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The Journal of Immunology, 2002, 169: 3661-3666.
Copyright © 2002 by The American Association of Immunologists

B6 Strain Ly49I Inhibitory Gene Expression on T Cells in FVB.Ly49IB6 Transgenic Mice Fails to Prevent Normal T Cell Functions1 ,2

Margaret A. Morris*,{dagger}, Jingxuan Liu*,{dagger}, Veera Arora*, Thaddeus C. George*,{dagger}, Jennifer Klem*,{dagger}, John D. Schatzle*, Vinay Kumar{ddagger} and Michael Bennett3,*

* Department of Pathology, Laboratory of Molecular Pathology, and {dagger} Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and {ddagger} Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitory Ly49 receptors expressed on NK cells provide a mechanism for tolerance to normal self tissues. The immunoregulatory tyrosine-based inhibitory motifs present in some Ly49s are able to transmit an inhibitory signal upon ligation by MHC class I ligands. In our system, as well as others, mice transgenic for inhibitory Ly49 receptors express these receptors on both NK and T cells. FVB (H2q) mice transgenic for the B6 strain Ly49I (Ly49IB6) express the inhibitory Ly49 receptor on the surface of both T and NK cells. Although Ly49I functions to prevent NK-mediated rejection of H2b donor bone marrow cells in this transgenic mouse strain, the T cells do not appear to be affected by the expression of the Ly49I transgene. FVB.Ly49I T cells have normal proliferative capabilities both in vitro and in vivo in response to the Ly49I ligand, H2b. In vivo functional T cell assays were also done, showing that transgenic T cells were not functionally affected. T cells in these mice also appear to undergo normal T cell development and activation. Only upon stimulation with suboptimal doses of anti-CD3 in the presence of anti-Ly49I is T cell proliferation inhibited. These data are in contrast with findings in Ly49A, and Ly49G2 receptor transgenic models. Perhaps Ly49I-H2b interactions are weaker or of lower avidity than Ly49A-H-2Dd interactions, especially in T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine NK and some T cells express a family of receptors termed Ly49 receptors. Within this family of receptors is a subset of inhibitory receptors that contain immunoregulatory tyrosine-based inhibitory motifs in their cytoplasmic domain (1). Upon ligation by MHC class I ligands, these inhibitory receptors can prevent NK cell-mediated lysis of the target cell bearing the ligand (2). This inhibition occurs due to binding of downstream signaling molecules to the phosphorylated immunoregulatory tyrosine-based inhibitory motif consensus sequence (I/VxYxxL/V) of the inhibitory receptor. The phosphatase SHP-1 is key in transmitting the inhibitory signal to the NK cell (2, 3). Once activated, the phosphatase can then cleave phosphorylated products resulting from activation pathways in the NK cell (2, 3).

Although the role of inhibitory receptors in NK cell function has been well defined, the function of these inhibitory receptors in T cells is not well understood. The functional homologs killer Ig-related receptors in humans and inhibitory Ly49 receptors in mice are expressed naturally on a small subset of T cells, including some memory CD8+ T cells (4, 5) and NK/T cells (6, 7). It has also been found that T and NK/T cell function can be affected by Ly49 expression (6, 7). Because these T cell populations are usually small and difficult to manipulate, several groups have concentrated on studying the T cell responses of Ly49 inhibitory receptor transgenic mice. All Ly49 receptor transgenic mice generated to date express the Ly49 receptors on both NK and T cells in abundance (8, 9, 10, 11). The development of these transgenic mouse lines has enabled the study of individual Ly49 inhibitory receptors apart from other members of the Ly49 receptor family. We have previously generated Ly49IB6 (Ly49I) (4) transgenic mice on the FVB (H2q) background. Using these mice, we determined that H2b is a ligand for Ly49I. The majority (95–98%) of the T cells in the FVB.Ly49I transgenic mice express the Ly49I receptor on their cell surface at high levels (10), similar to levels seen on B6 NK cells (12). In this study, we have looked at several aspects of T cell function and development to ascertain the function of Ly49I on T cells in FVB.Ly49I transgenic mice. Other groups have noted that T cell function and proliferation are altered in Ly49A and Ly49G2 transgenic mice (8, 9, 13, 14, 15). Contrary to findings in other inhibitory Ly49 transgenic systems, we do not find defects in T cell function in Ly49I transgenic FVB mice.


