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The Journal of Immunology, 2003, 171: 69-77.
Copyright © 2003 by The American Association of Immunologists

Thy-1 Signaling in the Context of Costimulation Provided by Dendritic Cells Provides Signal 1 for T Cell Proliferation and Cytotoxic Effector Molecule Expression, but Fails to Trigger Delivery of the Lethal Hit1

S. M. Mansour Haeryfar*, Monther M. Al-alwan*, Jamie S. Mader{ddagger}, Geoffry Rowden{dagger},{ddagger}, Kenneth A. West*,{dagger},{ddagger} and David W. Hoskin2,*,{ddagger}

Departments of * Microbiology and Immunology, {dagger} Medicine, and {ddagger} Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fc{gamma} receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell activation is a well-coordinated process that is initiated and regulated by multiple signals provided upon the engagement and reorganization of certain cell surface molecules. The Ag-specific signal, which is also referred to as signal 1, is supplied by the TCR recognizing an antigenic peptide displayed in the groove of a self MHC molecule on the surface of an APC (reviewed in Ref.1). Alternatively, signal 1 can be mimicked when the TCR/CD3 complexes on T cells are ligated and cross-linked by anti-CD3 mAbs (2, 3). Although necessary, signal 1 is typically not sufficient for optimal T cell activation, which requires an additional costimulatory signal or signal 2 that is delivered by the APC. Signal 2 is clearly important in determining the fate of a T cell responding to Ag because TCR triggering in the absence of signal 2 results in anergy rather than activation (4). The CD28-B7-1 (CD80)/B7-2 (CD86) system is arguably the most prominent source of costimulatory interactions (5). CD28 ligation leads to up-regulation of the Bcl-xL antiapoptotic protein (6), and enhancement of IL-2 synthesis at both transcriptional (7) and posttranscriptional levels (8). Once properly signaled, T cells undergo intracellular changes that ultimately culminate in T cell proliferation and differentiation into effector cells capable of providing help to other immunocytes or detecting and destroying specific targets.

Thy-1 (CD90) is a small GPI-anchored glycoprotein that is heavily expressed by mouse thymocytes and peripheral T lymphocytes (9). Although a physiological counterreceptor for Thy-1 has yet to be identified, cross-linking of Thy-1 by certain mAbs results in IL-2 production and T cell proliferation (10, 11), suggesting that Thy-1 may be a potential source of signal 1 for T cell activation. Several other roles have been proposed for Thy-1 in the process of T cell activation, ranging from negative regulation (12) to accessory function (13). However, whether Thy-1 can in fact substitute for signal 1 in the context of optimal costimulation is not yet known.

Dendritic cells (DCs)3 are the most potent APCs owing to their high expression levels of class II MHC as well as costimulatory molecules (reviewed in Ref.14). T cell-dependent immune responses initiated by DCs depend highly on the DC-mediated provision of B7-1 and/or B7-2 costimulatory signals. The specialized ability of DCs to bind and form immunological synapses with resting T cells allows DCs to prime naive T cells both in vivo and in vitro, and distinguishes DCs from other professional APCs (15, 16). Given the importance and efficiency of DCs in costimulating T cell responses, we asked whether T cell activation via Thy-1 as a potential source of signal 1 could be enhanced by a strong, DC-generated signal 2. Our data indicate that Thy-1 triggering can substitute for signal 1 for driving T cell proliferation and IL-2 synthesis when DCs are present to provide CD28-dependent costimulation. However, in contrast to signaling through the TCR, Thy-1-mediated signal 1 failed to induce cytotoxic effector function, even in the presence of strong costimulation. This reflects a hitherto unrecognized fundamental difference between Thy-1- and the TCR-associated T cell signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Adult (6- to 8-wk-old) FcR {gamma}-chain knockout (KO) mice (FcR{gamma}-/-) on BALB/c background and the wild-type (WT) controls (FcR{gamma}+/+) matched for age and sex were purchased from Taconic Farms (Germantown, NY). BALB/c mice carrying the I-Ad-restricted rearranged TCR transgene TgN (DO11.10) were obtained from the National Institutes of Health (Bethesda, MD). The transgenic TCR expressed by these mice recognize aa 323–339 of OVA (17). The expression of the transgenic TCR by the majority (typically greater than 82%) of TgN T cells was confirmed via staining by the mAb KJ1-26 (Caltag Laboratories, Burlingame, CA). Mice were housed in the Carleton Animal Care Facility of Dalhousie University and maintained on standard rodent chow and water supplied ad libitum.

Cell lines

The murine mastocytoma cell line P815, the B lymphoma cell line A20, and the IL-2-dependent CTLL-2 T cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The Fas-transfected P815 cell line (18) was kindly provided by E. Podack (University of Miami, Miami, FL). P815, P815-Fas, and A20 cell lines were all maintained in RPMI 1640 medium (Sigma-Aldrich, Mississauga, Ontario, Canada) supplemented with 5% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 mM HEPES buffer (pH 7.4; all from Invitrogen Canada, Burlington, Ontario, Canada), which will, hereafter, be referred to as complete RPMI 1640 medium. CTLL-2 cells were grown in complete RPMI 1640 medium containing 10% FCS and 20 U/ml (~4 ng/ml) mouse rIL-2 (PeproTech, Rocky Hill, NJ).

