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The Journal of Immunology, 2000, 165: 1871-1876.
Copyright © 00 by The American Association of Immunologists

Transgenic Expression of Ly49A on T Cells Impairs a Specific Antitumor Response1

Pierre Brawand*, François A. Lemonnier{dagger}, H. Robson MacDonald*, Jean-Charles Cerottini* and Werner Held2,*

* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and {dagger} Unité d’Immunité Cellulaire Antivirale, Département SIDA-Rétrovirus, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Receptors specific for MHC class I play important roles in regulating the activity of NK cells. Inhibitory MHC receptors allow NK cells to react to and eliminate autologous cells that lack expression of MHC class I molecules on target cells. This so-called "missing self" recognition may provide protection in situations that result in down-regulation of MHC class I molecules due to infection or tumor development and that would circumvent T cell-mediated immunity (1).

Inhibitory receptors for classical MHC class I molecules in the human belong to the killer inhibitory receptor (KIR)3 family that are Ig-like, type I cell surface receptors (2). In contrast, mice use Ly49 receptors that are type II transmembrane glycoproteins with a C-type lectin-like domain (3). Irrespective of their structural differences, both types of receptors can discriminate alleles of MHC class I molecules (4). For example, Ly49A interacts with H-2Dd but not Db class I molecules. Consequently NK cells expressing Ly49A are unable to kill H-2Dd, whereas they kill Db-expressing target cells (5). In general, Ly49 receptors exhibit reactivities to many MHC class I haplotypes. Only few receptors are selective for certain MHCs (6). In addition, both human and mouse NK cells express lectin-like inhibitory receptors specific for the nonclassical class I molecules HLA-E and Qa-1b, respectively (7, 8).

MHC class I-specific receptors in the mouse were first defined in the context of NK cell function (5). However, cell types other than NK cells also express these receptors. Indeed, both NK T and occasional NK1.1- T cells display Ly49 receptors (9, 10) (P. Brawand and W. Held, unpublished observation). Ly49 expression regulates NK T cell development and can influence T cell functions in vitro (9, 10).

In the human, the expression and function of KIR on T cells have been analyzed in more detail. These cells represent a subset of memory phenotype, mostly CD8+ T cells (11, 12). Interestingly, KIR-positive and -negative T cells were shown to share Ag-specificities (13), supporting the view that T cells expressing inhibitory MHC receptors represent a particular state of T cell activation (14).

Very little information is currently available regarding the reactivity of T cells expressing MHC inhibitory receptors in vivo. Here we have studied an antitumor response that is directed against a dominant CD8+ T cell epitope in mice expressing the Ly49A receptor. The presence of this receptor on all conventional T cells completely prevented the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor.


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

The generation and analysis of Ly49A transgenic (Tg) mice was described before (15, 16). Here we have used Tg and non-Tg littermate mice from C57BL/6 (B6) backcross (bc) 7 and 8. The generation of Db-/- (B6 bc12), Kb-/- (B6 bc12), and Db-/-Kb-/- mice (B6 bc6) was described before (17). All the above mice had a NK gene complex of B6, and their MHC class I status was controlled by flow cytometry using specific mAbs. B6 mice deficient for ß2-microbulin (B6.ß2m-/-) were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 and B10.D2 (H-2d) mice were purchased from Harlan Olac (Bicester, U.K.).

Immunizations

Moloney murine leukemia virus (M-MuLV)-infected MBL-2 (H-2b) tumor cells were maintained by weekly passage in syngeneic B6 mice (18). Rauscher murine leukemia virus-infected RMA (H-2b) tumor cells transfected with the human B7.1 gene (RMA/B7.1) were kindly provided by Dr. P. Dellabona (19).

For primary immunization, 40 x 106 irradiated (10,000 rad) MBL-2 cells were injected i.p. After 3–4 wk, secondary responses were elicited by i.p. injection of 10 x 106 viable MBL-2 cells. For RMA/B7.1 immunizations, recipient mice were anesthetized by injecting i.p. 200 µl of PBS containing Ketalar (Parke-Davis, Morris Planes, NJ) (2 mg) and Dormicum (Roche Pharma, Reinach, Switzerland) (0.1 mg). After shaving the back of the mice, 10 x 106 viable RMA/B7.1 tumor cells were injected s.c. into four different sites.

In vivo depletions

Mice were injected i.p. with purified mAb to CD4 (GK1.5) (600 µg), CD8 (H35) (600 µg), or NK1.1 (PK136) (100 µg) in 500 µl PBS 2 days before priming and/or challenge. At the time of immunization, residual CD4+, CD8+, or NK cells usually represent <1–2% of the initial T or NK cells population. Control mice were injected with PBS.

