Ly49A expression on T cells alters T cell selection

Linda Fahlén, Linda Öberg, Thomas Brännström, Nelson K. S. Khoo, Urban Lendahl and Charles L. Sentman

Umeå Center for Molecular Pathogenesis and
1 Department of Pathology, Umeå University, 901 87 Umeå, Sweden
2 Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden

Correspondence to: L. Fahlén


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ly49 receptors are inhibitory receptors expressed on subsets of both NK cells and NK1.1+ T cells. The function of these receptors on NK cells is believed to be important in maintaining self-tolerance, yet their role on T cells is unclear. In this report we investigated how an Ly49A transgene alters T and NK cell development in an in vivo environment, where a ligand for Ly49A is expressed. Ly49A transgenic mice that co-expressed an MHC ligand for Ly49A, H-2Dd, developed a severe inflammatory disorder that resulted in death within the first weeks of age. T cells expressing forbidden TCR Vß chains were found both in the thymus and periphery of transgenic mice, while non-transgenic littermates had successfully deleted these T cell subsets. These data indicate that the expression of Ly49A on T cells could alter T cell selection and allow survival of potentially self-reactive T cells.

Keywords: autoimmunity, NK cells, T lymphocytes, transgenic


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ly49 receptors are C-type lectin-like receptors and are expressed on subsets of NK cells (1). Ly49 molecules are a family of receptors with at least nine identified members (Ly49A–I) and their genes are clustered in the NK gene complex on murine chromosome 6 (2). The ligands for the Ly49 receptors are specific MHC class I molecules, when Ly49 receptors interact with MHC class I, cells receive inhibitory signals (3,4). These signals can overcome activating signals and prevent NK cells from killing target cells. Signals from Ly49 receptors can inhibit both NK cell- and T cell-mediated responses (48). Ly49A is a member of the Ly49 family, and binds specifically to H-2Dd and H-2Dk (4,9,10).

Some NKT cells also express Ly49 receptors (7,11), and the role of these receptors on NKT cells is not very well understood. NKT cells may play an important role in regulating the immune response through their production of various cytokines (12,13). NKT cells have an intermediate CD3 expression and a limited TCR V{alpha}Vß repertoire many of which are restricted to CD1 (1416). NKT cell development occurs mainly in the thymus and then NKT cells migrate to peripheral sites such as the liver (17).

Whether a T cell survives selection is determined by a balance between the different signals that the cell receives in the thymus. The selection process is designed to ensure that self-reactive T cells are eliminated and T cells restricted to self-MHC survive. This is believed to occur by the proper balance of positive and negative signals from various cell surface receptors. It is possible that inhibitory signals through Ly49 receptors could shift this balance and allow autoreactive T cells to develop. NKT cells with self-reactive TCR have been reported (1821). NKT cells express Ly49 receptors early during development (22) and previous data showed that Ly49 expression on NKT cells in the thymus was higher than on NKT cells in the periphery (23). One theory is that expression of Ly49 receptors may alter cell signaling during development and help CD1-reactive NKT cells to escape negative selection in the thymus. Results using common {gamma} chain-deficient mice may support this theory. The common {gamma} chain-deficient mice fail to express Ly49 molecules. These mice have NKT cells in the thymus but they are absent in the periphery, which suggests that expression of Ly49 receptors may be important early in development of NKT cells to allow maturation to occur or to help export these cells to the periphery (22).

In this report we have studied the role of Ly49 molecules on NK cells and T cells using Ly49A transgenic mice that expressed Ly49A on all NK cells, T cells and thymocytes. We found that mice expressing both the Ly49A transgene and a ligand for Ly49A, H-2Dd, had T cells that expressed normally deleted TCR specificities and developed a severe inflammatory disorder that was characterized by death within the first few weeks of age. T cells that expressed forbidden TCR Vß chains were found both in the thymus and in the periphery of these mice. Based on these data, we suggest that Ly49 signaling can alter T cell development and allow potentially autoreactive T cells to develop and may lead to an autoimmune-like disorder.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
C57BL/6 (B6) H-2b and BALB/c H-2d mice were purchased from Bomholtgård (Ry, Denmark). All mice were bred and maintained at the animal facility at Umeå University or at the animal house at FOA (NBC skydd, Umeå, Sweden). All animal work was approved by the Local Animal Ethical Committee (Umeå, Sweden).