Reagents and Abs

OVA peptides OVA323–339 (NH2-ISQAVHAAHAEINEAGR-COOH) and OVA324–334 (NH2-SQAVHAAHAEI-COOH) were from Bethyl Laboratories (Montgomery, TX). Hamster anti-mouse CD3{epsilon} mAb (3) was in the form of culture supernatant from hybridoma 145-2C11 kindly provided by J. Bluestone (University of Chicago, Chicago, IL). The hybridoma (clone 16-10A1) that produces hamster anti-mouse B7-1 mAb was obtained from ATCC, and the hybridoma-producing rat anti-mouse B7-2 (clone GL1) mAb (19) was a gift from K. Hathcock (National Cancer Institute, Bethesda, MD). Anti-mouse Thy-1 mAb (clone G7, rat IgG2c, {kappa}), rat IgG2c isotype control (clone A23-1 with unknown specificity), anti-mouse Fas mAb (clone Jo2), and purified rat anti-mouse CD16/CD32 (FcR{gamma}III/II) mAb (Fc block, clone 2.4G2, rat IgG2b) were purchased from BD PharMingen (Mississauga, Ontario, Canada). PE-conjugated anti-mouse {alpha}{beta} TCR mAb, hamster IgG PE, hamster anti-mouse CD28 mAb, rat IgG2b, and hamster IgG were obtained from Cedarlane Laboratories (Hornby, Ontario, Canada). FITC-conjugated mouse anti-rat IgG F(ab')2 and Cy3-conjugated donkey anti-rabbit IgG F(ab')2 were from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit anti-cathepsin D polyclonal Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

DC preparation

Mice were sacrificed by cervical dislocation, and DCs were generated from bone marrow precursors, as previously described (15, 20). The harvest typically consisted of >95% terminally differentiated DCs, as assessed by flow cytometric analysis of CD11c, MHC II, and B7-2 expression.

T cell isolation

Spleen cell suspensions were depleted of erythrocytes by hypo-osmotic shock and passaged through nylon wool (Polysciences, Warrington, PA) columns to remove most B cells and macrophages (21). T cell recovery column kits (Cedarlane) were then used to isolate total or CD4+ T cells. The resulting cell preparations typically contained 95–99% CD3+ or CD4+ T cells by flow cytometry.

B cell isolation

E-free spleen cells were depleted of T cells by a two-step treatment with anti-Thy-1.2 mAb and Low-Tox rabbit C (1:12; Cedarlane). The resulting cell suspension was subsequently incubated on a sterile plastic petri dish for 1 h at 37°C to remove macrophages. Plastic-nonadherent cells, which were highly enriched for B cells, were then harvested and used immediately. In some experiments, B cells were activated by culture for 72 h in the presence of 5 µg/ml LPS (Sigma-Aldrich).

T cell proliferation

T cells (2 x 105/well) and/or syngeneic DCs (8 x 103/well) were seeded into wells of U-bottom microtiter plates (Sarstedt, St. Leonard, Quebec, Canada) in a total volume of 200 µl/well. Optimal concentrations (determined in dose-response experiments) of soluble anti-CD3{epsilon} mAb (1/20 dilution of hybridoma supernatant that approximates the proliferative stimulus provided by 5 µg/ml of purified mAb) or anti-Thy-1 mAb (6 µg/ml) were used for polyclonal T cell activation. Ag-specific proliferation of CD4+ TCR transgenic T cells was induced with cognate peptide OVA323–339(300 nM) plus DCs. Noncognate peptide OVA324–334 was used as a negative control. Plates were incubated at 37°C and 5% CO2 in a 95%humidified atmosphere for 72 h. Cells were pulsed with 0.5 µCi of [3H]TdR (sp. act. 60 Ci/mmol; ICN Canada, Montreal, Quebec, Canada) for the final 18 h of the cultures. Cultures were then harvested onto glass fiber filter mats (Skatron, Sterling, VA) using a Titer-Tek multiple sample harvester, and [3H]TdR uptake was determined by liquid scintillation counting. In a number of experiments, cell-sized (10 µm in diameter) microbeads (Polysciences) were coated with the indicated mAbs and used as surrogate APCs at a 1:1 ratio with resting T cells. These cultures were performed in 96-well, flat-bottom plates (Sarstedt). Cells were pulsed with [3H]TdR during the final 8 h of a 48-h culture period.

IL-2 bioassay

IL-2 concentrations in culture supernatants were determined by a CTLL-2-based IL-2 bioassay, as previously described (22).

Cytotoxicity assay

T cells were harvested at 72 h poststimulation, washed, resuspended in complete RPMI 1640 medium, and seeded into wells of a 96-well, V-bottom plate (Sarstedt). Target cells were labeled with 100 µCi of Na251CrO4 (ICN Canada) for 1 h at 37°C, washed extensively, and added to the plate at 5 x 103 cells/well. Cell numbers were always corrected for viability using trypan blue exclusion. The plates were centrifuged at 400 x g for 5 min at the end of a 4-h incubation at 37°C. A 100-µl aliquot of supernatant was then harvested from each well, and the 51Cr content of the samples was determined by gamma counting. Specific lysis of the target cells was determined using following formula: percentage of specific lysis = ((ER - SR)/(TR - SR)) x 100, where ER (experimental release) is obtained from wells containing both T cells and target cells, while TR (total release) and SR (spontaneous release) are determined from wells receiving only target cells and medium or 10% SDS, respectively. A cytolytic anti-mouse Fas mAb (clone Jo2) was used as a positive control to induce cell death in Fas-expressing P815 cells. In some experiments, anti-CD3 mAb, anti-Thy-1 mAb, or Abs raised against rat IgG were present in the effector phase of killing for the assessment of redirected lysis of the target cells.

Conjugation assay

T cell adherence to P815 target cells was evaluated, as previously described (23).