Mixed lymphocyte:tumor cell cultures (MLTC)

Virus-specific CTL were generated in vitro in a 5-day MLTC (20). Responder spleen cells (25 x 106) from M-MuLV-immune mice and 1 x 106 irradiated MBL-2 cells were cocultured in 15 ml of DMEM (Life Technologies, Paisley, U.K.) supplemented with 2 x 10-3 M L-glutamine, 2 x 10-2 M HEPES, 3 x 10-5 M 2-ME, antibiotics, and 5% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA). Cells recovered from MLTC were washed and restimulated with irradiated MBL-2 and syngeneic feeder cells for a further 7 days in complete medium supplemented with 30 U/ml of IL-2 (EL-4 cell supernatant). CTL clones were established by plating MLTC cells at limiting dilution as described previously (21).

Cytotoxic assays

MLTC cells derived from M-MuLV-immune Ly49A Tg or control littermate mice were used as effector cells. Target cells were EL-4 lymphoma (H-2b). The Friend/Moloney/Rauscher gag-encoded epitope CCLCLTVFL (22) was synthesized and purified by standard procedures and dissolved in DMSO supplemented with 2-ME. For cytotoxic assays, effector cells and 51Cr-labeled target cells were mixed at different ratios in the presence or absence of various concentrations of peptide. Supernatants were harvested after 4 h, and specific 51Cr release was calculated as described previously (23).

Flow cytometry

Mice were tail-bled, and PBL were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden). PBL, nylon wool-nonadherent spleen cells, or MLTC were incubated with 2.4.G2 ({alpha}CD16/32) hybridoma supernatant to reduce background. Lymphocytes were stained using appropriately labeled mAbs to NK1.1 (PK136), H-2Db (KH-95), H-2Kb (AF6–88.5), H-2Dd (34.2.12), CD8{alpha} (53-6.7), CD62L (Mel-14), TCRß (H57), Vß5 (MR9–4), V{alpha}3.2 (RR3–16) (PharMingen, San Diego, CA), or Ly49A (JR-9.318) (24). Samples were run on a FACScalibur (Becton Dickinson, San Jose, CA) equipped with the CellQuest software, and viable cells were identified on the basis of light scatter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ly49A Tg mice

The ability of Ly49-expressing T cells to mount an Ag-specific immune response was studied using Ly49A Tg mice. As shown in Fig. 1Go, these mice express Ly49A on virtually all their CD8+ T cells (as well as CD4+ T, NK T, and NK cells, data not shown) at high (line 2) and intermediate (line 12) levels, respectively. Based on the mean fluorescence intensity (MFI) of Ly49A staining of PBL, Tg Ly49A expression on T cells in line 12 corresponds approximately to Ly49A levels on the occasional Ly49A+ CD8+ T cells and the respective NK and NK T cell subsets of non-Tg littermate-derived cells. Ly49A levels on T cells of line 2 were ~2-fold higher.



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  		FIGURE 1. Ly49A expression by CD8+ T cells from Ly49A Tg mice. Nylon wool-nonadherent spleen cells from Ly49A Tg (lines 2 and 12) and non-Tg littermate mice were stained with mAbs directed against CD8, NK1.1 and Ly49A, or a control (anti-H-2Dd) mAb. The percentage of positive cells among different lymphocyte subsets and the MFI of Ly49A staining with mAb JR-9.318 are indicated.

 
In Ly49A Tg mice of H-2b haplotype, i.e., in the absence of a known Ly49A ligand, the peripheral and thymic T cell compartments displayed normal cellularity and T cell subset composition (15) (data not shown). Thus, the presence of the transgene does not grossly perturb T cell development.

Ly49A Tg mice fail to reject M-MuLV-infected tumor cells

To study an Ag-specific immune response by Ly49-expressing T cells, Ly49A Tg mice (of H-2b haplotype) were injected with irradiated syngeneic M-MuLV-infected MBL-2 tumor cells. Two weeks later, the immunized mice were challenged with viable MBL-2 cells. Similar to B6 mice, most non-Tg littermate mice rejected the tumor. Surprisingly, however, Ly49A Tg mice were unable to eradicate the tumor cells (Table IGo). This is unlikely to be due to the transgene integration site because both Tg lines failed to reject the tumor cells. Rather, the expression of the Ly49A Tg on lymphoid cells prevented an effective antitumor response. This result was entirely unexpected because H-2b haplotype mice do not express a Ly49A ligand that is able to regulate NK cell function (25).


