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IL-2-Activated CD8⁺CD44^high Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells ¹

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

	Abstract

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CD8⁺ T cells depend on the TCR for Ag recognition and function. However, Ag-activated CD8⁺ T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8⁺ T cells expressing high levels of CD44 (CD8⁺CD44^high cells) from normal mice. These cells are distinct from conventional memory CD8⁺ T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8⁺ T cells, IL-2-activated CD8⁺CD44^high cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8⁺CD44^high cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.

	Introduction

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The cells of the adaptive immune system utilize functionally rearranged receptors to recognize foreign Ags. By contrast, cells of the innate immune system primarily use germline-encoded receptors to defend against infected or transformed cells. Interestingly, cells of the adaptive immune system can express some of these germline-encoded receptors (1). Furthermore, recent studies have indicated that in addition to providing immediate immune effector mechanisms to contain the spread of infections, the innate immune system also serves to prime the adaptive immune system to defend against infections that cannot be contained by the innate immune system (1).

NK cells are an integral part of the innate immune system, producing cytokines to activate other cells of the immune system as well as directly recognizing and killing infected or transformed cells (2). NK cells use a combination of activating and inhibitory receptors to perform these functions (3, 4, 5). These activating and inhibitory receptors set a threshold for the activation of NK cells, with inhibitory signals predominating in the absence of infection (6). When a host cell is transformed or infected, the balance shifts toward activation, allowing NK cells to eliminate these hazardous host cells (6).

TCR⁺CD8⁺ T cells play a crucial role in the adaptive immune system. The primary function of CD8⁺ T cells is the lysis of virally infected target cells via recognition of viral peptides that are presented by MHC class I molecules. Naive CD8⁺ T cells require two distinct signals for activation: signal one is provided by engagement of the TCR with its cognate ligand and signal two is provided by interaction of costimulatory receptors with their respective ligands on the APCs (7, 8).

Recent studies showed that normal mice possess a subset of CD8⁺ TCR⁺ T cells that express very high levels of CD44 (herein referred to as CD8⁺CD44^high cells). These CD8⁺CD44^high cells possess other markers of activated/memory cells such as the high expression of CD122 (IL-2R) and Ly6C and they can proliferate in response to IL-2 and IL-15 independently of TCR stimulation (9, 10, 11). The development of these CD8⁺ T cells appears to be driven by the interaction of the TCR with self-Ag and they can develop in the absence of a functional thymus (9).

CD8⁺CD44^high cells have also been shown to express receptors characteristic of NK cells (12). Both activating and inhibitory NK receptors can be found on these cells. Receptors such as 2B4, which can be either activating or inhibitory (13) and whose expression on T cells can be induced by activation with various cytokines (14), have been found on CD8⁺CD44^high cells (14). Inhibitory receptors such as the killer cell Ig-like receptor in humans and the lectin-like Ly49 family in mice have also been implicated in both the development and function of memory phenotype CD8 T cells (reviewed in Ref. 15). The observation that NK receptor expression by CD8 T cells is restricted to cells with an activated/memory phenotype and the fact that T cells do not express NK receptors in the thymus (16) strongly suggests that only cells that have encountered cognate Ag are capable of their expression.

In this report, we characterized the phenotype and function of CD8⁺CD44^high cells from normal mice. We showed that these cells only require IL-2 for proliferation and the acquisition of cytolytic activity. Interestingly, these IL-2-activated cells express both DAP10 and DAP12 adaptor molecules. DAP12 is normally expressed by activated NK, but not conventional CD8⁺ T cells (17). The activated cells demonstrate preferential killing of syngeneic tumor cells. Expression of the NKG2D ligand Rae-1 on the tumor cells led to greatly enhanced killing of the target cells.

	Materials and Methods

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Mice

Breeders for C57BL/6 (B6), BALB/c, and DBA/2 were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice were bred at the Animal Unit in our department. Mice 8–12 wk of age were used for the experiments described.

Abs and flow cytometry

The following mAbs were used: anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD8 (53.58), anti-CD3 (2C11), anti-CD44 (PGP1), anti-TCR (H57.597), anti- TCR, anti-CD43 (1B11), anti-CD94 (18D3), anti-NK-1.1 (PK136), anti-CD122 (TM-1), anti-Ly6C (AL-21), anti-CD244.2 (2B4), and anti-NKG2D (C7; Ref. 18). Biotinylated mAbs were detected using streptavidin-PE. All Abs were obtained from BD PharMingen (San Diego, CA) except anti-NKG2D (18), which was a kind gift from Dr. W. M. Yokoyama (Howard Hughes Medical Institute, Washington University, St. Louis, MO). Staining for NKG2D ligands was performed using the mNKG2D-Ig fusion protein (19), which was a kind gift from Dr. L. L. Lanier (University of California, San Francisco, CA) followed by staining with a FITC-labeled anti-human Ig mAb. Cell staining and flow cytometry were performed according to standard procedures. The CellQuest software program (BD Biosciences, Mountain View, CA) was used for data acquisition and analysis. For three-color analysis, a total of 20,000 live events were collected and analyzed.