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

FVB.Ly49I mice were generated as described (10). B6.Ly49A transgenic mice were kindly provided by C. L. Sentman (Uppsala, Sweden). All strains of mice used were maintained in the Department of Microbiology Animal Colony at University of Texas (Dallas, TX) under conventional conditions, unless otherwise indicated.

Abs and flow cytometry

Staining of fresh and cultured cells from FVB and FVB.Ly49IB6 mice for flow cytometric analysis was conducted as described (10, 16). We used Abs to the B6 strain Ly49I, either anti-5E6 (purified, FITC, PE, or biotin conjugated) or anti-8H7 (FITC or biotin conjugated) developed in our lab. T cells were stained with either anti-CD4 (PE conjugated), anti-CD8 (PE conjugated), or anti-CD3 (APC conjugated) mAbs. Lymph node cells (LNC)4 were stained with APC-labeled anti-CD3, PE-labeled anti-5E6, and FITC-labeled anti-V{beta} Abs. Thymocytes were stained with anti-CD44 and anti-Ly49I Abs (as above), and anti-CD69 and anti-CD25 mAbs. All Abs are from BD PharMingen (San Diego, CA), unless otherwise indicated.

Mixed lymphocyte reactions

Spleens were removed from FVB or FVB.Ly49IB6 mice, and single cell suspensions were made. Splenocytes used as stimulators were irradiated with 2900 cGy using 137Cs {gamma} rays. RBCs were lysed in Tris-ammonium chloride, and nucleated cells were washed and counted. Cultures were set up in either 96-well flat-bottom plates, or 24-well plates, with 7 x 106 responders/ml plated with 5 x 106 irradiated stimulators/ml. Control wells included responders plated with medium only, as well as stimulators plated with medium only. Depending upon the assay performed, cells were cultured from 3–5 days at 37°C and 5% CO2/air. To assay for proliferation, the cells were pulsed with 1 µCi [3H]thymidine after 72 h in culture. Nuclei of cells were harvested 18 h later using an automatic cell harvester; the filters were allowed to dry; scintillation fluid was added; and filters were counted in 2 ml scintillation fluid in a liquid scintillation counter (Beckman Instruments, Fullerton, CA). Uptake of [3H]thymidine allows for measurement of growth of responder cells in the MLR.

CFSE labeling of T cells

Fresh splenocytes were removed from either FVB or FVB.Ly49IB6 mice. T cells were enriched by negative selection over a VarioMacs CS column (Miltenyi Biotec, Auburn, CA). Briefly, FcR were blocked on splenocytes using purified anti-CD16/32 (2.4G2) before incubation with biotinylated Abs for 20 min at 4°C (anti-CD11b, anti-Gr1, anti-DX5, anti-B220, and anti-Ter119). Cells were washed and incubated with magnetic streptavidin microbeads (Miltenyi Biotec) for 15 min at 4°C. After washing, the cells were run over a magnetic depletion column. The negative selection process yielded T cells that were 95–98% pure. CFSE labeling of T cells was done by resuspending enriched T cells at 5–10 x 106 cells/ml in a 10 nM solution of CFSE. The cells were incubated at room temperature for 10 min, and washed twice.

Aliquots of the enriched CFSE-unlabeled and CFSE-labeled T cells were cultured on ELISA plates with plate-bound anti-CD3 at various concentrations. Groups also included wells that contained both plate-bound anti-CD3, as well as anti-Ly49I (8H7) mAbs. Proliferation of T cells (decrease in mean fluorescence intensity of CFSE) was assessed by staining cultured cells with APC-labeled anti-CD3 and PE-labeled anti-5E6 mAbs. The cells were then analyzed by flow cytometry.