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  		Table I. Impaired rejection of MBL-2 tumor cells by Ly49A transgenic mice

 
Ly49A Tg mice are unable to mount a response against the immunodominant M-MuLV epitope

We next wished to ascertain whether Tg mice were able to mount an antitumor T cell response. Thus we have assessed the expansion of tumor-specific CD8+ T cells that normally follows the immunization of B6 mice with MBL-2 tumor cells. These CD8+ T cells recognize a single immunodominant M-MuLV-derived epitope (CCLCLTVFL) in the context of H-2Db. They use TCR V{alpha}3.2 and Vß5 segments and are responsible for tumor rejection (23). Thus, MBL-2-challenged Ly49A Tg mice were bled 7 days after the second injection and PBL were analyzed for the expansion of V{alpha}3.2+Vß5+ CD8+ T cells. As expected, non-Tg mice showed a dramatic increase of their V{alpha}3.2+Vß5+CD8+ T cells (Fig. 2Go), which was confined to the activated (CD62L-) subset (not shown). In contrast, Tg mice did not reveal any sign of a specific response because V{alpha}3.2+Vß5+ cells represented only 1–2% of activated CD8+ T cells. Similar values were in fact observed in nonimmune mice (Table IIGo and Fig. 2Go). Thus, the failure to reject the tumor by Tg mice correlates with an inability to mount a specific CD8+ T cell response.



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  		FIGURE 2. Ly49A Tg mice immunized with M-MuLV-infected (MBL-2) tumor cells fail to mount a specific CD8+ T cell response. PBL from naive and M-MuLV-immune Ly49A Tg (lines 2 and 12) and non-Tg littermate mice were stained with mAbs against CD8, NK1.1, V{alpha}3.2, and Vß5. CD8+NK1.1- cells were analyzed for the expression of V{alpha}3.2 and Vß5. Numbers indicate percentage of positive cells in the respective quadrants.

 

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  		Table II. M-MuLV-specific CD8+ T cells fail to expand in Ly49A Tg mice1

 
The absence of a specific CD8+ T cell response in MBL-2-challenged Ly49A Tg mice is not simply due to the lack of V{alpha}3.2+Vß5+CD8+ T cells in these mice. The percentage of such T cells in naive Tg mice is at least as high or in line 2 even higher (2.7 ± 1.1% of CD8+) compared with non-Tg or B6 mice (1.1 ± 0.4 and 0.9 ± 0.2 of CD8+, respectively) (Table IIGo). These data further suggest that the TCR repertoire is significantly perturbed in Tg line 2.

Specific CD8+ cells arise in one Ly49A Tg line after multiple Ag exposures

Because Ly49A Tg mice were unable to reject viable MBL-2 tumor cells, these mice were immunized twice with 40 x 106 irradiated MBL-2 cells. Subsequently, spleen cells from immune mice were restimulated with Ag-bearing cells in vitro. As expected, all B6 and the vast majority of the non-Tg-derived MLTC contained a large fraction of V{alpha}3.2+Vß5+CD8+ T cells (data not shown). Interestingly, we also observed a significant expansion of V{alpha}3.2+Vß5+CD8+ cells in two of three cultures from Tg line 12. In contrast, no such response was observed using Tg line 2. V{alpha}3.2+Vß5+ T cell clones obtained from cultures of line 12 were in fact specific for the H-2Db-restricted nonapeptide CCLCLTVFL. The addition of this peptide promoted the lysis of EL-4 (H-2b) target cells in a dose-dependent manner by Tg line 12 similar to non-Tg littermate-derived clones (Fig. 3Go). No specific clones were obtained from line 2-derived cultures. Therefore, specific CD8+ T cells are present in Ly49A Tg mice; however, multiple exposures to Ag are required before a response is detected. Moreover, the specific response was completely prevented by high cell surface levels of the Ly49A receptor.



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  		FIGURE 3. V{alpha}3.2+Vß5+ CD8+ CTL clones expressing Ly49A recognize the immunodominant M-MuLV-derived epitope. MLTC were used to derive CTL clones from an immunized Ly49A Tg (line 12) and a control non-Tg littermate mouse. The cytotoxic activity of representative V{alpha}3.2+Vß5+ CD8+ clones was tested at an effector to EL-4 target cell ratio of 3:1 in the presence of increasing concentrations of the M-MuLV gag-derived peptide CCLCLTVFL.