Cell lines

Cell lines used were the RMA lymphoma (H-2^b+, Rae-1^-), RMA-Rae-1 transfectant (H-2^b+, Rae-1⁺), TAP-deficient RMAS (H-2^{b -} Rae-1^-), RMAS-Rae-1 transfectant (H-2^{b -} Rae-1⁺), A20 lymphoma (H-2^d), and P815 mastocytoma (H-2^d). The cell lines were cultured in IMEM (Life Technologies, Burlington, Canada), supplemented with 10% (v/v) FBS (Life Technologies, Grand Island, NY), 5 x 10⁵ µM 2-ME, and antibiotics (I-medium). The RMA-Rae-1 and RMAS-Rae-1 transfectants (20) were kind gifts from Dr. L. L. Lanier (University of California, San Francisco); these cell lines were passaged in I-medium and G418 (800 µg/ml) to maintain high levels of Rae-1 expression.

Ex vivo staining

Single-cell suspensions from the lymph nodes of mice were treated with anti-CD4 mAb and then depleted of CD4⁺Ig⁺ cells using Dynabeads M-450 sheep anti-mouse IgG (Dynal, Lake Success, New York) according to the manufacturer’s instructions. The cells were >95% CD8⁺ and were then stained with the appropriate mAbs and analyzed by FACS.

CD8⁺ T cell purification and sorting

Single-cell suspensions from the lymph nodes of mice were treated with biotinylated anti-CD8 mAb followed by positive selection using the MiniMACS system (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s specifications. The resulting cells were >95% pure CD8⁺TCR⁺ T cells. CD8⁺ T cells purified by this method contained 10% CD44^high cells. For purification of CD8⁺CD44^high and CD8⁺CD44^low T cells, MiniMACS-purified CD8⁺ T cells were stained with anti-CD8-FITC and anti-CD44-PE and sorted on a BD Biosciences FACSVantage SE Turbo sort cell sorter. Cell sorting was performed by A. Johnson (University of British Columbia, Vancouver, British Columbia, Canada) and the sorted CD8⁺CD44^high or CD8⁺CD44^low cells were >98% pure. For some assays, the following purification method was used to provide a source of CD8⁺CD44^low cells: lymph node cells were incubated with mAbs specific for CD4, mouse Ig, and CD44 followed by depletion with anti-mouse Ig-coated Dynabeads (Dynal). This method yielded a population of >98% CD8⁺CD44^- cells.

NK cell purification and activation

NK cells were isolated by treating spleen cells with anti-CD4, anti-CD8, and anti-CD3 mAbs followed by treatment with sheep anti-mouse Ig to deplete CD3⁺CD4⁺CD8⁺Ig⁺ cells. The cells were 60% CD3^-DX5⁺NK1.1⁺CD122⁺ and were then cultured in I-medium supplemented with IL-2 (200 U/ml) for 5 days. On day 5, the cells were >99% CD3^-DX5⁺NK1.1⁺2B4⁺CD122⁺.

CFSE labeling

Purified CD8⁺ T cells (1 x 10⁷/ml) were labeled with 1 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 8 min at room temperature. After stopping the reaction with the addition of an equal volume of FCS, cells were washed four times with complete medium before use.

Proliferation assays

For IL-2 proliferation, 1 x 10⁵ purified CD8⁺ T cells of the indicated CD44 phenotype were cultured in I-medium plus IL-2 (200 U/ml) in 96-well U-bottom plates for 5 days. Blocking mAbs were added to the indicated cultures at 10 µg/ml. The cells were pulsed with 1 µCi of [³H]thymidine for the final 6 h to assess proliferation. For anti-CD3 (2C11)-induced proliferation, 1 x 10⁴ purified CD8⁺ T cells of the indicated CD44 phenotype were cultured in 96-well flat-bottom plates coated with 2C11 (10 µg/ml) and IL-2 (20 U/ml) with or without the indicated blocking mAbs (10 µg/ml) for 4 days and pulsed with 1 µCi of [³H]thymidine for the final 6 h to assess proliferation. For CFSE proliferation assays, the same conditions as above were used except the cells were CFSE labeled and analyzed on day 4 by FACS.

CTL assays

Target cells (RMA, RMAS, RMA-Rae-1, RMAS-Rae-1, A20, or P815) were labeled with ⁵¹Cr (100 µCi) for 1 h at 37°C and then washed. Labeled target cells (1 x 10⁴) were added to 96-well U-bottom plates containing activated CD8 or NK cells at the indicated ratios in a final volume of 200 µl. After a 5-h incubation, the supernatants were collected and counted. Spontaneous release varied from 8 to 15% of the maximum. All assays were performed in triplicate. Percent specific lysis was calculated as 100% x (cpm (experimental well) - cpm (spontaneous release))/((cpm (maximum release) - cpm (spontaneous release)).