Immunoprecipitation and Western blotting

T cells were enriched as described above. Enriched cells were treated with a pervanadate solution (0.1 M sodium orthovanadate, 3% hydrogen peroxide) for 10 min at 37°C. Cells were harvested, washed, and lysed in 1% Triton X-100 at 4°C. Lysates were precipitated with anti-Ly49I (5E6, 5 µg/9 x 106 cells), and run on a 10% SDS-PAGE gel under reducing conditions. B6 lymphokine-activated killers (LAKs) were used as a positive control for phosphorylation. Briefly, B6 splenocytes were depleted of RBCs (described above), and cultured in the presence of 1 ng/ml murine rIL-12, 150 ng/ml human rIL-15, and 100 ng/ml murine rIL-18 at 3 x 106 cells/ml in complete DMEM for 5 days at 37°C and 10% CO2/air. Proteins were transferred to a nitrocellulose membrane and immunoblotted with HRP-conjugated antiphosphotyrosine.

Lymph node cell (LNC) and bone marrow cell (BMC) transplants

LNC and BMC transplants were conducted in a similar manner (17). For the LNC transplants, however, LNC harvested from donor mice were assayed for their ability to mount a graft-vs-host response. Host mice were irradiated with 800 cGy (lethal dose). They were then injected i.v. with 2–2.5 x 106 LNC from specified donors. After 5 days, host mice were injected i.p. with 10-7 M fluorodeoxyuridine to inhibit endogenous thymidine synthesis, followed 20–60 min later with 0.3 µCi thymidine analog, 125I-labeled iododeoxyuridine (125I-UdR). Spleens were removed from these mice at least 2 h later, and soaked in 70% ethanol to elute non-DNA-bound 125I. Percentage of 125I-UdR uptake was calculated, with higher percentage of 125I-UdR uptake indicating greater growth of donor cells. The graft-vs-host response of FVB and FVB.Ly49IB6 LNC, as well as of B6 and B6.Ly49A LNC, was compared in hosts bearing MHC class I ligands for the two Ly49 inhibitory receptors. Bone marrow transplantation was conducted as described (17), with slight modifications. Groups of mice were primed with 10 x 106 splenocytes from donor mice indicated to promote a memory T cell response during the bone marrow transplant. Some mice were treated with 1.25 mg/mouse with anti-CD8 (2.43) on days 4, 2, and 1 before transplantation, and 15 µl/mouse antiasialo GM1 serum at the time of transplantation, to deplete CD8+ T cells and NK cells, respectively. Host mice were irradiated with 800 cGy just before transplantation, and infused with 2 x 106 donor BMC i.v. Proliferation of transplanted BMC in recipients was judged in terms of splenic uptake (%) of 125I-UdR, as described above, 5 days after cell transfer (17). The percentage of injected 125I-UdR incorporated into each spleen was calculated and converted to log10 values. Geometric mean (95% confidence limits) values of groups (four to six mice) are presented. The significance of differences between any two groups was calculated by parametric and nonparametric methods, using the VAX computer UTSTAT NGROUP program provided by the Academic Computing Service at University of Texas.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of FVB.Ly49I T cells

Several groups studying Ly49A transgenic mice have found that T cells expressing NK cell inhibitory receptors are severely impaired in proliferation and function in response to MHC class I ligands. We have previously generated FVB.Ly49I mice in which the majority of T cells express Ly49I (10). To determine the effect of Ly49I expression on T cells of these mice, we assayed their proliferative response to ligand-bearing allostimulatory (H2b) cells in a one-way MLR. Fig. 1Go shows that bulk FVB.Ly49I responders are not inhibited from proliferating in response to alloantigens, even when they are exposed to known H2b MHC class I ligands for the Ly49I inhibitory receptor (10). Both FVB and FVB.Ly49I T cells proliferate equally well when assayed by MLR with [3H]thymidine uptake. These data differ from results using cells from B6.Ly49A transgenic mice (8, 9).