Production of B6VA49A transgenic mice
A 980 bp Ly49A cDNA clone as previously described (24), was cloned into VACD2 (25). A 8.5 kb fragment was used to produce B6VA49A transgenic mice using C57BL/6 as both egg donors and fertile males. Transgenic founder mice were identified by staining peripheral blood lymphocytes with anti-Ly49A antibodies.

Cell lines
Tumor cells used as targets in cytotoxicity assays (RMA, RMA-S, YAC-1 and RMA-Dd cells) were maintained in RPMI 1640 (Life Technologies, Täby, Sweden) supplemented with 5% FCS, 20 U/ml penicillin, 20 g/ml streptomycin and L-glutamine at 37°C, 5% CO2.

Antibodies
Antibodies used were as follows. 2.4G2 (anti-FcR{gamma}), biotin–YE1/48 (anti-Ly49A), biotin–5E6 (anti-Ly49C/I), biotin–4D11 (anti-Ly49G2). Biotin–KT 15 (anti-CD8) was purchased from Immunokontakt (Stockholm, Sweden). FITC–PK136 (anti-NK1.1), FITC–HB102 (anti-H-2Dd). FITC–anti-CD4 and FITC–Ly-5 (B220) were purchased from Caltag (Burlingame,CA). FITC–4E5 (anti-Ly49D) was a kind gift from Dr L. Mason (Frederick, MD). FITC–anti-Vß3 TCR, FITC–anti Vß5.1, 5.2 TCR, FITC–anti Vß6 TCR, FITC–anti-Vß8.1, 8.2 TCR, FITC–anti-Vß11 TCR were all purchased from PharMingen (La Jolla, CA). Phycoerythrin (PE)–anti-CD3{varepsilon} and PE–anti-CD4 were purchased from PharMingen. Red670–streptavidin was purchased from Life Technologies.

Flow cytometry
To inhibit non-specific binding of antibodies to the FcR{gamma}, spleen cells depleted of erythrocytes and lymph node (LN) cells were incubated with anti-FcR{gamma} antibodies for 20 min at 4°C prior to staining with specific antibodies. Staining was done as described (24). Briefly, cells (106) (spleen cells, thymocytes and LN cells) were incubated with primary antibodies for 30 min at 4°C. After washing with staining buffer (PBS containing 1% FCS), cells were incubated with secondary antibodies and incubated for 30 min at 4°C. After washing, cells were resuspended in PBS and analyzed using a FACSCalibur (Becton Dickinson). The FACSCalibur was calibrated on regular basis by Becton Dickinson personal using CaliBRITE beads. Plots were generated using the CellQuest software (Becton Dickinson). Viable lymphocytes are shown after gating on forward and side scatter.

Cytotoxicity assays
Lymphokine-activated killer (LAK) cells were generated as described and cytotoxicity was assessed in a standard 51Cr-release assay (26). When blocking assays were performed with F(ab')2 fragments of A1 (anti-Ly49A), effector cells were incubated with 2 µg/well of antibodies for 45 min at 37°C before target cells were added to the wells. For complement treatment, LAK cells (107) were incubated with anti-CD4 and anti-CD8 or with 3A4 (kind gift from Vinay Kumar, Dallas, TX) on ice for 30 min. Control cells were incubated in RPMI media. Cells were washed 3 times and resuspended in Baby Rabbit Complement (Cedarlane, Hornby, Ontario, Canada) or media. After 45 min at 37°C cells were washed, resuspended in media and directly used as effector cells.