 
The failure of specific Ly49A Tg CD8+ T cells to expand reflects deficient CD4+ T cell help

The Ly49A transgene is expressed by most lymphoid cells including all CD8+ as well as CD4+ T cells (15, 16). Thus, the failure to reject MBL-2 tumor cells by Ly49A Tg mice may be due to a defective CD8+ T cell compartment. Alternatively, CD8+ T cells may lack appropriate T cell help. The rejection of MBL-2 tumor cells depends indeed on T cell help, as it is completely prevented in the absence of CD4+ T cells (Table IGo) (26).

To discriminate between the above possibilities, we circumvented the requirement for T cell help by assessing the CD8+ T cell response to RMA tumor cells that are transfected with the gene encoding the costimulatory molecule B7.1 (RMA/B7.1) (19). Indeed, the expression of B7.1 is required for rejecting RMA cells (data not shown) (27, 28). Similar to MBL-2, RMA cells, which are infected with Rauscher MuLV, express the immunodominant CCLCLTVFL epitope (23). The s.c. injection of 10 x 106 live RMA/B7.1 tumor cells into B6 mice results in an expansion of V{alpha}3.2+Vß5+CD8+ cells (Fig. 4Go) and tumor rejection (Table IIIGo). In contrast to MBL-2, rejection of RMA/B7.1 does not require the presence of CD4+ T cells or NK cells (Table IIIGo) (19).



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  		FIGURE 4. V{alpha}3.2+Vß5+ CD8+ T cells in mice immunized with RMA/B7.1 tumor cells. PBL from Ly49A Tg (lines 2 and 12), non-Tg littermate, and B6 mice immunized with viable RMA/B7.1 tumor cells were stained with mAbs against CD8, CD62L, V{alpha}3.2, and Vß5. Columns indicate the presence of V{alpha}3.2+Vß5+ CD8+ cells among CD62L- ({square}) and (superimposed) CD62L+ ({blacksquare}) cells in individual immunized mice. Mice that failed to reject RMA/B7.1 tumor cells are indicated by an asterisk.

 

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  		Table III. Efficient rejection of RMA/B7.1 tumor cells by Ly49A transgenic mice1

 
Thus, Ly49A Tg mice were injected with live RMA/B7.1 cells. Tumor growth (Table IIIGo) and the CD8+ T cell response to the immunodominant epitope (Fig. 4Go) were monitored as before. As expected, non-Tg littermate mice rejected the tumor cells and showed an expansion of V{alpha}3.2+Vß5+CD8+ cells. In sharp contrast to the anti-MBL-2 response, a substantial fraction (3 of 6) of the Ly49A Tg mice of line 2 mice rejected the tumor. These mice mounted a significant V{alpha}3.2+Vß5+CD8+ T cell response. Although all Tg mice of line 12 rejected the tumor, we observed a relatively minor, but significant, accumulation of V{alpha}3.2+Vß5+ CD8+ T cells. We conclude that functional tumor-specific V{alpha}3.2+Vß5+CD8+ cells are present in Ly49A Tg mice. The failure to control the growth of MBL-2 tumor cells is thus likely due to the lack of T cell help, based on the presence of the Ly49A receptor on CD4+ T cells. In addition, alterations in the CD8+ T cell repertoire may contribute to the inability to control these tumor cells.

Ly49A interaction with H-2b class I molecules

The expression of a Ly49A transgene impairs an antitumor T cell response in B6 (H-2b) mice. These findings suggest the presence of a Ly49A ligand in these mice. It has been shown that Ly49A cell surface levels on NK cells are significantly (50%) lowered in the presence of its H-2Dd ligand (29). Moreover, in agreement with a previous report (30), we have found that Ly49A levels on NK cells were ~20% lower in B6 as compared with B6.ß2m-deficient (ß2m-/-) mice (Fig. 5Go), suggesting that a putative Ly49A ligand in H-2b mice depends on ß2m. To address this issue further, we have assessed Ly49A expression levels on NK cells from B6 mice deficient for either one or both of the classical MHC class I molecules Db or Kb (17). Similar to ß2m deficiency, the lack of class Ia molecules in B6.Db-/-Kb-/- mice resulted in a 20% increase of the MFI of staining with mAb JR-9.318 compared with wild type (Fig. 5Go). The presence of either Kb or Db was sufficient to reduce Ly49A expression to wild-type levels. These observations suggest that Ly49A interacts weakly with the class I molecules Db (or Qa-1b, which binds leader peptides derived from Db (31, 32, 33)) and Kb. Thus, these findings may provide the molecular basis for the perturbed antitumor T cell response observed in Ly49A Tg mice.