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  		FIGURE 1. Proliferative response of T cells to alloantigens in vitro. Single cell suspensions of whole spleens from nonimmunized FVB or FVB. Ly49IB6 mice were cultured in the presence of irradiated stimulator splenocytes from FVB (H2q) or B6 (H2b) mice. After 72 h in culture, cells were pulsed with [3H]thymidine, and harvested 18 h later. Data are expressed as mean cpm ± SD of [3H]thymidine uptake, and are representative of three experiments.

 
To determine whether the T cells of these mice responded differently in vivo, we performed LNC transplants to assess the graft-vs-host response of B6 and B6.Ly49A T cells exposed to a known ligand for Ly49I (Fig. 2GoA). The B6 LNC proliferate strongly in response to H2d alloantigens. However, B6.Ly49A LNC are significantly inhibited from proliferating after exposure to the Ly49A ligand, H2-Dd, expressed by BALB/c mice (p < 0.05). Also studied were FVB and FVB.Ly49I LNC to emphasize the difference seen in the two transgenic models (Fig. 2GoB). There was no difference in the proliferative capabilities of FVB vs FVB.Ly49I T cells in response to H2b alloantigens.



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  		FIGURE 2. Proliferative response of T cells to alloantigens in vivo. Donor LNC were assayed for proliferation in H2-identical and allogeneic irradiated hosts, as described in Materials and Methods. A, Growth of B6 and B6.Ly49A donor LNC is assessed in syngeneic B6 and allogeneic BALB/c hosts. B6.Ly49A LNC are significantly impaired in their response to H2d hosts. B, Growth of FVB and FVB.Ly49I donor LNC is assessed in syngeneic FVB and allogeneic B6 hosts. *, Mean values are statistically different (p < 0.05). Data are representative of three experiments.

 
To determine whether FVB.Ly49I T cells are inhibited from proliferating in response to anti-CD3 stimulation, we used CFSE-labeled responder T cells, and plated them with varying levels of anti-CD3 in the presence of a fixed concentration of anti-Ly49I (8H7). At optimal doses of anti-CD3 stimulation, proliferation of FVB and FVB.Ly49I T cells was equivalent (Fig. 3GoB). However, as the dose of anti-CD3 was decreased to suboptimal levels, we observed a slight inhibition of FVB.Ly49I T cells compared with FVB T cells (Fig. 3GoC). Negative (CFSE labeled without stimulation) and positive (CFSE labeled with anti-CD3 stimulation alone) controls for proliferation are shown in Fig. 3GoA. Upon careful titration of anti-CD3 and anti-Ly49I against one another, we saw similar results to those shown in Fig. 3Go (data not shown). High doses of anti-Ly49I (20–25 µg/ml) were needed to show partial inhibition of suboptimal anti-CD3 stimulation. Inhibition by anti-Ly49I cross-linking was not seen in doses of anti-CD3 higher than 5 µg/ml (data not shown).



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  		FIGURE 3. Response of FVB and FVB.Ly49I T cells when exposed to anti-CD3 and anti-Ly49I mAbs. Enriched T cells were labeled with CFSE and plated on ELISA plates. A, Positive and negative controls, with each indicated by arrows. Negative controls are cells left untreated, and both FVB and FVB.Ly49I T cells are shown with high levels of CFSE present. Positive controls, also shown in A, are cells treated with a high dose of plate-bound anti-CD3. Multiple peaks represent multiple cell divisions of both FVB and FVB.Ly49I T cells. B, Histograms of an optimal (20 µg/ml) dose of plate-bound anti-CD3. C, Histograms of a suboptimal (1 µg/ml) dose of plate-bound anti-CD3. Both B and C show cells cultured in the presence of a fixed concentration (20 µg/ml) of anti-Ly49I. In each histogram, FVB cells are gated on CD3+Ly49I- cells, FVB.Ly49I cells are gated on CD3+Ly49Ibright, and CFSE levels are compared. Dotted lines represent CFSE levels of FVB.Ly49I T cells, while solid lines represent FVB T cells. Data are representative of three experiments.