Histopathology
Organs (liver, heart, kidney and spleen) were removed and fixed in 4% paraformaldehyde for 1–2 h at 4°C and then transferred to 30% sucrose solution overnight at 4°C. The organ pieces were mounted in OCT tissue Tek medium and frozen at –70°C where they were stored until sectioning. Sections of 8–9 µm were placed on slides and fixed in 4% paraformaldehyde for 5 min, washed with 3xPBS, followed by 1xPBS and dried with increasing concentration of ethanol, ranging from 30 to 100%. Slides were stored in –70°C until stained with hemotoxylin & eosin (H & E), periodic acid–Schiff (PAS), Van Gieson (VG) or Laidlaw following standard protocols.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ly49A transgene is expressed on all NK cells, T cells and thymocytes, and inhibits killing of target cells expressing H-2Dd
We produced Ly49A transgenic mice using a modified CD2 promoter (25) to obtain high expression of Ly49A on all T cells, NK cells and thymocytes at an early stage during development of the cells. The data in Fig. 1Go(A) show the expression of Ly49A on splenic NK cells and T cells from these B6VA49A transgenic mice. All NK1.1+CD3 cells, NK1.1CD3+ cells and thymocytes expressed high levels of Ly49A, comparable to endogenous Ly49A expression found on NK cells from non-transgenic littermates. The expression pattern of the Ly49A transgene in different tissues was similar to our previous B649A transgenic mice, with highest transgene expression in the thymus (Fig. 1AGo) (24)(data not shown). Five independent founders were obtained with high transgene expression and the data presented in this paper were collected using mice derived from two founders, #26 and #49. To confirm that the Ly49A transgene was able to inhibit cell cytotoxicity, we performed cytotoxicity assays using target cells that did or did not express H-2Dd. LAK cells from B6VA49A transgenic mice killed RMA cells (H-2b) well but did not kill RMA-Dd (H-2b, H-2Dd) cells (Fig. 1BGo), while LAK cells from non-transgenic mice killed RMA and RMA-Dd cells equally well (Fig. 1CGo). When the interaction between Ly49A and H-2Dd was blocked using F(ab')2 fragments of anti- Ly49A antibodies, the killing of RMA-Dd cells by the B6VA49A LAK cells increased to similar levels as the killing of RMA cells (Fig. 1BGo), indicating that the Ly49A transgene was responsible for the lack of killing of the RMA-Dd cells. An interesting feature was that B6VA49A transgenic LAK cells killed RMA cells much better than RMA-S (MHC class I deficient) or YAC-1 cells (Fig. 2Go). Cytotoxicity assays, after deletion of T cells or NK cells with antibody and complement, indicated that T cells in the B6VA49A LAK cultures were responsible for the killing of RMA while NK cells were responsible for killing of RMA-S and YAC-1 (Table 1Go) (data not shown). Treatment using anti-CD4 and anti-CD8 antibodies and complement deleted most of the CD3+NK1.1 cells but few of the CD3+NK1.1+ cells. After incubating cells with 3A4 antibodies and complement, CD3NK1.1+ cells were deleted but the CD3+NK1.1+ cells were not depleted (data not shown).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Ly49A transgene inhibits lysis of RMA-Dd but not RMA by LAK cells. (A) Expression of Ly49A on splenic NK1.1+CD3 cells (solid line), NK1.1CD3+ cells (dashed line) or thymocytes (dotted line) from B6VA49A transgenic mice and on splenic NK1.1+CD3 cells from non-transgenic mice (thin line). (B and C) Specific lysis of RMA ({blacksquare}) and RMA-Dd ({square}) by 4 day LAK cells from (B) B6VA49A transgenic mice and (C) non-transgenic mice. Specific lysis of RMA-Dd including the presence of F(ab')2 fragments of anti-Ly49A antibodies (A1) to block Ly49A receptors ({circ}) is shown for transgenic LAK cells.

 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2. Transgenic LAK cells kill RMA better than RMA-S. Specific lysis of RMA ({blacksquare}), RMA-S ({lozenge}) and YAC-1 ({blacktriangleup}) by 4 day LAK cells from (A) B6VA49A transgenic mice (Tg) and (B) non-transgenic mice (NTg).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Cytotoxicity data from LAK cells depleted of T cells or NK cells
 
Ly49A transgenic mice co-expressing H-2Dd develop a severe inflammatory disorder
To study how an Ly49A transgene would alter NK cell and T cell function and development in an in vivo environment where a ligand for Ly49A is expressed, Ly49A transgenic mice (B6VA49A, H-2b) were bred to BALB/c (H-2d) and D8 (H-2b, H-2Dd) mice. D8 mice are B6 mice that express an H-2Dd transgene. The offspring that expressed both the Ly49A transgene and H-2Dd developed an autoimmune-like inflammatory disorder. Although the lifespan of these mice varied, most of the Ly49A transgenic H-2Dd-expressing mice (Ly49ATg/H-2Dd) died between 3 and 5 weeks of age. Body weight of these sick transgenic mice was significantly lower compared to healthy non-transgenic littermates, especially if the Ly49ATg/H-2Dd mice survived >35 days (Fig. 3Go). The physical manifestations of the Ly49ATg/H-2Dd mice included swollen and closed eyes, dried and flaking skin on the ears and tail, and swollen peritoneum. Occasionally a few mice that were Ly49A transgene positive showed little external signs of illness; however, gross examination of the internal organs revealed abnormalities. The internal organs were quite pale, especially the heart, liver, kidneys and spleen. The liver was often shivelled and the thymus virtually absent. This was true for offspring derived from both BALB/c and D8 crosses with two independent Ly49A transgenic lines. Since the only difference between B6 and D8 mice is the presence of an H-2Dd transgene, we concluded that the early onset of death was due to the presence of both Ly49A transgene and H-2Dd, an MHC class I ligand for Ly49A, and not due to another MHC molecule or a different background gene.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Ly49A transgenic mice that express H-2Dd show decreased growth. Data represent total body weight of (D8xB6VA49A)F1 mice (H-2b, H-2Dd) that express the Ly49A transgene ({square}) or non-transgenic littermates ({blacksquare}). Each square represents an individual mouse. Animals are derived from B6VA49A founders #26 or #49.