RT-PCR

NK cells and CD8⁺CD44^high T cells were activated with IL-2 (200 U/ml) and CD8⁺CD44^low cells were activated with anti-CD3 and IL-2 (20 U/ml) for 5 days. Cells were then harvested and total RNA was prepared according to the manufacturer’s recommendations using the RNeasy Mini kit (Qiagen, Valencia, CA). cDNAs were generated from total RNA using the Protoscript cDNA synthesis kit according to the manufacturer’s recommendations (NEB, Beverly, MA). Left and right primer sequences, respectively, were as follows: DAP10, 5'-CAGGCTACCTCCTGTTCCTG-3' and 5'-GCCAGGCATGTTGATGTAGA-3'; DAP12, 5'-CTGGTGTACTGGCTGGGATT-3' and 5'-CTGGTCTCTGACCCTGAAGC-3'; and GAPDH, 5'-TGC(A/C)TCCTGCACCACCAACT-3' and 5'-(C/T)GCCTGCTTCACCACCTT-3'. The PCR products were subjected to electrophoresis on a 2% agarose gel and visualized by ethidium bromide.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypic characterization of CD8⁺CD44^high cells from normal mice

CD8⁺ T cells from normal mice express varying levels of CD44. We first determined the cell surface phenotype of CD8⁺ T cells from B6 mice that expressed either high (CD44^high) or low (CD44^low) levels of CD44. The CD8⁺CD44^high T cells comprised 10–20% of total CD8⁺ T cells in B6 mice, with older mice possessing an increased proportion of these cells (n = 40; the mice vary between 6 and 12 wk of age). Fig. 1 shows the relative expression of various cell surface markers by the CD8⁺CD44^high and CD8⁺CD44^low cells from the same B6 mouse. These data indicate that the CD44^high cells expressed elevated levels of CD44, Ly6C, CD122, and 1B11, a CD43 isoform characteristic of activated CD8⁺ T cells (21). This activated/memory cell surface phenotype suggests that there is prior recognition of cognate Ag by the CD44^high cells. Interestingly, CD44^high cells expressed lower levels of the TCR than the CD44^low cells. The lower expression of the TCR is characteristic of NKT cells (22) as well as the self-specific CD8⁺ T cells that can develop via an extrathymic pathway (11). However, unlike NKT cells, immediately ex vivo CD8⁺CD44^high cells do not express NK1.1 or significant levels of other NK receptors such as CD94, 2B4 or NKG2D (Fig. 1).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Cell surface phenotype of CD8+CD44high and CD8+CD44low T cells ex vivo. Lymph node cells from B6 mice were depleted of CD4+ and lg+ cells and stained with Abs against various cell surface markers. The filled histograms represent expression of the indicated cell surface molecule by gated CD8+CD44high cells. The unfilled histograms represent expression of the indicated cell surface molecule by gated CD8+CD44low cells from the same mouse.

The expression of CD122 (IL-2R) on CD8⁺CD44^high T cells suggests that they might be capable of proliferating in response to cytokines such as IL-2 or IL-15. To test for this possibility, we purified CD8⁺CD44^high and CD8⁺CD44^low T cells from B6 mice by cell sorting and labeled these cells with the fluorescent dye CFSE and then cultured them with IL-2. The CFSE data in Fig. 2A indicate that only the CD8⁺CD44^high proliferated in response to IL-2. Proliferation is relatively rapid, as some of the cells have undergone more than six rounds of cell division by 72 h. By contrast, the vast majority of IL-2-activated CD8⁺CD44^low cells did not divide during the 48- to 96-h observation period. Measurement of cell proliferation by following the incorporation of [³H]thymidine during the last 6 h of a 96-h culture period also indicates that only the CD8⁺CD44^high cells are capable of IL-2-induced proliferation (Fig. 2B). These results are consistent with a previous report showing that only CD8⁺ T cells expressing high levels of CD44, but not Ag-specific memory CD8⁺ T cells, can proliferate in response to IL-2 or IL-15 (23).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Only CD8+CD44high cells proliferate in response to IL-2. A, FACS-sorted CD8+CD44high and CD44low T cells from B6 mice were labeled with CFSE and cultured with IL-2-supplemented medium. Cell division, as assessed by CFSE dilution, was determined at 48, 72, and 96 h by FACS analysis. B, Sorted CD8+CD44high and CD44low T cells were cultured with IL-2 for 4 days and pulsed with [3H]thymidine for the last 6 h of culture. Error bars represent SDs of triplicate cultures.