Semiquantitative RT-PCR

Total RNA was extracted at the indicated time points using TRIzol reagent, and reverse transcribed by 200 U of Moloney murine leukemia virus reverse transcriptase in the presence of 0.5 mM dNTPs, 1 µg of random hexanucleotide primers, and 10 mM DTT. The resulting cDNA samples were diluted 1/10 in pyrogen-free water and subjected to PCR in an automatic thermal cycler (MJ Research, Watertown, MA). Each PCR mixture also included 2.5 U of native Taq DNA polymerase, 0.2 mM dNTPs, and primer pairs at 0.5 µM each in a 1/10 dilution of the PCR buffer. Amplification of the housekeeping gene GAPDH cDNA was initially conducted to determine sample volumes giving equal amounts of cDNA in PCR mixtures. All primers were designed to bind intron-bridging exons of their corresponding genes. The following primer sequences were used for the PCR (the amplicon size is given after the reverse primer): GAPDH (forward), 5'-ACTCACGGCAAATTCAACGGC-3' and GAPDH (reverse), 5'-ATCACAAACATGGGGGCATCG-3' (246 bp); perforin (forward),5'-TCAATAACGACTGGCGTGTGG-3' and perforin (reverse), 5'-GTGGAGCTGTTAAAGTTGCGG-3' (252 bp); granzyme B (forward), 5'-GCCCACAACATCAAAGAACAG-3' and granzyme B (reverse), 5'-GAGAACACATCAGCAACTTGGG-3' (889 bp); Fas ligand (FasL) (forward), 5'-ATGGTTCTGGTGGCTCTGGT-3' and FasL (reverse), 5'-GTTTAGGGGCTGGTTGTTGC-3' (362 bp). The amplification protocols for GAPDH (28 cycles), perforin (32 cycles), granzyme B, and FasL (30 cycles) were as follows: denaturation at 92°C for 30 s, annealing at 57°C for 30 s, and primer extension at 72°C for 90 s (for granzyme B, primer extension was performed for 2 min). The number of PCR cycles was initially determined to yield PCR products during the exponential phase of amplification. RT-PCR performed under these conditions provides reliable detection of greater than 2-fold differences in mRNA levels (24). All of the reagents used for RNA isolation and RT-PCR were from Invitrogen Canada, except for Taq DNA polymerase and PCR buffer, which were purchased from Amersham Biosciences (Baie d’Urfé, Quebec, Canada).

Immunofluorescence

Freshly isolated DCs were incubated for 30 min on ice with 1 µg of rat anti-mouse CD16/CD32 mAb in a 100 µl vol of PBS containing 1% BSA and 0.2% sodium azide. DCs were next washed and incubated in dark with FITC-conjugated mouse anti-rat IgG F(ab')2 at 1 µg/100 µl for another 30 min, followed by washing and fixation with 1% paraformaldehyde. For TCR/CD3 staining of resting and activated T cells, PE-conjugated anti-mouse {alpha}{beta} TCR mAb was used in parallel with hamster IgG-PE as an isotype control. The expression of CD16/CD32 by DCs or TCR by T cells was determined by analysis of 104 cells with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). In some experiments, PE-conjugated anti-CD3 mAb (5 µg/ml) was used to activate T cells and surface-bound anti-CD3 mAb that remained after 72 h of priming was measured by flow cytometry. Anti-Thy-1 mAb that remained on the surface of Thy-1-stimulated T cells after 72 h of priming was determined by flow cytometric analysis of staining with FITC-conjugated mouse anti-rat IgG F(ab')2. CTL lytic granules were visualized with anti-cathepsin D Ab, as described by Stinchcombe et al. (25). Briefly, CTL-target cell conjugates were centrifuged onto poly(L-lysine)-coated glass slides and incubated at 37°C for 30 min. Samples were fixed for 5 min with methanol precooled to -20°C or 4% paraformaldehyde, permeabilized for 10 min with 0.2% Triton X-100, and blocked in PBS and 5% BSA (Sigma-Aldrich) for 2 h. Samples were incubated with rabbit anti-cathepsin D Ab (1/10) for 1 h, washed, and stained by incubation with Cy3-conjugated F(ab')2 anti-rabbit IgG (1/800) for 1 h. Samples were examined under a Zeiss (Oberkochen, Germany) LSM510 confocal laser-scanning microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DCs augment Thy-1-driven T cell proliferation and IL-2 production

The requirement for concurrent stimulation with phorbol ester in order for Thy-1 signaling to elicit IL-2 synthesis and substantial T cell proliferation in the absence of APCs (10, 11) suggests a need for costimulation. Because DCs are a potent source of costimulatory molecules (14), we stimulated mouse T cells with a mitogenic anti-Thy-1 mAb (clone G7) in the presence of mature, syngeneic DCs. The T cells responded by vigorously proliferating and producing substantial amounts of IL-2 in comparison with T cells activated with anti-Thy-1 mAb in the absence of DCs (Fig. 1, A and B). The presence of IL-2 bioactivity in cultures was judged by the ability of culture supernatants to support the growth of the IL-2-dependent CTLL-2 cells. Anti-Thy-1 mAb alone neither induced CTLL-2 proliferation nor modulated the CTLL-2 response to IL-2 (data not shown). The enhanced Thy-1-driven responses were clearly dependent on the number of DCs present in the cultures (Fig. 1C). In contrast, even high numbers of freshly explanted B cells failed to appreciably enhance T cell activation through Thy-1 triggering. We also examined the effect of LPS-activated B cells, on which costimulatory molecule expression is increased (19), on anti-Thy-1-induced T cell activation. Although LPS-activated B cells were slightly more effective than unactivated B cells in costimulating Thy-1-driven T cell proliferation (stimulation index of 1.7 vs 1.1 at 10,000 APC/well), they remained inferior to DC (stimulation index of 6.0 at 10,000 APC/well). Collectively, these data indicate a unique role for DCs in the activation of T cells through Thy-1. DCs did not induce measurable T cell proliferation in the presence of irrelevant IgG of the same isotype, a nonstimulatory mAb to Thy-1 (clone 30-H12), or mAbs to other T cell surface molecules such as class I MHC, CD2, or CD28 (data not shown). This indicates that the DC enhancement of Thy-1-driven T cell proliferation is not simply a general phenomenon that might occur in response to the presence of IgG or any T cell-specific mAb in the cultures.