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  		FIGURE 5. MHC class I-dependent down-modulation of Ly49A in H-2b mice. Nylon wool-nonadherent spleen cells from the indicated strains of mice were stained with mAbs directed against TCRß, NK1.1, and Ly49A. The MFI of staining with the anti-Ly49A mAb JR-9.318 was determined among TCR{alpha}ß-NK1.1+ NK cells. For each experiment, MFI values were normalized to the value obtained in B6 mice (=100%) Data are derived from four or more independent experiments. Significance of differences was determined using the two-tailed Student’s t test: *, significant difference (p < 0.05) compared with Db-/-Kb-/- or ß2m-/-; **, significant difference (p < 0.05) compared with Db-/-Kb-/- but not to ß2m-/-.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have investigated the consequences of Ly49 expression on the T cell response to a well-defined tumor Ag. We show that the T cell response is not induced due to the presence of the Tg Ly49A receptor and consequently that these mice fail to reject the transplanted tumor cells. Unexpectedly, these effects are observed in H-2b mice, i.e., that lack a Ly49A ligand known to inhibit the function of NK cells.

Ly49A specificity

H-2b haplotype mice are thought to lack an inhibitory ligand for Ly49A. Thus, the fact that transgene expression completely prevented the antitumor T cell response in H-2b mice suggested the existence of a Ly49A ligand in these mice. Using cellular adhesion assays or class I tetramers, it has been found that Ly49A binds to H-2Dd but not Kb or Db class I molecules (6, 34, 35, 36), even though one study has reported Db tetramer binding to Ly49A (37). However, alterations in the endogenous Ly49 receptor repertoire were documented in Ly49A Tg H-2b compared with class I (ß2m)-deficient mice (16). The latter results suggested a weak interaction between the Ly49A receptor and a ß2m-dependent molecule in H-2b mice. These findings were confirmed and extended here. Based on the down-modulation of Ly49A cell surface levels, we conclude that Ly49A can weakly interact with either Db or Kb class I molecules. In the case of Kb-deficient mice, it is also possible that Ly49A interacts with the nonclassical class I molecule Qa-1b, which binds leader peptides derived from the class Ia molecule Db (31, 32, 33). Clearly, however, such interactions are insufficient to inhibit NK cell function upon encounter of syngeneic (H-2b)-expressing target cells (25). We show in this study that for T cells expressing the Ly49A transgene, there is a relevant Ly49A ligand in B6 (H-2b) mice because Ly49A Tg mice of H-2b haplotype were unable to control syngeneic MBL-2 tumor cells in vivo. Indeed, the T cell response was inversely correlated with Ly49A expression levels. Intermediate Ly49A levels allowed a T cell response following repeated immunizations, while high Ly49A levels prevented any response.

Therefore, although a particular MHC background may not play a significant role in inhibiting NK cells via some Ly49 receptors, such an "irrelevant" specificity of Ly49 receptors may not exist in the context of T cell responses. Thus, T cell responses may be significantly more susceptible to regulation by inhibitory receptors than NK cells.

T cell response

The Ly49A transgene is expressed by most lymphoid cells. Therefore, it was important to examine which lymphocyte compartment was impaired. In normal mice, the rejection of MBL-2 tumor cells requires both CD4 and CD8+ T cells (Table IGo). In contrast, the rejection of RMA/B7.1 cells is mediated exclusively by CD8+ T cells with little or no contribution from CD4+ T cells or NK cells (Table IIIGo) (19). Although the latter tumor cells were rejected by a significant number of Ly49A Tg mice, MBL-2 tumor cells were not rejected. Therefore, the CD4+ T cell compartment expressing the Ly49A receptor may not provide appropriate T cell help. Because tumor-specific Th cells do not expand like CD8+ T cells (23) we cannot assess whether CD4+ T cells fail to be activated. However, it has been shown that CD4+ T cells from Ly49A Tg mice can mount a proliferative response to allogeneic MHC in vitro (15). Alternatively, the development of Ag-specific CD4+ T cells may be impaired due to the presence of Ly49A. Although the TCR repertoire was not assessed directly for CD4+ T cells, we provide evidence that the CD8+ T cell compartment of naive Ly49A Tg mice indeed shows alterations of the TCR repertoire. As shown in Table IIGo, the percentage of V{alpha}3.2+Vß5+ cells among CD8+ T cells is significantly increased in the high Tg line 2 compared with Tg line 12 (p < 0.01) or to non-Tg mice (p < 0.001) based on data evaluation using Student’s t test. Indeed, the increased representation of both V{alpha}3.2 and Vß5 TCR segments contributes to this repertoire alteration in line 2 (data not shown). In addition, two recent reports have provided additional evidence for a shift in the TCR repertoires of two independent lines of Ly49A Tg mice (38, 39).

