marrow cells were i.v. injected into sublethally irradiated B6 mice that had been thymectomized or sham-thymectomized as a control. a and b, Spleen cells were stained with anti-1B2-TCR, CD4, and CD8 mAbs 50 days after reconstitution. a, Representative flow plot showing splenic T cells in B6 mice reconstituted with 2C bone marrow in the presence (left panel) or absence (right panel) of a thymus. Cells are gated on the 1B2-TCR+ population. b, Total numbers of splenic 1B2+CD8+ (left panel) or 1B2+DN (right panel) T cells that develop in the presence () or absence () of a thymus at 50 days following bone marrow reconstitution. c, GFP+ spleen cells were stained with anti-TCR-, -CD4, -CD8, and -NK1.1 mAbs 50 days after the reconstitution. Total numbers of splenic CD4+ and CD8+ (left panel) or DN (right panel) T cells that develop in the presence (, n = 5) or absence (, n = 4) of a thymus at 50 days following bone marrow reconstitution. Value of p = 0.0003 for the number of CD4+ and CD8+ T cells within the spleens of sham vs thymectomized mice.

Extrathymically derived DN T cells possess enhanced regulatory function

Both murine and human DN Tregs can suppress immune responses mediated by syngeneic T cells that are activated by the same Ags as those that activate the DN Tregs (11, 14). To further study the relative ability of DN T cells that had developed in the presence or absence of a thymus to regulate immune responses, GFP⁺DN T cells were purified from the spleens and lymph nodes of mice that had been thymectomized or sham-thymectomized, then B6GFP bone marrow-reconstituted. DN T cells were then used as putative suppressor cells for naive syngeneic T cell proliferation toward alloantigen stimulation. We found that DN T cells that had developed in either the presence or absence of a thymus could suppress syngeneic T cell proliferation in a dose-dependent fashion (Fig. 5a). Interestingly, the DN T cells that had developed in the absence of a thymus had a more potent ability to suppress T cell proliferation than those that had developed in the presence of a thymus.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Extrathymically developed DN Tregs have enhanced ability to suppress and kill syngeneic T cells compared with those that have developed in the presence of a thymus. DN T cells that had developed in thymectomized or sham-thymectomized mice were isolated from the spleens and lymph nodes at 50 days following B6GFP (a) or B62C (b) bone marrow reconstitution. a, B6GFP+ DN T cells from either thymectomized () or sham-thymectomized () mice were incubated either alone or in the presence of 104 CD8+ T cells from naive B6 mice. CD8+ T cells were also plated alone as a control (). Each well received 105 irradiated allogeneic splenocytes (H-2b/d), 50 U/ml IL-2, and 30 U/ml IL-4. Cell proliferation was measured on day 4 after pulsing cells with [3H]TdR for 18 h. b, B62C (H-2b) DN T cells from either thymectomized (hatched line) or sham-thymectomized (solid lines) mice were activated for 4 days with irradiated allogeneic splenocytes (H-2b/d), 50 U/ml IL-2, and 30 U/ml IL-4. Viable DN T cells were used as effector cells and cocultured with [3H]TdR-labeled syngeneic CD8 T cells that were activated with Ld+ alloantigen at the indicated ratios. Percentage of specific lysis was determined 18 h later. The data shown represent the results of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Recent studies into the developmental requirements of Tregs have been focused largely on the CD4⁺CD25⁺ Treg subset. The development of naturally occurring CD4⁺CD25⁺ Tregs, which have been shown to be potent inhibitors of autoimmune diseases, requires thymic maturation (7, 39, 42). Neonatal thymectomy significantly reduces the number of CD4⁺CD25⁺ Tregs and promotes the development of autoimmune diseases (43). In addition, data have shown that some CD4⁺CD25⁺ Tregs undergo a positive selection process in the thymus that involves interaction with self-peptides presented in the context of MHC class II (44, 45, 46). On the other hand, studies have also shown that adult thymectomy does not prevent the development of CD4⁺CD25⁺ Tregs from CD4⁺CD25^– cells (47). This suggests that, although the thymus may be required for the initial development of some CD4⁺CD25⁺ Tregs, the gain of Treg function can also happen in the absence of the thymus. In addition to CD4⁺CD25⁺ Tregs, several other subtypes of Tregs have also been shown to play an important role in regulating immune responses (1, 3, 4, 5). However, there have been conflicting data regarding the requirement for the thymus during their development. Recently, several studies have demonstrated that DN Tregs from both mice and humans can inhibit immune responses in an Ag-specific fashion (3, 10, 11, 12, 48), indicating that DN Tregs represent another important subset of Tregs. Similar to naturally occurring CD4⁺CD25⁺ Tregs, DN Tregs exist in naive animals and can readily suppress immune responses both in vitro (14, 17) and in vivo (10, 12, 16) upon activation with alloantigens. Whereas the ontogeny and maturation pathways for NK⁺ DN T cells have been well studied, the origin and developmental requirements for functional NK^– DN Tregs remain elusive.