 
Due to the inhibition seen above, we wanted to ensure that Ly49I expressed by T cells could be phosphorylated. To answer this question, we isolated T cells and treated the cells with pervanadate. Cells were lysed and lysates were precipitated with the anti-Ly49C/I mAb 5E6 (Fig. 4Go). The 5E6 mAb was used to precipitate Ly49I in lieu of 8H7 because it has been previously shown to work in immunoprecipitations. Although this Ab recognizes Ly49C and I of the B6 strain, expression of Ly49I dominates over that of Ly49C. In previous studies, we have shown that the Ly49C-specific Ab does not react with FVB cells (10). Therefore, Ly49I should be the only receptor precipitated from FVB.Ly49I cells using the 5E6 mAb. Pervanadate-treated B6 LAK lysates were used as a control for the immunoprecipitation of phosphorylated Ly49I. Western blots of both samples were probed with HRP-conjugated anti-phosphotyrosine mAbs, followed by detection with chemiluminescent reagents. The data indicate that the B6 strain Ly49I can be phosphorylated in both T and NK cells.



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  		FIGURE 4. Immunoprecipitation of Ly49I from pervanadate-treated T cells. B6 LAKs and enriched T cells were treated with pervanadate. Cells were lysed and precipitated with anti-Ly49I (5E6), run on a 10% SDS-PAGE gel, and blotted with antiphosphotyrosine. Data are representative of three experiments.

 
Memory T cell function of FVB.Ly49I mice

To assay for memory T cell function in our transgenic mice, we performed bone marrow transplantation assays using groups of FVB and FVB.Ly49I mice primed with alloantigens before transplantation. Nontransgenic FVB mice were primed with the BALB/c (H-2d) donor splenocytes 5–7 days before transplantation. To determine whether antiasialo GM1 is depleting T cells in addition to NK cells, we studied the effect of the Ab on T cell responses to BALB/c alloantigens, because H2d BMC are not rejected by FVB NK cells (10). One group of these primed mice was treated with anti-CD8 before transplantation to deplete CD8+ T cells. Another group of the primed mice was treated with antiasialo GM1 at the time of transplant. It appears that treatment with antiasialo GM1 has little effect on the T cell response of primed FVB mice, and that the T cell response is mediated mainly by asialo GM1-CD8+ T cells (Fig. 5Go).



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  		FIGURE 5. Bone marrow transplantation assay to assess memory T vs NK cell function. Groups of wild-type FVB mice were primed, as indicated with 10 x 106 BALB/c splenocytes 5–7 days before BMC graft. Anti-CD8 (2.43) mAbs were injected i.p. on days 4, 2, and 1 before transplant to the indicated group. A total of 2 x 106 BALB/c BMC was injected i.v. on day 0. The indicated group was given antiasialo GM1 at the time of transplant. Statistical significance between mean values is denoted by *, p < 0.05. Data are representative of four experiments.

 
To determine whether FVB.Ly49I T cells respond functionally to alloantigens on cells bearing MHC class I ligands for Ly49I, we performed a bone marrow transplant similar to that performed in Fig. 5Go. We used B6 (H-2b) splenocytes to prime the indicated groups of mice, and B6 donor BMC to assay the memory T cell response (Fig. 6Go). Nontransgenic FVB mice elicit a strong NK cell response to B6 donor BMC, as there is no growth of B6 donor cells in FVB mice after 5 days. Rejection of B6 BMC is more vigorous in FVB mice primed with B6 splenocytes before transplant. We used antiasialo GM1 serum to abrogate the NK cell response without inhibiting the T cell response. Because the NK cells of FVB.Ly49I mice are unable to respond to B6 alloantigens, the response of primed FVB.Ly49I mice is due solely to the primed T cells. The T cell response of FVB.Ly49I mice equals that seen in primed FVB mice treated with antiasialo GM1 serum. Therefore, these data suggest that Ly49I is not functionally inhibiting T cell responses to alloantigens bearing MHC class I ligands in FVB.Ly49I transgenic mice.