Because tumor-specific CD8+ T cells make up only a small fraction of the V{alpha}3.2+Vß5+ cells in naive mice, it is not possible to directly assess the presence or absence of specific CD8+ T cells. However, 50% of Ly49A Tg mice of line 2 are able to control RMA/B7.1 tumor growth and mount a CD8+ T response. The variability of an Ag-specific CD8+ T cell response has previously been used to calculate the repertoire size of reactive cells (40). These calculations are based on the assumption that 1) all clones of the repertoire are of equal size and 2) the observed variations in the V{alpha}3.2+Vß5+ population are due to oligoclonal sampling effects (for details see Ref. 40). These calculations indicate a repertoire size in B6 or non-Tg littermate mice of ~23 Ag-specific CD8+ T cells. In contrast, the presence of Ly49A in Tg line 2 reduces the repertoire to 10 specific clones. The sample size of Tg line 12 was too small to obtain an accurate estimation. Therefore, the presence of the Tg Ly49A may alter T cell development and result in a smaller number of tumor-specific CD8+ T cells compared with littermate mice.

Similar to the antitumor T cell response investigated here, Zajac et al. reported adverse effects of Ly49A transgene expression on the clearance of lymphocytic choriomeningitis virus infections (41). Although all the mice were able to control the infection with the Armstrong isolate of lymphocytic choriomeningitis virus, a more virulent variant resulted in viremia in both H-2d and H-2b Ly49A Tg mice. Because viral clearance largely depends on virus-specific CTLs, these findings also suggest a defect in the CD8+ T cell compartment of Ly49A Tg H-2b mice.

Selection and activation of T cells is tightly regulated by the avidity of the TCR. Expression of inhibitory MHC receptors may increase the threshold for activation (42). Indeed, Ly49 receptors modulate TCR-mediated cytotoxicity and cytokine production by NK1.1+ T cells (10) and prevent T cell proliferation in responses to alloantigens (15). Therefore, signaling via the TCR is clearly susceptible to a Ly49-dependent regulatory mechanism. These regulatory effects may also perturb T lymphocyte development as described above and evidenced by the impaired development of NK T cells in Ly49A Tg mice that express the inhibitory MHC ligand (9).

A small population of Ly49-expressing CD8+ T cells, some of which are NK1.1 negative, is present in normal mice (43, 44) (P. Brawand and W. Held, unpublished observation). It is interesting to note that these cells display a somewhat skewed TCR repertoire with an overrepresentation of Vß5 TCR segment usage. This finding together with a memory phenotype and the accumulation of these cells with age (44) is compatible with the notion that they are driven by some form of Ag. Like conventional {alpha}ß T cells, the emergence of Ly49+ CD8+ T cells predominantly depends on MHC class I expression. However, MHC class I expression on hemopoietic cells may play a more important role for Ly49+ as compared with Ly49- CD8+ T cells (44). Thus, it remains to be determined whether Ly49-expressing T cells in normal mice represent a particular state of T cell activation as proposed for human T cells expressing KIR (14) or alternatively belong to a distinct T cell lineage.


   Acknowledgments
 
We thank D. H. Raulet for Ly49A Tg mice and J. Roland for providing the Ly49A (JR-9.318) mAb.


   Footnotes
 
1 This work was supported in part by Grant 31-48871.96 and a Swiss Talents for Academic Research and Teaching (START) fellowship from the Swiss National Science Foundation (to W.H.). Back

2 Address correspondence and reprint requests to Dr. Werner Held, Ludwig Institute for Cancer Research, Lausanne Branch, 155 Ch. des Boveresses, 1066 Epalinges, Switzerland. Back

3 Abbreviations used in this paper: KIR, killer inhibitory receptor; MFI, mean fluorescence intensity, Tg, transgenic; B6, C57BL/6; bc, backcross; M-MuLV, Moloney murine leukemia virus; MLTC, mixed lymphocyte:tumor cell cultures; ß2m, ß2-microglobulin. Back