Several studies have shown that activated CD8⁺ T cells can express various types of NK receptors (24). However, these studies do not distinguish between the expression of NK receptors by either activated CD8⁺CD44^high cells or CD8⁺CD44^low cells. To determine whether CD8⁺CD44^high cells express NK receptors after IL-2 activation, we examined the expression of NK receptors on IL-2-activated CD8⁺ cells. Since only CD8⁺CD44^high but not CD8⁺CD44^low cells can be activated by IL-2 (Fig. 2), these results were equated with IL-2-activated CD8⁺CD44^high T cells. Fig. 3 shows that IL-2 activated CD8⁺CD44^high cells expressed high levels of 2B4 and CD94 but relatively low levels of DX5 and NK1.1. The activated cells also uniformly expressed a low level of NKG2D. This pattern of NK receptor expression is distinct from IL-2-activated NK cells, which express high levels of these NK receptors (data not shown). This pattern is also distinct from Ag-activated conventional CD8⁺CD44^low T cells, which with the exception of NKG2D do not express the other NK receptors (25). Previous studies have shown that both the S and L isoforms of 2B4 are induced upon IL-2 activation of CD8⁺ T cells (14). In this study, we showed that IL-2-activated CD8⁺CD44^high cells expressed high levels of 2B4. By contrast, anti-TCR activated CD8⁺CD44^low cells did not express 2B4 (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Activated CD8+CD44high cells express several NK receptors. CD8+ T cells were purified from B6 lymph nodes as described in Materials and Methods. The purified CD8+ T cells were cultured with IL-2-supplemented medium for 5 days and then stained with Abs against various cell surface markers that are characteristic of NK cells. The filled histograms represent expression levels of the indicated NK receptor by IL-2 activated CD8+CD44high cells and the unfilled histograms represent unstained controls.

2B4 is a member of the CD2 subset of Ig superfamily molecules and is the high-affinity ligand for CD48 (26, 27). CD48 is also a member of the CD2 subset and is expressed by lymphocytes, monocytes, and endothelial cells (28). Members of the CD2 subset have been observed to interact with themselves or with other family members (28). CD48 has also been shown to function as a costimulatory molecule for T cells (29). Engagement of 2B4 on NK cell surfaces with CD48 can trigger cell-mediated cytotoxicity, IFN- secretion, phosphoinositol turnover, and NK cell invasiveness (28). During the course of our experiments, we noticed that IL-2-induced proliferation of CD8⁺CD44^high cells was dependent on cell-cell contact (data not shown). However, since highly purified CD8⁺CD44^high cells proliferated in response to IL-2, it is unlikely that contact with other lymph node cell types is required for IL-2-induced proliferation. As CD8⁺CD44^high cells express high levels of CD48 and CD2 (Fig. 4A) and these cells expressed high levels of 2B4 upon activation (Fig. 3), we determined whether CD48, 2B4, or CD2 is important in mediating IL-2-induced proliferation. To test the role of these molecules in IL-2-induced proliferation, we cultured purified CD8⁺ T cells in IL-2 in the presence or absence of anti-CD48, anti-CD2, or anti-2B4 mAbs. Proliferation was determined either by measuring CFSE fluorescence (Fig. 4B) or by the incorporation of radiolabeled thymidine (Fig. 4C). The data indicate that anti-CD48 and anti-CD2 inhibited IL-2-induced proliferation to the same extent, whereas the anti-2B4 mAb did not have any effect on the proliferative response. This observation indicates that CD48 and CD2, but not 2B4, are important in mediating IL-2-induced proliferation. We also determined the antiproliferative effect of the anti-CD48 mAb on anti-CD3 plus IL-2-induced proliferation, as assessed by CFSE dilution (Fig. 4B) or the incorporation of radiolabeled thymidine (Fig. 4C). Under these conditions, both the CD8⁺CD44^high as well as the CD8⁺CD44^low populations will be activated. However, since the CD8⁺CD44^low cells comprise 90% of the starting population these cells constitute the vast majority of the responding cells. By contrast to IL-2-induced proliferation, the anti-CD3-induced proliferation was not inhibited by the anti-CD48 mAb demonstrating that anti-CD3-induced activation of CD8⁺CD44^low cells is resistant to inhibition by anti-CD48 mAb. These data support the hypothesis that IL-2-induced activation of CD8⁺CD44^high cells and anti-CD3-induced activation of CD8⁺CD44^low cells occur via CD48-dependent and CD48-independent mechanisms, respectively.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. CD48-CD2 interactions are required for IL-2-induced proliferation of CD8+CD44high cells. A, The expression of CD48 and CD2 on B6 CD8+CD44high cells ex vivo (filled histograms) vs unstained controls (unfilled histograms). B, Purified CD8+ T cells from B6 mice were labeled with CFSE and cultured in either IL-2-supplemented medium (left panel) or plate-bound anti-CD3 Ab (2C11) plus 20U/ml IL-2 (right panel) in the presence of 10 µg/ml anti-CD48 mAb (solid line), anti-CD2 (dashed line), anti-2B4 (dotted line), or without any Ab (filled histogram) for 4 days and analyzed by FACS. C, Purified CD8+ T cells from B6 mice were cultured in either IL-2-supplemented medium (left panel) or plate-bound anti-CD3 Ab (2C11) plus 20U/ml IL-2 (right panel) in the presence or absence of 10 µg/ml anti-CD48 mAb for 4 days and then pulsed with [3H]thymidine for the final 6 h of culture. The error bars represent the SD of triplicate cultures.