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  		FIGURE 1. DCs selectively enhance Thy-1-driven T cell responses. A, T cells were incubated with or without anti-Thy-1 mAb (6 µg/ml) in the absence or presence of DCs for 72 h. T cells were pulsed with [3H]TdR 18 h before the culture was harvested. [3H]TdR incorporation was then determined as a measure of T cell proliferation. B, Supernatants from 24-h T cell cultures (or increasing doses of murine rIL-2 as a positive control) were added to IL-2-dependent CTLL-2 cells that were then cultured for 24 h. CTLL-2 cells were pulsed with [3H]TdR 8 h before the culture was harvested. [3H]TdR incorporation by CTLL-2 cells was then quantified as an indicator of IL-2 bioactivity in the supernatants. C, The indicated numbers of DCs or B cells were incubated with T cells and anti-Thy-1 mAb for 72 h, and T cell proliferation was determined by [3H]TdR incorporation. Data are given as mean ± SD from quadruplicate cultures, and are representative of three (A) or two (B, C) independent experiments yielding similar results.

 
DC enhancement of Thy-1-triggered T cell proliferation is not dependent on FcR{gamma}

Because T cell activation by mitogenic Abs normally requires cross-linking of Abs by Fc{gamma}Rs expressed by accessory cells (26), we next examined bone marrow-derived DCs for Fc{gamma}R expression. As depicted in Fig. 2A, freshly isolated DCs showed substantial reactivity with 2.4G2 mAb (Fc block), which is routinely used to detect Fc{gamma}RII (CD32)/Fc{gamma}RIII (CD16) (27, 28). We then used FcR{gamma} gene KO mice that lack Fc{gamma}RI (CD64) and Fc{gamma}RIII (29), as well as Ab-mediated blockade of Fc{gamma}RII and Fc{gamma}RIII to determine whether or not DC Fc{gamma}Rs play a role in anti-Thy-1-induced T cell activation. The absence and/or blockade of Fc{gamma}Rs did not affect T cell proliferation in response to anti-Thy-1 mAb (Fig. 2B). In contrast, anti-CD3-induced T cell proliferation was marginally attenuated (23% inhibition) by the absence of Fc{gamma}RI and Fc{gamma}RIII on FcR{gamma}-/- DCs, while the Ab-mediated blockade of Fc{gamma}RII/III nearly completely inhibited anti-CD3-induced T cell proliferation (92% inhibition). Moreover, the use of Fc block with DCs from KO mice abolished anti-CD3-induced T cell proliferation. We did not detect any differences between DCs from FcR{gamma}-/- mice and DCs isolated from their WT littermates in terms of cell yields and gross morphology in cultures. Furthermore, the KO DCs were as potent as WT DCs in presenting an OVA peptide to T cells rendered transgenic for the corresponding TCR, and initiating an Ag-specific T cell response (Fig. 2C). Collectively, our data demonstrate that although mouse bone marrow-derived DCs express Fc{gamma}Rs, Fc{gamma}Rs are not needed for DC-mediated enhancement of T cell proliferation induced by Thy-1 triggering.



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  		FIGURE 2. Fc{gamma}Rs on bone marrow-derived DCs do not contribute to the DC enhancement of Thy-1-driven T cell activation. A, Cell surface expression of Fc{gamma}Rs by mouse bone marrow-derived DCs was assessed by flow cytometric analysis after a two-step staining with a rat anti-mouse Fc{gamma}RIII (CD16)/Fc{gamma}RsII (CD32) mAb (clone 2.4G2) and FITC-conjugated mouse anti-rat IgG F(ab')2 (open peak), in comparison with background staining using no primary Ab (filled peak). B, T cells were stimulated with anti-Thy-1 mAb plus DCs obtained from wild-type (WT) or Fc{gamma}R KO mice in the absence or presence of 2.4G2 mAb (Fc block). Parallel cultures of T cells were stimulated with anti-CD3 mAb as a positive control. T cell proliferation was then measured by [3H]TdR incorporation. C, Resting CD4+ T cells expressing I-Ad-restricted TCR transgene TgN (DO11.10) were combined with syngeneic DCs obtained from wild-type (FcR+/+) or FcR{gamma} knockout (FcR-/-) mice and pulsed with a cognate (OVA323–339) or control (OVA324–334) peptide. T cell proliferation was measured after 72 h by [3H]TdR incorporation. Data are representative of three (A, B) or two (C) independent experiments yielding similar results.

 
Costimulation through CD28 enhances T cell activation triggered by Thy-1-driven signal 1

Although B7 family members are important costimulators of TCR-driven T cell proliferation (30), little is known about the role of CD28/B7 interactions during Thy-1-mediated T cell activation. We, therefore, asked whether or not DC enhancement of Thy-1-driven T cell activation is a consequence of costimulation provided by DCs. As illustrated in Fig. 3A, anti-CD3- and anti-Thy-1-induced T cell proliferative responses were similarly inhibited by the inclusion of blocking Abs specific to B7-1 (31 and 33% inhibition, respectively) or B7-2 (34 and 45% inhibition, respectively). Blocking both B7-1 and B7-2 resulted in a more pronounced inhibition of T cell proliferation triggered by anti-CD3 (87% inhibition) or anti-Thy-1 (90% inhibition) mAb, emphasizing the importance of both costimulatory interactions in driving these responses. To confirm that Thy-1 is capable of providing a signal 1 substitute for T cell activation, we coated microbeads with anti-Thy-1 and/or anti-CD28 mAbs and used these microbeads as surrogate APCs to provide T cells with signal 1 and/or 2, respectively. The presence of anti-CD28 mAb on beads was adequate to replace CD28/B7 interactions and dramatically enhanced T cell proliferation when combined with anti-Thy-1 mAb (Fig. 3B). The presence of varying concentrations of anti-Thy-1 mAb together with a constant amount of anti-CD28 mAb on beads caused T cell proliferation in a dose-dependent manner (Fig. 3C). Taken together, these data demonstrate that Thy-1 signaling can substitute for signal 1, and leads to robust T cell activation upon coupling with a strong signal 2.