Received for publication September 1, 1999. Accepted for publication June 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ljunggren, H. G., K. Karre. 1985. Host resistance directed selectively against H-2-deficient lymphoma variants: analysis of the mechanism. J. Exp. Med. 162:1745.[Abstract/Free Full Text]
  2. Colonna, M., J. Samaridis. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268:405.[Abstract/Free Full Text]
  3. Yokoyama, W. M., W. E. Seaman. 1993. The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. Annu. Rev. Immunol. 11:613.[Medline]
  4. Raulet, D. H., W. Held, I. Correa, J. R. Dorfman, M. F. Wu, L. Corral. 1997. Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors. Immunol. Rev. 155:41.[Medline]
  5. Karlhofer, F. M., R. K. Ribaudo, W. M. Yokoyama. 1992. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358:66.[Medline]
  6. Hanke, T., H. Takizawa, C. W. McMahon, D. H. Busch, E. G. Pamer, J. D. Miller, J. D. Altman, Y. Liu, D. Cado, F. A. Lemonnier, P. J. Bjorkman, D. H. Raulet. 1999. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11:67.[Medline]
  7. Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. Soderstrom, A. D’Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, L. L. Lanier, A. J. McMichael. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795.[Medline]
  8. Vance, R. E., J. R. Kraft, J. D. Altman, P. E. Jensen, D. H. Raulet. 1998. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1b. J. Exp. Med. 188:1841.[Abstract/Free Full Text]
  9. MacDonald, H. R., R. K. Lees, W. Held. 1998. Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells. J. Exp. Med. 187:2109.[Abstract/Free Full Text]
  10. Ortaldo, J. R., R. Winkler-Pickett, A. T. Mason, L. H. Mason. 1998. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J. Immunol. 160:1158.[Abstract/Free Full Text]
  11. Phillips, J. H., J. E. Gumperz, P. Parham, L. L. Lanier. 1995. Superantigen-dependent, cell-mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 268:403.[Abstract/Free Full Text]
  12. D’Andrea, A., C. Chang, J. H. Phillips, L. L. Lanier. 1996. Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J. Exp. Med. 184:789.[Abstract/Free Full Text]
  13. Ikeda, H., B. Lethe, F. Lehmann, N. Vanbaren, J. F. Baurain, C. Desmet, H. Chambost, M. Vitale, A. Moretta, T. Boon, P. G. Coulie. 1997. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 6:199.[Medline]
  14. Mingari, M. C., A. Moretta, L. Moretta. 1998. Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunol. Today 19:153.[Medline]
  15. Held, W., D. Cado, D. H. Raulet. 1996. Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection. J. Exp. Med. 184:2037.[Abstract/Free Full Text]
  16. Held, W., D. H. Raulet. 1997. Ly49A transgenic mice provide evidence for a major histocompatibility complex-dependent education process in natural killer cell development. J. Exp. Med. 185:2079.[Abstract/Free Full Text]
  17. Perarnau, B., M. F. Saron, B. R. San Martin, N. Bervas, H. Ong, M. J. Soloski, A. G. Smith, J. M. Ure, J. E. Gairin, F. A. Lemonnier. 1999. Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur. J. Immunol. 29:1243.[Medline]
  18. Brunner, K. T., H. R. MacDonald, J. C. Cerottini. 1980. Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors. II. Analysis of the clonal progeny of CTL precursors stimulated in vitro with syngeneic tumor cells. J. Immunol. 124:1627.[Medline]
  19. Martin-Fontecha, A., F. Cavallo, M. Bellone, S. Heltai, G. Iezzi, P. Tornaghi, N. Nabavi, G. Forni, P. Dellabona, G. Casorati. 1996. Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors. Eur. J. Immunol. 26:1851.[Medline]
  20. Biasi, G., L. Saggin, F. Dazzi, A. De Rossi, D. Collavo. 1983. Role of accessory cells in the induction of a secondary cytotoxic response to Moloney murine sarcoma virus-induced tumors. J. Immunol. 130:1447.[Abstract]
  21. MacDonald, H. R., J. C. Cerottini, J. E. Ryser, J. L. Maryanski, C. Taswell, M. B. Widmer, K. T. Brunner. 1980. Quantitation and cloning of cytolytic T lymphocytes and their precursors. Immunol. Rev. 51:93.[Medline]