Previous studies suggested that the development of CD8⁺CD44^high cells is dependent on interaction with self-Ags in peripheral lymphoid organs (9, 30, 31). Therefore, it was of interest to determine whether IL-2-activated CD8⁺CD44^high cells demonstrate self-specificity as would be suggested by the killing of syngeneic tumor cells. RMA tumor cells are syngeneic to B6 mice and are lethal when injected into these mice (32). We first compared the ability of activated CD8⁺CD44^high and CD8⁺CD44^low cells from a B6 mouse to kill RMA target cells. Since only CD8⁺CD44^high cells can be activated by IL-2 whereas both the CD8⁺CD44^high as well as CD8⁺CD44^low cells can be activated by anti-CD3 plus IL-2, we used anti-CD3 plus IL-2 as a common means of activating FACS-purified CD8⁺CD44^high and CD8⁺CD44^low cells. The purpose of this experiment was to determine whether activated CD8⁺CD44^high and CD8⁺CD44^low cells differ in their ability to kill syngeneic tumor targets. We found that only anti-CD3-activated CD8⁺CD44^high cells demonstrate significant killing of RMA target cells (Fig. 5A) even though the CD8⁺CD44^low cells were activated to the same extent as assessed by the expression of CD25 and CD69 (data not shown). These results indicate that these two activated CD8⁺ subsets do indeed differ in their ability to kill syngeneic tumor target cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Activated CD8+CD44high cells preferentially killed syngeneic tumor target cells. A, FACS-purified CD8+CD44high and CD8+CD44low cells from a B6 mouse were activated with anti-CD3 (2C11) and IL-2 (20 U/ml) for 3 days and then used as effectors in a 51Cr release assay against RMA target cells, which are syngeneic to B6 mice. Error bars represent the SD for triplicate cultures. B, Left panel, purified CD8+ T cells from BALB/c mice were cultured in IL-2-supplemented medium for 5 days to activate CD8+CD44high cells. The cytolytic activities of the activated cells against A20 (syngeneic to BALB/c) or P815 (syngeneic to DBA/2) target cells were then determined. Right panel, Lymph node cells from BALB/c mice were depleted of Ig+, CD4+, and CD44+ cells. Cells purified in this manner were 98% CD8+CD44low. They were cultured with plate-bound 2C11 in IL-2-supplemented medium for 5 days and the cytolytic activities of the activated cells against A20 or P815 target cells were then determined. C, Same as B except that purified CD8+ or CD8+CD44low lymph node cells from DBA/2 mice were used as the responding population. Error bars represent the SD for triplicate cultures.

We also examined the sensitivity of these tumor target cells to killing by anti-CD3 plus IL-2-activated CD8⁺CD44^low T cells. To minimize the contribution of CD8⁺CD44^high cells in these studies, we depleted the CD8⁺ cells of CD44⁺ cells before culture (see Materials and Methods). We found that anti-CD3-activated CD8⁺CD44^low T cells from either BALB/c (Fig. 5B, right panel) or DBA/2 (Fig. 5C, right panel) did not kill either target, even though these cells were highly activated. This observation likely reflects differences in the TCR repertoire of the CD8⁺CD44^high and CD8⁺CD44^low cells. Our data also supports the hypothesis that the CD8⁺CD44^high population expresses TCRs with a strong bias toward self-Ag. By contrast, the TCRs of the conventional CD8⁺CD44^low population are expected to be purged of anti-self (H-2^d)-reactivity and therefore are unable to kill syngeneic (H-2^d) tumor target cells. These data also implied that the CD8⁺CD44^high and CD8⁺CD44^low cells have differential requirements for self-Ags for their selection and development.

IL-2-activated CD8⁺CD44^high cells express DAP12

The expression of NKG2D on IL-2-activated NK and CD8⁺CD44^high cells as well as anti-CD3 plus IL-2-activated CD8⁺CD44^low cells were determined by staining with an anti-NKG2D mAb (18). Consistent with previous reports, we found that IL-2-activated NK cells expressed high levels of NKG2D whereas anti-CD3 plus IL-2-activated CD44^low cells expressed lower levels (Fig. 6A). IL-2-activated CD44^high cells also expressed lower levels of NKG2D (Fig. 6A). Recent studies showed that activated NK cells express two alternative splice variants of NKG2D that associate differentially with DAP10 and DAP12 (17, 33). In NK cells, association of NKG2D with DAP10 provides a costimulatory signal, whereas association of NKG2D with DAP12 confers a direct stimulatory signal (17, 33). Furthermore, activated CD8⁺ cells from DAP10-deficient mice lack NKG2D expression, suggesting that only DAP10 but not DAP12 is expressed by conventional CD8⁺ T cells (34). We determined the expression of DAP10 and DAP12 in activated NK cells, IL-2-activated CD8⁺CD44^high, and anti-CD3 plus IL-2-activated CD8⁺CD44^low cells from B6 mice (Fig. 6B). Consistent with previous reports, activated NK cells expressed both DAP10 and DAP12 whereas anti-CD3-activated CD8⁺CD44^low cells expressed only DAP10. Interestingly, IL-2-activated CD8⁺CD44^high cells expressed both DAP10 and DAP12. Thus, CD8⁺CD44^high cells are more like NK cells with regard to DAP12 expression.