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  		FIGURE 3. DC enhancement of Thy-1-driven T cell proliferation is dependent on B7 costimulatory molecules. A, T cells were stimulated with anti-Thy-1 mAb plus syngeneic DCs in the absence or presence of anti-B7-1 and/or anti-B7-2 mAb (1:10 hybridoma supernatants) or irrelevant IgGs. Parallel cultures of T cells stimulated with anti-CD3 mAb served as a positive control. T cell proliferation after 72 h of culture was determined by [3H]TdR incorporation. B, T cells were mixed with anti-Thy-1 and/or anti-CD28 mAb-coated microbeads as surrogate APCs, and T cell proliferation was measured 48 h poststimulation by [3H]TdR incorporation. C, T cells were stimulated with microbeads coated with a combination of anti-CD28 (10 µg) and anti-Thy-1 mAb at the indicated doses. T cell proliferation was determined by [3H]TdR incorporation after 48 h of culture. Results are presented as mean ± SD obtained from quadruplicate cultures, and are representative of three independent experiments.

 
T cells stimulated by anti-Thy-1 in the presence of DCs do not exhibit cytotoxic effector function

CD28-B7 costimulatory interactions are known to play an important role in the generation of Ag-specific (31), allogeneic (32), or anti-CD3-induced (33) CTL. We have previously observed that T cells stimulated with anti-Thy-1 mAb alone or in the presence of weak costimulation by B cells and macrophages failed to develop cytotoxic effector function (data not shown). Because DCs potently enhanced Thy-1-driven T cell proliferation and IL-2 synthesis in an Fc{gamma}R-independent and costimulation-dependent manner, it was of interest to determine whether DCs could also contribute to the generation of a cytotoxic phenotype following Thy-1 triggering. Anti-CD3-induced CTL were used as a positive control (33). As expected, anti-CD3-activated T cells exhibited potent cytotoxicity against P815 mastocytoma cells (Fig. 4A). CTL induced with anti-CD3 mAb interact and kill Fc{gamma}R+ P815 cells via anti-CD3 mAb that remains on the T cell surface following the 72-h priming period (Fig. 4B), because the addition of Fc block (5 µg/ml) to the killing assay virtually abrogated lysis of P815 cells (93% reduction in cytotoxicity) by anti-CD3-activated T cells (data not shown). In contrast, T cells stimulated with anti-Thy-1 in the presence of DCs failed to kill P815 mastocytoma cells (Fig. 4A), even though anti-Thy-1 mAb remained on the T cell surface after 72 h of priming (Fig. 4C) and should, therefore, allow interactions with Fc{gamma}R+ P815 cells consistent with previous reports that the G7 anti-Thy-1 mAb redirects CTL clones to lyse Fc{gamma}R+ target cells (34, 35). The failure of Thy-1-triggered T cells to kill target cells was not due to insufficient time for activation because extending the priming time to 5 or 7 days did not result in the development of lytic function (data not shown). Because P815 cells express only minute amounts of Fas (CD95/APO-1) and are resistant to Fas-mediated killing (36), we used Fas-transfected P815 cells (P815-Fas) to rule out the possibility that Thy-1-stimulated T cells may use FasL rather than perforin and granzyme B to induce cell death in target cells. As shown in Fig. 4D, P815-Fas cells were lysed by an agonistic anti-Fas mAb (clone Jo2), and were also more susceptible than their wild-type counterparts to cell death induced by anti-CD3-activated T cells. In contrast, Thy-1-stimulated T cells did not lyse P815-Fas cells, leading us to conclude that Thy-1 triggering does not result in the generation of cytotoxic effector function that is dependent on either cytolytic pathway. This was not a cell line-specific phenomenon because Thy-1-stimulated T cells also failed to kill Fc{gamma}R+ A20 B lymphoma cells (data not shown).



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  		FIGURE 4. Thy-1 stimulation of T cells in the presence of DCs does not generate cytotoxic effector function. A, T cells were stimulated with anti-Thy-1 or anti-CD3 mAb (as a positive control) in the presence of DCs for 72 h. Activated T cells were then harvested, washed, and combined at the indicated E:T ratios with 51Cr-labeled P815 mastocytoma cells. After 4 h, a 100-µl aliquot of the culture supernatants was collected from each well, and specific 51Cr release from the target cells was calculated. B, T cells were incubated with PE-conjugated anti-CD3 mAb (5 µg/ml) and DCs for 72 h. The presence of surface-bound anti-CD3 mAb on activated T cells (open peak) was then determined by flow cytometry in comparison with T cells cultured in the absence of anti-CD3 PE (filled peak). C, T cells were stimulated with anti-Thy-1 mAb plus DCs for 72 h, followed by staining with (open peak) or without (filled peak) FITC-conjugated mouse anti-rat IgG F(ab')2. D, T cells activated with anti-Thy-1 or anti-CD3 mAb (as a positive control) in the presence of DCs were combined with P815 or P815-Fas cells at a 50:1 E:T ratio, and specific 51Cr release from the target cells was used to determine percentage of specific lysis. An agonistic anti-Fas mAb was used as an additional positive control to induce cell death in Fas-positive P815 cells. Data in A and D are given as mean percentage of lysis (±SD) in triplicate wells, and are representative of three similar experiments.

 
The failure of Thy-1-stimulated T cells to destroy P815 cells was despite their ability to efficiently conjugate with P815 target cells (Fig. 5A), and also to express substantial amounts of mRNA transcripts coding for perforin, granzyme B, and FasL (Fig. 5, B and C). However, examination of T cell-target cell conjugates stained with anti-cathepsin D Ab to detect lytic granules by immunofluorescence (25) revealed that, unlike anti-CD3-activated T cells of which 50% exhibited granule polarization, Thy-1-stimulated T cells consistently failed to polarize their lytic granules to the point of contact with target cells (Fig. 6). Collectively, our data indicate that T cells that have received a signal 1 substitute through Thy-1 triggering in combination with a strong, DC-generated signal 2 acquire cytotoxic effector molecules and adhere to target cells, but do not exhibit cytolytic function due to a failure to reorganize T cell secretion machinery.