 
Analysis of lymphoid organs revealed that the total number of cells in the spleen and LN did not show significant differences between these mice, although the Ly49ATg/H-2Dd mice tended to have fewer cells. The number of thymocytes were dramatically reduced, so that 75% of the Ly49ATg/H-2Dd mice analyzed had a thymus with a total number of cells <3x106 cells compared to 1–3x108 cells for their non-transgenic H-2Dd littermates. There was a decrease in the number of T cells in the LN with the Ly49ATg/H-2Dd mice having only 34% of the number of CD4+ cells compared to non-transgenic littermates. The expression of CD8 and CD3 was reduced on T cells. There was a 2- to 4-fold increase in the number of splenic B cells in the Ly49ATg/H-2Dd mice. The percentage of NK cells in the spleen was similar; however, the percentage of other Ly49 receptors (C/I, G2 or D) on the Ly49ATg/H-2Dd NK cells was much lower and sometimes almost undetectable (data not shown). These data agree with previous findings where the expression of an Ly49A transgene in the presence of its MHC class I ligand resulted in a decrease in the percentage of NK cells that expressed other Ly49 receptors (27). Ly49A transgene expression was down-regulated in mice expressing the ligand H-2Dd. The Ly49A levels found on peripheral T cells in H-2d mice was ~20 % of the levels on peripheral T cells in H-2b mice.

We performed histological analysis of the heart, kidney and liver of Ly49ATg/H-2Dd mice and non-transgenic littermates. H & E stainings showed a massive increase in cells infiltrating the heart and liver from the transgenic mice compared to the organs from non-transgenic littermates (Fig. 4Go). The heart was mostly infiltrated by lymphocytes and had activated fibroblasts. In the liver the infiltrating cells consisted of lymphocytes, granulocytes and plasma cells. These cells were mainly seen next to or in close contact with large vessels. No extramedullar hematopoesis was seen. PAS staining did not indicate glycogen depletion. Laidlaw staining showed a disruption of tissue architecture and cell loss (data not shown). VG staining of collagen demonstrated extensive periportal fibrosis in the heart and liver. These histological data are consistent with a long-term, mainly chronic inflammatory disease. Since these mice were only 3–4 weeks of age, this suggests that the inflammatory reaction started before or around the time of birth.



View larger version (128K):
[in this window]
[in a new window]
 
Fig. 4. Histological analysis of heart and liver tissue from (BALB/cxB6VA49A)F1 mice. Analysis of frozen tissue sections taken from heart (A–D) or liver (E–H) of (BALB/cxB6VA49A)F1 mice (B, D, F and H) or non-transgenic littermates (A, C, E and G). Tissue sections stained with H & E (A, B, E and F) or VG (C, D, G and H) methods.