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  		FIGURE 6. Bone marrow transplantation to assess memory T vs NK cell responses. B6 donors (known ligand) were used for priming and challenge. Shown were both primed and unprimed groups of FVB and FVB.Ly49I mice (indicated in graph). Antiasialo GM1 serum treatment of primed FVB mice is indicated. Statistical significance is indicated by *, p < 0.05. Data are representative of three experiments.

 
T cell repertoire of FVB.Ly49I T cells

Several groups have also looked at the process of T cell selection in Ly49 receptor transgenic mice. Ly49A transgenic T cells developing in the presence of MHC class I ligands are unable to go through proper negative selection, leading to the development of autoreactive T cells in the periphery (18, 19). Skewing of the V{beta} repertoire (18) is also seen in Ly49A transgenic mice. To look for alterations in T cell repertoire, we stained LNC from wild-type FVB and FVB.Ly49I mice with Abs against the V{beta} Ags (Fig. 7GoA). There are no major alterations in the V{beta} repertoire of our transgenic mice. This is especially intriguing in light of the evidence indicating that MHC class I Ags of FVB mice (H-2q) are ligands for Ly49I (11, 20). Thymocytes from the two strains were analyzed for phenotypic differences in T cell development (Fig. 7GoB). No significant phenotypic differences were seen in the development of FVB.Ly49I T cells when compared with wild-type FVB T cells. Splenic T cells were tested for expression of markers of memory or activation. FVB and FVB.Ly49I mice had very similar phenotypes (Table IGo).



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  		FIGURE 7. Phenotyping of FVB vs FVB.Ly49I LNC and thymocytes. A, LNC of wild-type and transgenic mice were stained with anti-CD3 and a panel of anti-V{beta} Abs. Shown are the percentages of V{beta}+ cells when gated on CD3+Ly49I- (FVB) or CD3+Ly49I+ (FVB.Ly49I) cells. Percentage of FVB LNC stained is indicated by open bars, whereas FVB.Ly49I LNC are represented by shaded bars. B, Thymocytes of FVB and FVB.Ly49I mice were stained with various Abs indicated to look for skewing of memory vs activated T cell repertoire. FVB thymocytes are gated on Ly49I- populations; FVB.Ly49I thymocytes are gated on Ly49I+ populations. Data are representative of four experiments.

 

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  		Table I. Comparison of splenic T cell activation in FVB and FVB.Ly49I micea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of transgenic Ly49 inhibitory receptor has been shown to result in a loss of T cell function and tolerance. Although Ly49A serves as the strongest example of this inhibition (8, 14, 18, 19, 21, 22), Ly49G2 has also been shown to inhibit T cell function (15). Overexpression of Ly49A on T cells leads to impairment of T cell response to allogeneic cells bearing ligands for Ly49A (8, 22), impaired antitumor responses (14), altered cytokine production (13, 21), and impaired antiviral responses (21). Several groups have also shown that T cell development is also affected in Ly49A transgenic mice (18, 19), which can lead to severe autoimmune disorders.