View larger version (48K): [in this window] [in a new window]   FIGURE 6. Activated CD8+CD44high cells express DAP12. NK cells were enriched by depleting B6 spleen cells of CD4+, CD8+, Ig+, and CD3+ cells by negative selection and the negatively selected cells were activated with IL-2-supplemented medium. Purified CD8+ cells from B6 mice were activated with IL-2-supplemented medium and provided a source of IL-2-activated CD8+CD44high cells. CD8+CD44low cells were purified as described in Fig. 5 legend and activated with anti-CD3 plus IL-2. mRNA was extracted from activated cells and RT-PCR for DAP10, DAP12, and GAPDH were performed. The data indicate that activated NK and CD8+CD44high cells express both DAP10 and DAP12, whereas activated CD8+CD44low cells only express DAP10.

Expression of Rae-1 enhances the lysis of syngeneic tumors by IL-2-activated CD8⁺CD44^high cells

We next determined whether NK receptors participate in this propensity to kill syngeneic tumor cells by IL-2-activated CD8⁺CD44^high cells. Recent studies have shown that the activating NKG2D receptor plays a crucial role in the killing of syngeneic tumor cells (20, 32). The ligands for NKG2D, Rae-1, and H60 are expressed on infected or transformed cells (34). However, RMA tumor cells from B6 mice do not express Rae-1 (34). Rae-1 is normally expressed in B6 mice (20) and we used RMA-Rae-1 transfectants (20) to determine whether NKG2D participates in the killing of syngeneic tumor cells. We tested the ability of IL-2-activated CD8⁺CD44^high and anti-CD3 plus IL-2-activated CD8⁺CD44^low cells from B6 mice to kill RMA and RMA-Rae-1 target cells. We also used the peptide transporter (TAP)-deficient cell lines, RMAS and RMAS-Rae-1, as sources of MHC class I-deficient target cells that do or do not express Rae-1. The expression of Rae-1 on transfectant cell lines was confirmed by staining with a murine NKG2D-Ig fusion protein (Ref. 19 and data not shown). As an additional control for the specificity of killing, we determined the cytolytic activity of IL-2-activated NK cells against these same target cells. NK cells were enriched by depleting B6 spleen cells of CD4⁺, CD8⁺, Ig⁺, and CD3⁺ cells by negative selection and the negatively selected cells were activated with IL-2. These activated cells were of the CD4^-CD8^-CD3^-NKG2D⁺DX5⁺NK1.1⁺2B4⁺CD94⁺ cell surface phenotype (data not shown), consistent with the conclusion that this purification and activation scheme led to a pure population of effector NK cells.

The data in Fig. 7 indicate that the activated NK cells killed RMAS target cells to a greater extent than RMA cells. This is consistent with the conclusion that engagement of MHC class I molecules by inhibitory NK receptors likely contribute to the poorer killing of RMA target cells. More interestingly and consistent with the observations of others (20, 25, 32), expression of Rae-1 on either RMA or RMAS cells greatly increased their susceptibility to NK killing (Fig. 7). By contrast, IL-2-activated CD8⁺CD44^high cells were relatively inefficient in killing either RMA or RMAS target cells (Fig. 7). This observation indicates that the TAP mutation has differential effects on target cell susceptibility to killing by NK or IL-2-activated CD8⁺CD44^high cells. Interestingly, the presence of Rae-1 on either RMA or RMAS target cells also led to greatly enhanced killing by IL-2-activated CD8⁺CD44^high cells. This observation suggests that the interaction of NKG2D with its ligand, Rae-1, greatly enhanced killing of syngeneic tumor cells by IL-2-acitvated CD8⁺CD44^high cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. NKG2D-Rae-1 interaction enhances killing of syngeneic tumor target cells by IL-2 activated CD8+CD44high cells. NK, CD8+CD44high, and CD8+CD44low cells were purified and activated as described in Fig. 6. The ability of IL-2-activated NK cells (upper panel), CD8+CD44high cells (middle panel), or 2C11 plus IL-2-activated CD8+CD44low cells to kill syngeneic target cells expressing a ligand for NKG2D was determined. The cells were >99% CD3-NK1.1+DX5+ 2B4+, CD94+NKG2D+ for the purified NK cells, and >98% TCR+CD8+ for both groups of CD8+ T cells. The cells were used as effectors in a 51Cr release assay against RMA, RMAS, RMA-Rae-1, and RMAS-Rae-1 tumor cells. Error bars represent the SD for triplicate cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this report, we described a population of CD8⁺CD44^high cells in normal mice that possess properties distinct from conventional memory T cells. These cells comprise between 10 and 20% of total CD8 T cells of normal mice. They proliferated in response to stimulation by exogenous IL-2 and differentiated into potent killer cells that demonstrate specificity for syngeneic tumors. These cells also up-regulate several NK receptors including NKG2D upon activation with IL-2. Interestingly, IL-2-activated CD8⁺CD44^high cells express DAP12, an adaptor molecule that is normally found in activated NK cells (35). Furthermore, tumor targets expressing a NKG2D ligand are exquisitely sensitive to killing by IL-2-activated CD8⁺CD44^high cells.