 
(BALB/cxB6VA49A)F1 mice fail to negatively select forbidden TCR Vß clones
To understand the reason for this inflammatory disease, we examined T cell development. To analyze whether T cell selection was altered in the (BALB/cxB6VA49A)F1 transgenic mice (H-2d/b), flow cytometric analysis of LN cells and thymocytes from B6VA49A and (BALB/cxB6VA49A)F1 Ly49A transgenic and non-transgenic littermates were performed to examine the percentage of T cells expressing TCR Vß3, Vß5, Vß6, Vß8 or Vß11 receptors (Fig. 5AGo). (BALB/cxB6)F1 mice normally eliminate T cells expressing TCR chains Vß3, Vß5 or Vß11 because of virally encoded superantigens in the genome (28,29), while CD4+ T cells from B6 mice express all of these TCR Vß chains. B6VA49A transgenic and non-transgenic littermates mice did not show any differences in their percentages of T cells that expressed TCR Vß chains except for Vß5, where there was a consistently higher percentage of Vß5+ CD4+ T cells in both thymus and LN in B6VA49A transgenic mice. When comparing LN cells from the (BALB/cxB6VA49A)F1 Ly49A transgenic and non-transgenic littermates, we readily detected T cells that expressed Vß3, Vß5 and Vß11 TCR in the (BALB/cxB6VA49A)F1 transgenic mice where they were deleted in their non-transgenic littermates. All (BALB/cxB6VA49A)F1 mice had an increased level of cells that expressed TCR Vß6 compared to the B6VA49A mice, suggesting that positive selection was intact (28).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. (BALB/cxB6VA49A)F1 transgenic mice fail to negatively select T cells that express Vß3, Vß5 or Vß11 TCR chains. Percentage of (A) CD4+ LN cells and (B) single-positive (SP) thymocytes that expressed Vß3, Vß5, Vß6, Vß8 and Vß11 TCR chains. Cells analyzed were derived from B6VA49A non-transgenic mice (H-2b, {square}), B6VA49A transgenic mice (H-2b, {blacksquare}), (BALB/cxB6VA49A)F1 non-transgenic mice (H-2d/b, {blacksquare}) or (BALB/cxB6VA49A)F1 transgenic mice (H-2d/b, {blacksquare}). Error bars represent SD. Each group consisted of five to eight mice in (A) and four to eight mice in (B).

 
To test whether negative selection was altered as opposed to accumulation of rare surviving T cells in the periphery, we examined the presence of Vß3, Vß5 or Vß11 TCR among single-positive thymocytes. As the data in Fig. 5Go(B) demonstrate, we observed that the percentage of single-positive thymocytes that expressed TCR Vß3, Vß5 or Vß11 in the (BALB/c xB6VA49A)F1 Ly49A transgenic (H-2d/b) mice was similar to the percentages observed in B6 (H-2b) and B6VA49A (H-2b) mice. These data indicate that the expression of the Ly49A transgene in the presence of its MHC class I ligand was able to alter T cell selection and allow the survival of potentially self-reactive T cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have used Ly49A transgenic mice where all T cells and NK cells expressed an Ly49A transgene similar to endogenous Ly49A levels to study the effect of Ly49A on T cell development and function in vivo. The percentage of T cells that normally express Ly49 receptors is small so a transgenic system is useful to investigate the role of these receptors on T cells. Ly49 expression on T cells has been shown to be able to interfere with the signaling pathway mediated through the TCR, including the inhibition of both cytotoxicity and cytokine production (5,8), yet the normal function of Ly49 receptors on T cells remains unclear. F1 progeny from crosses of Ly49A transgenic mice with BALB/c or D8 mice that expressed both the Ly49A transgene and an MHC class I ligand, H-2Dd, developed a severe inflammatory disorder at an early age that affected multiple organs, particularly the heart and liver. Histological analysis showed massive infiltration of cells in both heart and liver as evidence for a mainly chronic inflammatory reaction in the liver and a chronic inflammatory reaction in the heart. These observations were quite striking considering the young age of the mice, and were dependent upon the expression of both the Ly49A transgene and H-2Dd.

The numbers of thymocytes in Ly49ATg/H-2Dd mice were dramatically reduced. Small thymi have also been described in motheaten mice (30) which also suffer from an autoimmune-like disorder. One explanation for the diminished thymus could be an enhanced output of mature T cells. Another more likely explanation could be an enhancement in glucocorticoids due to stress caused by the inflammatory response that can lead to killing of thymocytes. Down-regulation of CD8 and CD3 on peripheral T cells was observed in the Ly49ATg/H-2Dd mice. It has been suggested that decreased surface expression of CD8 and CD3 could be a mechanism to induce lack of responsiveness (3134).

The balance between positive and negative signals during thymocyte selection is thought to be critical in deciding which cells survive. T cells expressing forbidden TCR Vß chains were found both in the thymus and periphery in (BALB/c xB6VA49A)F1 mice while the non-transgenic littermates had successfully deleted the corresponding T cells. Thus expression of inhibitory receptors having an ITIM motif in the cytoplasmic tail during T cell selection altered the TCR repertoire and allowed potentially self-reactive T cells to escape to the periphery. High Ly49 expression early in T cell development may be necessary to alter T cell selection since other Ly49A transgenic mice with lower transgene expression did not develop an inflammatory disorder when bred to mice expressing H-2Dd (24). In H-2b/d F1 mice with a lower expression of transgenic Ly49A, we did not detect a significant increase in T cells expressing forbidden TCR Vß chains (unpublished data).