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  		FIGURE 5. T cells stimulated with anti-Thy-1 mAb in the presence of DCs adhere to target cells, and express cytotoxic effector molecules. A, T cells activated with anti-Thy-1 or anti-CD3 mAb in the presence of DCs were incubated in a 1:1 ratio with neutral red dye-stained P815 cells. Conjugates were then enumerated under a light microscope. The percentage of T cells conjugated with P815 cells (±SD) from triplicate tubes was calculated. B and C, Total RNA was isolated from T cells activated with anti-Thy-1 or anti-CD3 mAb in the presence of DCs after 72 h of culture for detection of perforin (PFN) and granzyme B (GzmB) mRNA transcripts, or after 4 h for detection of FasL mRNA transcripts. Cytotoxic effector molecule mRNA levels were determined by RT-PCR in comparison with the steady state expression of GAPDH. Data are representative of two independent experiments yielding similar results.

 


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  		FIGURE 6. Thy-1-stimulated T cells fail to polarize lytic granules to the T cell-target cell interface. Anti-CD3-activated (A, B) or anti-Thy-1-activated (C, D) T cells were allowed for 30 min to form conjugates with P815 target cells. T cells were then stained with anti-cathepsin D Ab plus Cy3-conjugated anti-rabbit Ab (B, D) or the secondary Ab alone (A, C). Staining of T cell lytic granules was visualized by confocal laser-scanning microscopy. Arrows denote T cells bound to target cells. Results are representative of two independent experiments.

 
TCR signaling redirects Thy-1-stimulated T cells to lyse target cells

We next used anti-CD3 mAb in a redirected lysis assay to determine whether the cytotoxic machinery of Thy-1-stimulated T cells was in fact functional. Fig. 7A illustrates that the TCR/CD3 complex was still present on the surface of anti-Thy-1-activated T cells at the end of the culture period. In contrast, anti-CD3-activated T cells exhibited relatively low immunoreactivity with anti-{alpha}{beta} TCR PE due to the known internalization of the TCR/CD3 complex. Thy-1-stimulated T cells showed significant cytotoxic capability against Fc{gamma}R-bearing P815 cells when anti-CD3 mAb was present during the effector phase of killing (Fig. 7B). A20 B lymphoma cells that are also Fc{gamma}R+ were similarly lysed by anti-Thy-1-activated T cells when anti-CD3 mAb was present in the killing assay (data not shown). In contrast, mitogenic anti-Thy-1 mAb failed to cause the redirected lysis of Fc{gamma}R+ P815 target cells by Thy-1-stimulated T cells (Fig. 7B), even when mouse, rabbit, or goat anti-rat IgG Abs of the IgG isotype that are known to interact with Fc{gamma}Rs were added to the killing assay to maximize cross-linking of surface-bound anti-Thy-1 mAb by target cell Fc{gamma}Rs (data not shown). Several different mAbs to other T cell surface molecules, including hamster anti-mouse CD28 mAb, which has the same isotype as hamster anti-mouse CD3 mAb, also failed to trigger redirected lysis of target cells by Thy-1-stimulated T cells (data not shown). This demonstrates a unique requirement for TCR signaling in the induction of a fully functional cytotoxic phenotype, and suggests that Thy-1 triggering in the absence of TCR ligation is not able to provide a complete form of signal 1 for T cell activation.



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  		FIGURE 7. Thy-1-stimulated T cells exhibit cytotoxic effector function following TCR, but not Thy-1 triggering in a redirected lysis assay. A, Cell surface expression of the TCR/CD3 complex was determined by flow cytometric analysis at 72 h poststimulation with anti-CD3 mAb or anti-Thy-1 mAb in the presence of DCs. PE-labeled anti-{alpha}{beta} TCR mAb was used to stain the TCR/CD3 complex (open peaks) in comparison with background staining (filled peaks) with PE-labeled hamster IgG. B, T cells were stimulated with anti-Thy-1 or anti-CD3 (as a positive control) mAbs in the presence of DCs. Stimulated T cells were harvested at 72 h, washed, and combined with Fc{gamma}R+ P815 target cells at a 50:1 ratio in the presence or absence of anti-CD3 mAb, anti-Thy-1 mAb, or isotype controls. 51Cr release from target cells was used to determine percentage of specific lysis. Data are representative of three similar experiments.

 
Thy-1 triggering can generate signal 2 in the presence of concomitant TCR signaling

Because Thy-1 has been suggested to serve as an accessory molecule for T cell responses (13, 37), we also asked whether Thy-1 triggering might synergize with TCR signaling to induce T cell activation. We stimulated resting T cells with microbeads coated with a suboptimal concentration (0.1 µg/ml) of anti-CD3 mAb plus G7 anti-Thy-1 mAb (10 µg/ml). T cells were also stimulated in parallel with microbeads coated with a combination of anti-CD3 and anti-CD28 mAbs. Beads coated with anti-CD3 mAb plus isotype controls did not stimulate T cell proliferation beyond that induced by anti-CD3 mAb alone (data not shown). Anti-Thy-1 mAb in combination with anti-CD3 mAb induced substantial T cell proliferation similar to that induced following coligation of the TCR/CD3 complex and CD28 (Table I). This finding suggests that Thy-1 stimulation may also act in a costimulatory capacity when signal 1 is supplied via the TCR.


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  		Table I. Thy-1 stimulation enhances T cell activation through TCR

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although T cell activation occurs normally in response to TCR triggering, signaling through Thy-1 has been suggested also to result in T cell activation. Thy-1 cross-linking with certain mAbs leads to T cell proliferation and IL-2 production (10, 11), which are considered to be hallmarks of T cell activation. However, the exact function of Thy-1 in the context of T cell activation has remained puzzling and highly controversial. In this study, we show for the first time that Thy-1 triggering can substitute for signal 1 for T cell activation, which, in combination with a strong signal 2 (delivered by B7 family members on DCs, or by anti-CD28 mAb coated on microbeads), leads to robust T cell proliferation and IL-2 synthesis. Thy-1-driven signal 1 is, however, insufficient to trigger cytotoxic effector function, pointing to a fundamental difference between conventional, TCR-mediated signal 1 and the form of signal 1 that is generated by Thy-1 triggering.