In this study, we have investigated the effect of Ly49I expression on T cells in FVB.Ly49I transgenic mice. Although many labs have shown that T cell responses are inhibited in Ly49 receptor transgenic mice, we have not found that to be the case in FVB.Ly49I transgenic mice. This could be due in part to the mechanism by which the Ly49 receptors bind MHC class I molecules. Ly49A is the most widely studied Ly49 receptor with regard to its binding to MHC class I molecules (23, 24, 25). There is some controversy concerning the binding site of Ly49A. Matsumoto et al. (24) suggested that the functional binding site of Ly49A overlaps with that of the CD8 receptor, while others have suggested that Ly49A binds to the {alpha}1 and {alpha}2 regions of the MHC class I molecules (26). It has been documented that Ly49A and Ly49D recognize and presumably bind different epitopes of H2d MHC class I molecules (27). There is some evidence that each of the Ly49 receptors binds to unique sites on the MHC class I molecule, as some of the Ly49 receptors can differentiate the peptides bound in the groove of the MHC class I molecules (11, 28), while other receptors cannot. It is possible that Ly49I does not bind to this site and is therefore unable to prevent binding of the CD8 receptor to MHC class I molecules. This would allow the T cell to function in a normal manner. Ly49A, in contrast, may prevent CD8-MHC class I interactions and be cross-linked by its binding to MHC class I ligands, leading to T cell inhibition. Although differences in binding sites may be an attractive explanation for differences in T cell responses of Ly49 receptor transgenic mice, it does not provide an explanation for the inhibition seen in CD4+ T cells.

Another reason Ly49IB6 might not be able to inhibit T cell responses is due to strength of signal and/or binding. It is possible that Ly49I is able to bind to the same site as Ly49A on the MHC class I molecule, but its binding avidity is lower than that of Ly49A, leading to transmission of a weaker inhibitory signal. This may be a reasonable explanation, given the difficulty studying Ly49I in some in vitro binding assays (29). Daws et al. (30) have shown that engagement of Ly49A by H2-Dd leads to phosphorylation of the receptor. We detected modest inhibition of proliferation upon cross-linking of FVB.Ly49I T cells with the 8H7 mAb and phosphorylation of Ly49I following pervanadate stimulation. Thus, Ly49I might be functional in T cells of FVB.Ly49I mice. However, Ly49I may not bind strongly enough to yield a signal capable of overcoming signals transmitted by the TCR. Ly49I may also transmit a weaker signal upon cross-linking than the signals transmitted by Ly49A.

Further studies must be completed to determine whether Ly49I functions on memory T cells. Although studies comparing naturally expressed Ly49A and Ly49I inhibitory receptors on memory T cell function would be ideal, they would also be difficult undertakings. These studies will be made easier by crossing FVB.Ly49I transgenic mice with TCR transgenic mice. These mice will provide us with a highly enriched population of memory T cells recognizing a specific Ag. We are also in the process of backcrossing FVB.Ly49I transgenic mice to the B6 background. It is possible that strain differences might affect T cell as well as NK cell function.

Although the studies of T cells in FVB.Ly49I transgenic mice are by no means exhaustive, they suggest that Ly49I, while strong enough to signal and function in NK cells, may not be able to signal with enough vigor to inhibit T cell signaling through the TCR. We did not obtain evidence that T cells in transgenic mice are inhibited under a variety of experimental conditions, other than Ab cross-linking. Our data also suggest that each Ly49 receptor must be considered on an individual basis when considering its function.


   Acknowledgments
 
We thank Silvio and Maria Peña for their excellent animal husbandry.


   Footnotes
 
1 This work was supported by Grants CA36922, AI38938, CA70134, AI20451, and CA09082 from the National Institutes of Health. Back

2 A preliminary report of this work appeared in FASEB J., 2001. Back

3 Address correspondence and reprint requests to Dr. Michael Bennett, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9072. E-mail address: Michael.Bennett{at}UTSouthwestern.edu Back

4 Abbreviations used in this paper: LNC, lymph node cell; BMC, bone marrow cell; 125I-UdR, 125I-labeled iododeoxyuridine; LAK, lymphokine-activated killer. Back

Received for publication December 5, 2001. Accepted for publication July 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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