It may seem curious that the Ly49A transgene did not turn off the inflammatory response in the periphery of mice that expressed H-2Dd yet altered the TCR repertoire of developing thymocytes. Thymocytes and peripheral T cells do not respond in a similar manner to receptor stimulation, and thymocytes have been shown to be more sensitive to stimuli by low-affinity peptides than mature T cells (3538). The amount of, and sensitivity to, different TCR signaling molecules has been shown to vary between the stages of T cell development (39). These observations suggest that there are different signaling pathways or outcomes of those pathways that operate in thymocytes and mature T cells. Hence the effects of Ly49 receptor signaling on thymocyte development may not be similar to those in peripheral T cells. Data indicate that signaling via TCR and Ly49 receptors are quantitative phenomenon such that a threshold level of ligand must be present on target cells for sufficient signals to be generated to affect cell function. Recent data from our laboratory have demonstrated that by increasing the presence of MHC–peptide ligands for a specific TCR it is possible to overcome the inhibitory signaling of Ly49A (L. Öberg and C. L. Sentman, in preparation). Thus Ly49A receptors may alter the signaling balance during T cell development but be unable to modify very strong signals via the TCR in mature T cells. Another possible explanation for the severe inflammatory disorder is that Ly49A expression on all thymocytes may have altered the TCR repertoire of some of the regulatory T cells that have been postulated as important in maintaining immunologic self-tolerance so that they were lost or unable to suppress self-reactive T cells (40). The complexities of interactions between signaling pathways at different stages of T cell development have yet to be unraveled; however, it is clear that Ly49A expression on T cells from an early stage led to survival of cells with TCR specificities that would normally be deleted in H-2b/d F1 mice.

A paradoxical observation was that LAK cells from B6VA49A transgenic mice (H-2b) killed RMA cells (H-2b) better than the ordinarily more NK-sensitive target cells, such as RMA-S and YAC-1. Cytotoxicity assays, after deletion of specific subsets of cells with antibodies and complement, indicated that T cells were responsible for the killing of RMA while NK cells were responsible for killing of RMA-S and YAC-1 in these assays (Table 1Go.) (data not shown). These data are difficult to understand without the presence of some type of ligand for Ly49A in H-2b mice. However, recent data indicate that there may be a ligand for Ly49A in H-2b mice. H-2Db tetramers have been shown to bind to cells that express Ly49A (J. Michaelsson and K. Kärre, pers. commun.). It is well known that Ly49 receptors are generally down-regulated in mice that express MHC class I ligands for them and NK cells from B6 mice have lower Ly49A expression than NK cells from ß2-microglobulin-deficient mice (41). IL-2 treatment in vitro during LAK cell generation may activate T cells from the B6VA49A mice sufficiently to induce a high degree of lysis against cells that normally would be rather resistant to lysis. However, in the absence of strong activation signals, this self-reactivity may not be seen in vivo. The fact that H-2Db can bind to Ly49A, although perhaps with a lower affinity than H-2Dd, and the high transgene expression of Ly49A in H-2b mice may explain why highly activated T cells in the LAK cultures from H-2b mice seem to have autoreactive phenotype and kill RMA target cells so well. The alteration in Vß5-expressing T cells may also reflect the interaction of H-2Db and Ly49A.

In this report, we show that expression of high levels of an Ly49A transgene on all T cells led to the development of a severe inflammatory disorder and the survival of potentially autoreactive T cells. Although it may be likely, we have not presented direct evidence that the peripheral T cells are themselves the cause of the autoimmune-like inflammatory disorder. A delicate balance of signaling events is necessary for the development and selection of a useful pool of peripheral T cells and Ly49 receptors can mediate inhibitory signals that can over-ride activation signals through a variety of receptor pathways. Our data demonstrate that Ly49 receptors can alter T cell selection. Irregularities in the expression of inhibitory receptors may be associated with altered T cell regulation and autoimmunity.


    Acknowledgments
 
The authors would like to thank Erik Nilsson for excellent technical assistance in generating the transgenic mice and Karin Wallgren for help with animal care. This work was supported by grants to C. L. S. from the Swedish Medical Research Council (project nos 11598 and 11324) and the Swedish Cancer Society. L. F. was partly supported by a grant from the Kempe Foundation.