Several reports have shown that after stimulation of splenocytes with Ags in vitro (28, 29) or injection of Ags into TCR Tg mice (14, 18, 30, 31, 37), there was a decrease of CD8⁺ T cells concomitant with an increase of DN T cells. Therefore, this suggested that DN T cells may be derived from activated CD8⁺ T cells as a consequence of down-regulation of CD8 during immune responses (30). However, in the previous experiments, both the responder cells used in cultures as well as the TCR Tg mice that received Ag stimulation in vivo contained a pre-existing population of DN T cells. Thus, it is possible that the increased number of DN T cells in those studies was due to direct activation and expansion of pre-existing DN T cells. To distinguish these two possibilities and directly address the question of whether CD8⁺ T cells can covert to DN Tregs, instead of using a mixed whole splenic population, we used purified Ag-specific CD8⁺ T cells in this study. We found neither a down-regulation of CD8 expression nor an increase in the number of Ag-specific DN T cells when purified L^d-specific CD8⁺ T cells were stimulated in vitro with L^d+ allogeneic cells (data not shown) or adoptively transferred into alloantigen-expressing recipients (Fig. 1). Our findings are consistent with some reports from others that demonstrate that CD8⁺ T cells do not convert to DN T cell after Ag stimulation (49, 50). These data suggest that Ag exposure does not cause down-regulation of CD8 expression but rather the increase in DN T cells is the result of direct activation and proliferation of a pre-existing DN T cell population. This notion is further supported by the finding that CD8 RNA was undetectable by either northern blot or RT-PCR analysis in DN Tregs that have been cloned from allotransplant-tolerant mice (10).

In addition, our study demonstrates that the percentage and total number of DN T cells does not differ significantly between wild-type and CD8a^–/– mice (Fig. 2). This suggests that CD8 expression is not required for the development of DN T cells in the peripheral lymphoid organs. Furthermore, since the DN T cells from CD8^–/– mice can suppress allogeneic immune responses as effectively as those obtained from wild-type mice (Fig. 3), it suggests that the regulatory subset of DN T cells do not develop from CD8⁺ T cell precursors.

Although the thymus has been shown to support the maturation of natural occurring CD4⁺CD25⁺ Tregs (39) and NKT cells (9), whether this organ is also required for the development of DN Tregs has not been investigated previously. We found that DN Tregs could develop in sublethally irradiated thymectomized B6 mice following reconstitution with bone marrow cells from both B62C as well as B6GFP mice (Fig. 4). These results clearly demonstrate that the development of at least some subsets of DN Tregs does not require the thymus. Whereas the total number of splenic DN T cells reached a similar level in both thymectomized and nonthymectomized mice after reconstitution with 2C TCR Tg bone marrow (Fig. 4b), there was a reduction in DN T cell number in thymectomized compared with nonthymectomized recipients that were reconstituted with bone marrow cells from B6GFP mice (Fig. 4c). This discrepancy could be due to Tg expression of the TCR inducing aberrant development of DN T cells within nonthymic tissues. The fact that the number of DN T cells within the lymphatic tissues of 2C mice is significantly higher than that found in B6 mice suggests that 2C TCR expression may preferentially promote the development of DN T cells. Importantly, although mice reconstituted with 2C bone marrow had increased overall development of DN T cells, the 1B2⁺DN T cells showed similar functional characteristics to GFP⁺DN T cells. Both 1B2⁺DN T cells and GFP⁺DN T cells that had developed in the absence of a thymus could inhibit immune responses to a greater extent than those that had developed in the presence of a thymus (Fig. 5). This finding suggests the possibility that the nonregulatory DN T cell subset develops and matures through the thymus, whereas regulatory DN T cells may preferentially develop outside the thymus. Further studies are needed to identify cellular markers to differentiate between regulatory DN T cells and nonregulatory DN T cells.

Currently, the specific location for the maturation of DN Tregs remains unclear. Studies have suggested that some DN T cells may develop in the bone marrow (51, 52, 53), appendix (32), female genital tract (20), NALT (33) and liver (34, 35). In addition, the levels of DN T cells in the female genital tract or the NALT of nude mice was found to be similar to that found in wild-type mice, indicating that development of DN T cells within these tissues does not require the thymus (20, 33, 51). Strober’s group (54) has also shown that precursors to functional CD4⁺ and CD8⁺ T cells are found in the bone marrow, not the thymus, and that functional T cells could develop from these precursors in athymic nude mice. Similarly, Blais et al. (55) showed that development of CD4⁺ and CD8⁺ T cells could occur in the lymph node of nude mice. However, although these extrathymically developed T cells could recognize and expand in response to viral Ag, they were unable to clear the infection at the dose where thymically developed T cells were effective at clearing the virus (55). In the present study, we found that the DN T cells obtained from thymectomized mice are more potent in both suppression and killing of syngeneic CD8⁺ T cells when compared with the DN T cells obtained from nonthymectmized mice (Fig. 5). These findings suggest that T cells that develop extrathymically may have different functions from those that develop through the thymus.

Taken together, our data demonstrate that peripheral DN Tregs are not derived from CD8⁺ T cells as a consequence of down-regulation of CD8 upon Ag stimulation and do not require CD8 expression to develop in vivo. In addition, we show that the development of functional DN Tregs does not require the thymus, indicating that selection events that occur in the thymus are not essential for the development of DN Tregs. These data suggest that regulatory DN T cells may represent a unique cell lineage that can develop extrathymically through a pathway that is independent of CD8 expression. Further investigation into the molecular mechanisms involved in the extrathymic selection and maturation of DN Tregs in vivo may provide the means to manipulate their functions and improve immunotherapy.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work is supported by Canadian Institutes of Health Research Grant MOP 14431 and National Cancer Institute of Canada Grant 15067 (to L.Z.). Additional funding is provided by Wyeth-Ayerst Canada.

² M.S.F. and Z.-X.Z. contributed equally to this paper.

³ Current address: Department of Surgery, University of Western Ontario, London, Ontario, Canada.

⁴ Address correspondence and reprint requests to Dr. Li Zhang, Toronto General Research Institute, University Health Network, 101 College Street, TMDT 2-807, Toronto, Ontario M5G 1L7, Canada. E-mail address: lzhang{at}uhnres.utoronto.ca

⁵ Abbreviations used in this paper: Treg, regulatory T cell; DN, double negative; NALT, nasal-associated lymphoid tissue; Tg, transgenic; DLI, donor lymphocyte infusion.

Received for publication December 29, 2005. Accepted for publication June 6, 2006.

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