We initially discovered that DCs dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering. To delineate the mechanism(s) accounting for this phenomenon, we first examined the possibility that DCs enhanced Thy-1-driven responses by cross-linking the anti-Thy-1 mAb via their Fc{gamma}Rs. DC expression of Fc{gamma}Rs is known to depend on the DC subtype and state of maturation, the method of cell preparation, and the species from which the DCs are obtained (38, 39, 40). Murine DC lines and primary DCs of the Langerhans cell type reportedly potentiate anti-TCR/CD3-mediated T cell proliferation in an FcR-dependent manner (39, 41). Our results indicate that murine bone marrow-derived DCs also express functional Fc{gamma}Rs, and confirm that DC Fc{gamma}Rs are required for anti-CD3-driven T cell proliferation. Anti-CD3-induced T cell proliferation was largely retained in the presence of FcR{gamma}-/- DCs, which are devoid of Fc{gamma}RI/III, but was almost completely inhibited by mAb-mediated blockade of Fc{gamma}RII/III, suggesting that Fc{gamma}RII is the major Fc{gamma}R used by bone marrow-derived DCs to cross-link anti-CD3 mAb. Interestingly, DC Fc{gamma}Rs were not required for anti-Thy-1-driven T cell proliferation. One possibility is that anti-Thy-1 mAb can by itself cross-link sufficient Thy-1 to elicit an activating signal due to the very heavy expression of Thy-1 on the T cell surface in comparison with the TCR (9). Alternatively, this may simply be a reflection of the physicochemical characteristics of the G7 anti-Thy-1 mAb per se.

Ruling out a possible role for Fc{gamma}Rs in DC enhancement of Thy-1-driven T cell proliferation, we hypothesized that DCs augment Thy-1-mediated responses by providing costimulation through B7 interactions with CD28. Bone marrow-derived DCs display abundant levels of both B7-1 and B7-2 (20), which participate in the costimulation of T cell responses to stimuli such as Con A and anti-{alpha}{beta} TCR mAb (39). Thy-1-driven T cell proliferation similarly depended on both B7-1- and B7-2-mediated costimulation. Our results are in stark contrast with an earlier report that relatively inefficient CD4+ T cell proliferation and IL-2 synthesis occur in response to the G7 anti-Thy-1 mAb in a xenogeneic system that used B7-transfected Chinese hamster ovary cells as accessory cells (42). However, because B7-2 was not identified at the time, the failure of B7-1-transfected Chinese hamster ovary cells to optimally costimulate Thy-1-driven responses could be at least in part due to a lack of CD28-B7-2 interactions. In any case, our use of syngeneic DCs, which naturally express ample B7-1 and B7-2 together with other potentially important costimulatory molecules, provides a more physiologically relevant system for T cell activation. The requirement for B7 costimulation in Thy-1-stimulated responses may explain the failure of freshly explanted B cells to augment T cell proliferation in response to anti-Thy-1 mAb because the B7 expression by B cells is modest in comparison with DCs (19, 43). In fact, LPS-activated B cells, in which the expression of B7-1 and B7-2 is substantially up-regulated (19, 33), could costimulate Thy-1-driven T cell responses, albeit less efficiently than DCs.

Our overall observations are consistent with the hypothesis that Thy-1 triggering can substitute for signal 1 for T cell activation. We have confirmed our findings by using anti-Thy-1 and anti-CD28 mAb-coated microbeads as surrogate APCs and showed that the combination of immobilized anti-Thy-1 and anti-CD28 mAbs could provide signals 1 and 2, respectively, for T cell activation. A costimulatory role for Thy-1 during T cell activation has also been suggested (13, 37). Although we found that Thy-1 triggering enhanced anti-CD3-induced T cell proliferation in the absence of CD28 stimulation, it is very unlikely that T cell activation by immobilized anti-Thy-1 and anti-CD28 mAbs was caused by the combination of two costimulatory signals because we have observed that coligation of multiple costimulatory molecules in the absence of signal 1 does not lead to T cell activation. For example, microbeads coated with mAbs to CD28 and CD2, two known costimulatory molecules, failed to trigger T cell proliferation (our unpublished data). Nevertheless, it is intriguing that Thy-1 stimulation can provide either signal 1 in the absence of TCR signaling or signal 2 in the absence of CD28 signaling.

We have demonstrated that both proliferative and IL-2 responses of resting T cells to Thy-1 triggering are greatly increased by the DC contribution of costimulatory B7 molecules. Because signal 2 delivered through B7-CD28 interactions is known to be important in the generation of CTL responses (30, 33), we analyzed T cell cytotoxic responses following stimulation with anti-Thy-1 mAb in the presence of DCs. Several studies have pointed to a role for Thy-1 in the generation of CTL responses. Preincubation with the G7 anti-Thy-1 mAb was reported by Ozery et al. (44) to enhance the cytolytic activity of several memory-like CTL hybridomas generated in vivo or in MLR. The same anti-Thy-1 mAb was also reported to boost the cytolytic function of OVA-specific CD4+ Th1 clones, as well as alloreactive CD8+ T cell clones in a redirected lysis assay (34). Our data indicate that Thy-1 triggering of primary T cells in the presence of DCs leads to the expression of granzyme B, perforin, and FasL, all of which function as CTL effector molecules (reviewed in Ref.45). The granule-dependent cytolytic pathway relies on the expression and function of granzyme B (and related serine proteases) and perforin, while the death receptor pathway employs FasL to cross-link Fas and activate the death program in susceptible target cells. The up-regulation of perforin and granzyme B gene expression as a result of Thy-1 triggering that we observed conflicts with a recent report that the G7 anti-Thy-1 mAb selectively activates FasL-, but not granule-mediated cytotoxicity by cloned CTL (35). The discrepancies between our findings and those of Kojima et al. may reflect distinct activation requirements for resting T cells vs CTL clones that have already been exposed to Ag at some point and are therefore considered to be primed. As a result, such cells may not accurately represent naive T lymphocytes, which are likely to differ in activation requirements and the capacity to acquire cytotoxic activity. Furthermore, the changes observed by Kojima et al. may not necessarily represent events taking shape during the induction phase of a CTL response. In this regard, up-regulation of FasL expression and that of perforin and granzyme B do not follow the same kinetics. FasL induction takes place within hours of TCR ligation, whereas up-regulation of perforin and granzyme B gene expression is a later event that is at least in part secondary to the production of cytokines such as IL-2 (46, 47). Therefore, short-term exposure to anti-Thy-1 mAb in a redirected lysis assay may not be sufficient to induce perforin and granzyme B expression, while a longer period of T cell incubation with anti-Thy-1 mAb and DCs may allow sufficient costimulation and exposure to cytotoxicity-inducing cytokines to induce the granule-dependent pathway of cytolysis.