    Abbreviations
 
B6 C57BL/6
H & E hemotoxylin & eosin
LAK lymphocyte-activated killer
LN lymph node
PAS periodic acid–Schiff
VG Van Gieson

    Notes
 
Transmitting editor: G. Klein

Received 30 April 1999, accepted 27 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lanier, L. L. and Phillips, J. H. 1998. Inhibitory MHC class I receptors on NK cells and T cells. Immunol. Today 17:86.
  2. Yokoyama, W. M. 1995. Natural killer cell receptors. Curr. Opin. Immunol. 7:110.[ISI][Medline]
  3. Ljunggren, H. G. and Kärre, K. 1990. In search of the `missing self': MHC molecules and NK cell recognition. Immunol. Today 11:237.[ISI][Medline]
  4. Karlhofer, F. M., Ribaudo, R. K. and Yokoyama, W. M. 1992. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358:66.[ISI][Medline]
  5. Held, W., Cado, D. and Raulet, D. H. 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]
  6. Yu, Y. Y., George, T., Dorfman, J. R., Roland, J., Kumar, V. and Bennett, M. 1996. The role of Ly49A and 5E6(Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts. Immunity 4:67.[ISI][Medline]
  7. Roland, J. and Cazenave, P. A. 1992. Ly-49 antigen defines and {alpha}ß TCR population in i-IEL with and extrathymic maturation. Int. Immunol. 4:699.[Abstract]
  8. Ortaldo, J. R., Winkler-Pickett, R., Mason, A. T. and Mason, L. H. 1998. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J. Immunol. 160:1158.[Abstract/Free Full Text]
  9. Kane, K. P. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011.[Abstract]
  10. Daniels, B. F., Nakamura, M. C., Rosen, S. D., Yokoyama, W. M. and Seaman, W. E. 1994. Ly-49A, a receptor for H-2Dd, has a functional carbohydrate recognition domain. Immunity 1:785.[ISI][Medline]
  11. MacDonald, H. R. 1995. NK1.1+ T cell receptor-{alpha}+ cells: new clues to their origin, specificity, and function. J. Exp. Med. 182:633.[ISI][Medline]
  12. Arase, H., Arase, N., Nakagawa, K., Good, R. A. and Onoe, K. 1993. NK1.1+ CD4+ CD8 thymocytes with specific lymphokine secretion. Eur. J. Immunol. 23:307.[ISI][Medline]
  13. Vicari, A. P. and Zlotnik, A. 1996. Mouse NK1.1+ T cells: a new family of T cells. Immunol. Today 17:71.[ISI][Medline]
  14. Smiley, S. T., Kaplan, M. H. and Grusby, M. J. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275:977.[Abstract/Free Full Text]
  15. Bendelac, A., Killeen, N., Littman, D. R. and Schwartz, R. H. 1995. A subset of CD4+ thymocytes selected by MHC class I molecules. Science 263:1774.[ISI]
  16. Chen, Y. H., Chiu, N. M., Mandal, M., Wang, N. and Wang, C. R. 1998. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459.[ISI]
  17. Schulz, R.-J., Parkes, A., Mizoguchi, E., Bhan, A. K. and Koyasu, S. 1996. Development of CD4CD8 {alpha}ßTCR+NK1.1+ T lymphocytes: thymic selection by self antigen. J. Immunol. 157:4379.[Abstract]
  18. Takahama, Y., Atsushi, K. and Singer, A. 1991. Phenotype, ontogeny, and repertoire of CD4CD8 T cell receptor {alpha}ß+ thymocytes. J. Immunol. 146:1134.[Abstract/Free Full Text]
  19. Kariv, I., Hardy, R. R. and Hayakawa, K. 1993. Selective enrichment of major histocompatibility complex class II-specific autoreactive T cells in the thymic Thy0 subset. J. Exp. Med. 177:1429.[Abstract]
  20. Kariv, I., Hardy, R. R. and Hayakawa, K. 1994. Altered major histocompatibility complex restriction in the NK1.1+Ly-6Chi autoreactive CD4+ T cell subset from class II-deficient mice. J. Exp. Med. 180:2419.[Abstract]
  21. Arase, H., Arase, N., Ogasawara, K., Good, R. A. and Onoe, K. 1992. An NK1.1+ CD4+8 single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor Vß family. Proc. Natl Acad. Sci. USA 89:6506.[Abstract]
  22. Lantz, O., Sharara, L. I., Tilloy, F., Andersson, Å. and DiSanto, J. P. 1998. Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules. J. Exp. Med. 185:1395.[Abstract/Free Full Text]