Other studies also demonstrate that the CD8⁺CD44^high cells that are present in normal mice are distinct from conventional memory T cells in many aspects. In contrast to MHC class I-restricted CD8⁺ T cells, the development of CD8⁺CD44^high cells can be thymus independent (9, 11). The frequency of CD8⁺CD44^high cells increases with age even when the mice are maintained under germfree conditions, suggesting that the development of these cells is not foreign Ag driven (36, 37). Memory CD8⁺ T cells and CD8⁺CD44^high cells also differ in their activation threshold. Whereas memory CD8⁺ cells possess a lower activation threshold compared with naive T cells, CD8⁺CD44^high cells were shown to require a higher activation threshold relative to naive CD8⁺ T cells (11). Memory CD8⁺ T cell also expressed lower levels of CD44 and CD122 compared with the CD8⁺CD44^high (38) cells and the growth of CD8⁺CD44^high cells, but not memory CD8 T cells, can be supported by IL-2 or IL-15 (11). These differences between CD8⁺CD44^high cells and memory CD8⁺ T cells strongly suggest that the CD8⁺CD44^high cells are of a lineage that is distinct from conventional CD8⁺ T cells.

TCR-transgenic mice provide a defined system for determining the developmental requirement of CD8⁺CD44^high cells. In TCR-transgenic mice these cells express only the transgenic TCR and can be easily tracked. It was found that the development of CD8⁺CD44^high cells in TCR-transgenic mice is independent of a thymus but is dependent on interaction with self-Ag in extrathymic tissues (9). Our observation that IL-2-activated CD8⁺CD44^high cells demonstrate a preference in the killing of syngeneic tumor cells is also consistent with the notion that the development of these cells is dependent on selection by self-Ags. It was shown that in vivo delivery of large amounts of IL-2 to athymic nude mice results in an autoimmune disease (39). This observation is consistent with the hypothesis that the autoimmune disease is mediated by IL-2 activation of self-specific extrathymic CD8⁺CD44^high cells. Large numbers of extrathymic T cells developed in oncostatin M-transgenic mice (31). Interestingly, the presence of cognate Ag is required for these CD8⁺ T cells to acquire a memory phenotype (31). In normal mice, the self-ligands that are required for the development of CD8⁺CD44^high cells remain to be determined. We found that small numbers of CD8⁺CD44^high cells are also present in ₂-microglobulin and TAP-deficient mice (our unpublished observations), suggesting that these cells may be restricted to nonclassical MHC molecules. The report by Urdahl et al. (40) demonstrating that CD8⁺ T cells with a memory phenotype can be positively selected on MHC class I^b molecules by hemopoietic cells is consistent with this notion. Several reports have also suggested that CD8⁺ T cells that are specific for self-Ags, such as melanocyte differentiation Ags, can be isolated from healthy donors (41, 42). Interestingly, it was found that CD8⁺ T cells that are capable of initiating tumor regression could also induce autoimmune reactions. This finding supports the hypothesis that self-specific CD8⁺CD44^high cells may also contribute to autoimmune diseases (43).

The preferential killing of syngeneic tumor target cells by IL-2-activated CD8⁺CD44^high and anti-CD3-activated CD8⁺CD44^low cells may reflect differences in the TCR repertoire of these two cell populations. However, analysis of TCR V usage using the BD PharMingen mouse V TCR screening panel (a collection of mAbs to 17 Vs) by these two populations before and after activation reveals no significant differences in TCR V usage by these cells (data not shown). This result indicates that there is no preferential usage of V gene segments by these two populations. More importantly, they suggest that the TCR repertoire of CD8⁺CD44^high cells is likely to be very heterogeneous and more sophisticated analyses are required to determine whether there is a bias toward self-Ags in this heterogeneous TCR repertoire.