Surprisingly, Thy-1-stimulated T cells in our system were unable to lyse Fc{gamma}R+ P815 target cells, despite expressing cytotoxic effector molecules and retaining surface-bound anti-Thy-1 mAb that should allow for redirected lysis of Fc{gamma}R+ target cells. Indeed, the same G7 anti-Thy-1 mAb used in our studies has previously been shown by other investigators to redirect mouse CTL to kill Fc{gamma}R-bearing target cells (34, 35). Moreover, anti-Thy-1-activated T cells readily formed conjugates with P815 target cells with efficiency equal to that of anti-CD3-activated T cells, which exhibited potent cytotoxicity against P815 target cells that was entirely dependent on interactions between T cell-bound anti-CD3 mAb and target cell Fc{gamma}Rs. Importantly, anti-Thy-1 mAb failed to trigger redirected lysis of Fc{gamma}R+ target cells, even when T cell-bound anti-Thy-1 mAb was extensively cross-linked by FcR-reactive IgG molecules (mouse, rabbit, or goat) with specificity for rat IgG. The lack of cytotoxic effector function by Thy-1-stimulated T cells was, therefore, not due to a failure of T cell surface-bound anti-Thy-1 mAb to interact with the target cell Fc{gamma}Rs and thereby bridge the T cell and the target cell. Rather, the defect in cytotoxicity resulted from an inability of Thy-1-stimulated T cells to reorganize their secretion machinery following binding to target cells, as revealed by immunofluorescence examination of T cell-P815 cell conjugates stained with anti-cathepsin D Ab, which localizes to lytic granules (25). In contrast, anti-CD3-activated T cells exhibited strong polarization of lytic granules to the effector-target cell interface. Interestingly, when we added anti-CD3 mAb into the killing assay to redirect the lysis of Fc{gamma}R+ target cells by Thy-1-stimulated T cells, very potent lytic activity was observed. This finding, on one hand, ruled out the unlikely failure of Thy-1-stimulated T cells to express cytotoxic effector molecules at the protein level, and, in contrast, pointed to a requirement for TCR/CD3 triggering in the development of a fully functional cytotoxic phenotype. Thy-1 stimulation in the absence of TCR signaling is therefore not sufficient to induce fully functional CTL. One possible explanation for the failure of Thy-1-stimulated T cells to exhibit cytotoxic effector function is the absence of TCR internalization following Thy-1 triggering. Internalization of the TCR/CD3 complex normally takes place following stimulation with specific Ag or anti-CD3 mAb (48, 49), and may, therefore, be a critical step in the intracellular events that eventually give rise to fully functional CTL.

Our results are particularly important considering the speculations that signaling through Thy-1 occurs via a structural and/or functional association of Thy-1 with the TCR (50, 51). The failure of Thy-1-stimulated T cells to down-regulate TCR and to kill Fc{gamma}R+ target cells provides new evidence for the existence of fundamental differences between TCR- and Thy-1-mediated pathways of T cell activation. Whether such differences are a consequence of distinct intracellular signaling cascades associated with the two pathways is not clear, although differential requirements for the protein tyrosine kinase fyn and the mitogen-activated protein kinase p38 in cytotoxic and proliferative responses associated with the two pathways have been reported (34, 52).

In summary, we have shown that Thy-1 triggering provides an incomplete form of signal 1, which can be coupled with a strong signal 2 provided by DCs to induce T cell proliferation and IL-2 synthesis. This provides a useful model system in which to study the consequences of T cell activation in the absence of traditional TCR signaling. In addition, our data reveal that T cell expansion in the absence of conventional signal 1 is not associated with the development of a complete cytolytic phenotype. This may constitute a mechanism for the maintenance of T cell homeostasis without the risk of developing promiscuous cytotoxicity. Furthermore, we have shown that Thy-1 triggering can also provide signal 2 in the context of TCR-induced T cell activation. To our knowledge, this is the first report that a T cell surface GPI-anchored protein can provide either signal 1 or signal 2 for T cell activation, depending on the availability of other signaling molecule ligands.


   Acknowledgments
 
We thank Julie Bunker for technical assistance.


   Footnotes
 
1 This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Kidney Foundation of Canada. S.M.M.H. was a Killam Scholar and recipient of postgraduate scholarships from the Natural Sciences and Engineering Research Council of Canada and the Dalhousie Inflammation Group. J.S.M. is the recipient of a Cancer Research Training Program Studentship with funding from the Dalhousie Cancer Research Program. Back

2 Address correspondence and reprint requests to Dr. David W. Hoskin, Department of Microbiology & Immunology, Faculty of Medicine, Sir Charles Tupper Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail address: d.w.hoskin{at}dal.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; FasL, Fas ligand; KO, knockout; WT, wild type. Back

Received for publication August 27, 2002. Accepted for publication April 21, 2003.


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 Results
 Discussion
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S. M. M. Haeryfar and D. W. Hoskin
Thy-1: More than a Mouse Pan-T Cell Marker
J. Immunol., September 15, 2004; 173(6): 3581 - 3588.
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