  22. Chen, W., H. L. Qin, B. Chesebro, M. A. Cheever. 1996. Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. J. Virol. 70:7773.[Abstract]
  23. Brawand, P., G. Biasi, C. Horvath, J. C. Cerottini, H. R. MacDonald. 1998. Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo. J. Immunol. 160:1659.[Abstract/Free Full Text]
  24. Roland, J., P. A. Cazenave. 1992. Ly-49 antigen defines an {alpha} ß TCR population in i-IEL with an extrathymic maturation. Int. Immunol. 4:699.[Abstract/Free Full Text]
  25. Dorfman, J. R., D. H. Raulet. 1996. Major histocompatibility complex genes determine natural killer cell tolerance. Eur. J. Immunol. 26:151.[Medline]
  26. Ossendorp, F., E. Mengede, M. Camps, R. Filius, C. J. Melief. 1998. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J. Exp. Med. 187:693.[Abstract/Free Full Text]
  27. Chen, L., P. McGowan, S. Ashe, J. Johnston, Y. Li, I. Hellstrom, K. E. Hellstrom. 1994. Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J. Exp. Med. 179:523.[Abstract/Free Full Text]
  28. Cavallo, F., A. Martin-Fontecha, M. Bellone, S. Heltai, E. Gatti, P. Tornaghi, M. Freschi, G. Forni, P. Dellabona, G. Casorati. 1995. Co-expression of B7-1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response. Eur. J. Immunol. 25:1154.[Medline]
  29. Sykes, M., M. W. Harty, F. M. Karlhofer, D. A. Pearson, G. Szot, W. Yokoyama. 1993. Hematopoietic cells and radioresistant host elements influence natural killer cell differentiation. J. Exp. Med. 178:223.[Abstract/Free Full Text]
  30. Held, W., J. R. Dorfman, M. F. Wu, and D. H. Raulet. 1996. Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor repertoire. [Published erratum appears in 1996 Eur. J. Immunol. 26:3242.] Eur. J. Immunol. 26:2286.
  31. Aldrich, C. J., A. DeCloux, A. S. Woods, R. J. Cotter, M. J. Soloski, J. Forman. 1994. Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell 79:649.[Medline]
  32. DeCloux, A., A. S. Woods, R. J. Cotter, M. J. Soloski, J. Forman. 1997. Dominance of a single peptide bound to the class I(B) molecule, Qa-1b. J. Immunol. 158:2183.[Abstract]
  33. Cotterill, L. A., H. J. Stauss, M. M. Millrain, D. J. Pappin, D. Rahman, B. Canas, P. Chandler, A. Stackpoole, E. Simpson, P. J. Robinson, P. J. Dyson. 1997. Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide. Eur. J. Immunol. 27:2123.[Medline]
  34. Kane, K. P.. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011.[Abstract/Free Full Text]
  35. Brennan, J., D. Mager, W. Jefferies, F. Takei. 1994. Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties. J. Exp. Med. 180:2287.[Abstract/Free Full Text]
  36. Daniels, B. F., F. M. Karlhofer, W. E. Seaman, W. M. Yokoyama. 1994. A natural killer cell receptor specific for a major histocompatibility complex class I molecule. J. Exp. Med. 180:687.[Abstract/Free Full Text]
  37. Michaelsson, J., A. Achour, M. Salcedo, A. Kase-Sjostrom, J. Sundback, R. A. Harris, K. Karre. 2000. Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers. Eur. J. Immunol. 30:300.[Medline]
  38. Fahlen, L., L. Oberg, T. Brannstrom, N. K. S. Khoo, U. Lendahl, C. L. Sentman. 2000. Ly49A expression on T cells alters T cell selection. Int. Immunol. 12:215.[Abstract/Free Full Text]
  39. Pauza, M., K. M. Smith, H. Neal, C. Reilly, L. L. Lanier, D. Lo. 2000. Transgenic expression of Ly-49A in thymocytes alters repertoire selection. J. Immunol. 164:884.[Abstract/Free Full Text]
  40. Walker, P. R., A. Wilson, P. Bucher, J. L. Maryanski. 1996. Memory TCR repertoires analyzed long-term reflect those selected during the primary response. Int. Immunol. 8:1131.[Abstract/Free Full Text]
  41. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205.[Abstract/Free Full Text]
  42. Lanier, L. L.. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Medline]
  43. Eberl, G., R. Lees, S. T. Smiley, M. Taniguchi, M. J. Grusby, H. R. MacDonald. 1999. Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells. J. Immunol. 162:6410.[Abstract/Free Full Text]
  44. Coles, M. G., C. W. McMahon, H. Takizawa, D. H. Raulet. 2000. Memory CD8 T lymphocytes express inhibitory MHC-specific Ly49 receptors. Eur. J. Immunol. 30:236.[Medline]



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