  23. Khoo, N. K. S., Fahlén, L. and Sentman, C. L. 1998. Modulation of Ly49A receptors on mature cells to changes in major histocompatibility complex class I molecules. Immunology 95:126.[ISI][Medline]
  24. Fahlén, L., Khoo, N. K. S., Daws, M. R. and Sentman, C. L. 1997. Location-specific regulation of transgenic Ly49A receptors by major histocompatibility complex class I molecules. Eur. J. Immunol. 27:2057.[ISI][Medline]
  25. Zhumabekov, T., Corbella, P., Tolaini, M. and Kiousis, D. 1995. Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. J. Immunol. Methods 185:133.[ISI][Medline]
  26. Sentman, C. L., Olsson, M. Y., Salcedo, M., Höglund, P., Lendahl, U. and Kärre, K. 1994. H-2 allele-specific protection from NK cell lysis in vitro for lymphoblasts but not tumor targets: protection mediated by {alpha}1/{alpha}2 domains. J. Immunol. 153:5482.[Abstract/Free Full Text]
  27. Held, W. and Raulet, D. H. 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]
  28. Okada, C. Y. and Weissman, I. L. 1989. Relative Vß transcript levels in thymus and peripheral lymphoid tissues from various mouse strains. J. Exp. Med. 169:1703.[Abstract]
  29. Bill, J., Kanagawa, O., Woodland, D. L. and Palmer, E. 1989. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of Vß11-bearing T cells. J. Exp. Med. 169:1405.[Abstract]
  30. Schultz, L. D. and Green, M. C. 1976. Motheaten, an immunodeficient mutant of the mouse. J. Immunol. 116:936.[Abstract]
  31. Schönrich, G., Kalinke, U., Momburg, F., Malissen, M., Schmitt-Verhulst, A. M., Malissen, B., Hämmerling, G. J. and Arnold, B. 1991. Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 65:293.[ISI][Medline]
  32. Auphan, N., Jézo-Brémond, A., Schönrich, G., Hämmerling, G., Arnold, B., Malissen, B. and Schmitt-Verhulst, A.-M. 1992. Threshold tolerance in H-2Kb-specific TCR transgenic mice expressing mutant H-2Kb: conversion of helper-independent to helper-dependent CTL. Int. Immunol. 4:1419.[Abstract]
  33. Cook, J. R., Wormstall, E.-M., Hornell, T., Russell, J., Connolly, J. and Hansen, T. H. 1997. Quantitation of the cell surface level of Ld resulting in positive versus negative selection of the 2C transgenic T cell receptor in vivo. Immunity 7:233.[ISI][Medline]
  34. Jameson, S. C., Hogquist, K. A. and Bevan, M. J. 1994. Specificity and flexibility in thymic selection. Nature 369:750.[ISI][Medline]
  35. Roehm, N., Herron, L., Cambier, J., DiGuisto, D., Haskins, K., Kappler, J. and Marrack, P. 1984. The major histocompatibility complex-restricted antigen receptor on T cells: distribution on thymus and peripheral T cells. Cell 38:577.[ISI][Medline]
  36. Lucas, B., Stefanová, I., Yasutomo, K., Dautigny, N. and Germain, R. N. 1999. Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire. Immunity 10:367.[ISI][Medline]
  37. Peterson, D. A., DiPaolo, R. J., Kanagawa, O. and Unanue, E. R. 1999. Negative selection of immature thymocytes by a few peptide–MHC complexes: differential sensitivity of immature and mature T cells. J. Immunol. 162:3117.[Abstract/Free Full Text]
  38. Davey, G. M., Schober, S. L., Endrizzi, B. T., Dutcher, A. K., Jameson, S. C. and Hogquist, K. A. 1998. Preselection thymocytes are more sensitive to T cell receptor stimulation than mature T cells. J. Exp. Med. 188:1867.[Abstract/Free Full Text]
  39. Chan, A. C., van Oers, N. S. C., Tran, A., Turka, L., Law, C.-L., Ryan, J. C., Clark, E. A. and Weiss, A. 1994. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J. Immunol. 152:4758.[Abstract/Free Full Text]
  40. Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
  41. Salcedo, M., Diehl, A. D., Olsson-Alheim, M. Y., Sundbäck, J., Van Kaer, L., Kärre, K. and Ljunggren, H.-G. 1997. Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J. Immunol. 158:3174.[Abstract]