Immediately ex vivo CD8⁺CD44^high cells do not express 2B4 (Fig. 1), but express high levels of 2B4 upon activation with IL-2 (Fig. 3). 2B4 has been shown to be expressed primarily on NK cells and a small subset of memory phenotype CD8⁺ T cells (44, 45, 46), some of which can mediate non-MHC-restricted cytotoxicity (44, 45). The expression of 2B4 by CD8⁺ T cells has been shown to correlate with the acquisition of effector functions (46) and was involved in proliferation (14). Since 2B4 is a high-affinity receptor for CD48, it may serve as a receptor for CD48 and participate in IL-2-induced proliferation. However, we found that IL-2-induced proliferation was only inhibited by the anti-CD48 but not the anti-2B4 mAb (Fig. 4). A trivial explanation for the lack of inhibition by the anti-2B4 mAb is that this mAb does not block CD48–2B4 interaction. Arguing against this explanation is the observation that either the anti-CD48 or the same anti-2B4 mAb suppressed Ag-induced proliferation of CD8⁺ T cells to the same extent; anti-CD2 mAb has no inhibitory effect in this system (14). Furthermore, there was no additive effect of the anti-CD48 and anti-2B4 mAbs in suppressing Ag-induced proliferation of CD8⁺ T cells in this system (14). These observations suggest that the anti-2B4 mAb acts by inhibiting CD48–2B4 interaction. Therefore, a more likely explanation of our data for the lack of inhibitory effect of the anti-2B4 mAb is the late induction of 2B4 in IL-2-activated cells. By contrast, CD2 is expressed at a high level in ex vivo CD8⁺CD44^high cells and anti-CD2 mAb inhibited IL-2-induced proliferation to the same extent as the anti-CD48 mAb. This observation is consistent with the hypothesis that CD48 and/or CD2 are important in mediating IL-2-induced proliferation. CD48 is a GPI-anchored molecule and it can aggregate lipid rafts when it is engaged (47). Thus, the anti-CD48 mAb could potentially exert its inhibitory effects by preventing the aggregation of lipid rafts. Mouse CD2 has also been shown to constitutively associate with lipid rafts (48) and therefore the anti-CD2 mAb may also exert its effect by preventing the aggregation of lipid rafts. Alternatively, the anti-CD48 mAb could serve as a ligand for the CD2 receptor. CD2 is implicated as an important costimulatory molecule in lymphocyte activation and proliferation (49). Furthermore, proline residues in the CD2 cytoplasmic domain have been shown to activate kinase activity such as phosphatidylinositol 3-kinase and the Tec family tyrosine kinase inducible T cell kinase (50, 51). In this alternative model, the anti-CD48 or the anti-CD2 mAb inhibits IL-2-induced proliferation by interfering with the CD2 signaling pathway. It is noted that the anti-CD48 mAb has no effect on anti-CD3-driven proliferation of CD8⁺ T cells (Fig. 4). This observation provides independent support that CD8⁺CD44^high cells and conventional CD8⁺ T cells are dependent on distinct signaling pathways for growth.

Our data clearly show that the activating NK receptor NKG2D plays an important role in the lysis of syngeneic tumor target cells by IL-2-activated CD8⁺CD44^high cells (Fig. 7). The expression of the ligands for NKG2D, Rae-1, and H60 on tumor cells leads to their rejection in vivo by both NK cells and CD8⁺ T cells (20, 32). Interestingly, the expression of the ligands for NKG2D on tumor cells in vivo results in protection from subsequent challenge from parental (ligand-negative) tumors, suggesting a role for NKG2D in the activation of tumor-specific CD8⁺ T cells (32). NKG2D has been shown to be exclusively costimulatory in CD8⁺ T cells and directly stimulating in NK cells (17, 33). These differences have been attributed to the differential recruitment of adaptor molecules in NK cells vs CD8⁺ T cells. Stimulation of NKG2D on NK cells primarily results in recruitment of DAP12 while stimulation in CD8 T cells leads to recruitment of DAP10 (17, 33). Interestingly, we found that in contrast to anti-CD3-activated CD8⁺CD44^low cells, which express only DAP10, IL-2-activated CD8⁺CD44^high cells express both DAP10 and DAP12. Thus, the IL-2-activated CD8⁺CD44^high cells are more NK-like in this regard. In this study, we have shown that IL-2-activated CD8⁺CD44^high cells from B6 mice can kill Rae-1^- RMA target cells, albeit with low efficiency. This observation suggests that the killing of syngeneic tumor target cells may be mediated in part by the TCR. The expression of Rae-1 on RMA cells resulted in greatly enhanced lysis by IL-2-activated CD8⁺CD44^high cells, suggesting that the NKG2D receptor plays an important role in the killing of syngeneic tumor cells by these cells. Interestingly, deficiency in the TAP peptide transporter did not enhance killing of syngeneic tumor target cells by IL-2-activated CD8⁺CD44^high cells. This observation is consistent with the notion that these activated cells either lack the inhibitory receptors that are expressed by NK cells and/or have a distinct combination of activating/inhibitory receptors from the activated NK cells. Alternatively, the ligands that are recognized by the TCR on IL-2-activated CD8⁺CD44^high cells may be independent of the TAP peptide transporter. The existence of CD8⁺CD44^high cells in TAP-deficient mice (our unpublished observations) is consistent with this notion. Collectively, our data support the hypothesis that IL-2-activated CD8⁺CD44^high cells express TCRs that are specific for syngeneic tumor target cells. In the absence of Rae-1 expression, the tumor target cells are killed at relatively low efficiency. When the tumor target cells express Rae-1, then there is synergy between the TCR and the NKG2D in the killing of syngeneic tumor target cells. Such a synergy between the TCR and activating NK receptors would render these cells particularly adept in the surveillance of host cells that have been altered through infection or transformation.

	Acknowledgments

 
We thank Soo-Jeet Teh for excellent technical assistance. We are grateful to Dr. Lewis Lanier (University of California, San Francisco) for providing us with cell lines (RMA, RMA-Rae-1, RMAS-Rae-1) and the NKG2D-Ig fusion protein and Dr. Wayne Yokoyama (Howard Hughes Medical Institute, Washington University) for providing us with the anti-NKG2D mAb.

	Footnotes

 
¹ This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research (to H.S.T.). S.D. is a recipient of a University Graduate Fellowship from the University of British Columbia

² Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3. E-mail address: teh{at}interchange.ubc.ca

Received for publication April 1, 2003. Accepted for publication July 18, 2003.

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