Expression and selection of productively rearranged TCRß VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1+ {alpha}ß T cells

Nicole Baur, Gabi Nerz, Ahmed Nil and Klaus Eichmann

Max-Planck-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany

Correspondence to: K. Eichmann


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The generation of thymic NK1.1+{alpha}ßT (NKT) cells involves positive selection of cells enriched for V{alpha}14/Vß8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent ß selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3{zeta}/{eta}–/– and/or p56lck–/–) and TCR{alpha}-deficient mice. In contrast to wild-type mice, NK1.1+ thymocytes in CD3 signaling-deficient mice are ~10-fold reduced in number, do not exhibit V{alpha}14–J{alpha}281 rearrangements and fail to express {alpha}ßTCR at the cell surface. However, they exhibit TCRß VDJ rearrangements and pre-T{alpha} mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3{zeta}/Lck double-deficient mice fail to express TCRß mRNA and protein. Whereas in wild-type NKT cells TCRß VDJ junctions are selected for productive Vß8 and against productive Vß5 rearrangements, Vß8 and Vß5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCRß VDJ genes but before expression of TCRß proteins. Most NKT cells of TCR{alpha}-deficient mice exhibit cell surface {gamma}{delta}TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, ~25% of NKT cells of TCR{alpha}-deficient mice exhibit intracellular TCRß polypeptide chains. Moreover, both Vß8 and Vß5 families are selected for in-frame VDJ joints in the TCRß+ NKT cell subset of TCR{alpha}-deficient mice. The data suggest that CD3 signals regulate initial TCRß VDJ gene expression prior to ß selection in developing pre-NKT cells.

Keywords: CD3 complex, NKT cells, TCRß gene expression, TCRß gene rearrangement, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mainstream {alpha}ß T cell development encompasses two consecutive phases of thymic selection. The first, termed ß selection, takes place during the CD4CD8 double-negative (DN) or pre-T cell stage of thymocyte development, following rearrangement of the TCRß genes (reviewed in 1–3). Pre-T cells that have succeeded in generating a productive TCRß VDJ rearrangement express TCRß polypeptide chains which assemble with pre-T{alpha} (4,5) and clonotype-independent CD3 complexes (CIC) (6) at the cell surface to form the pre-TCR. Signals generated by the pre-TCR are essential for survival, proliferation and maturation to the CD4+CD8+ double-positive (DP) stage. The second phase of selection, termed repertoire selection, takes place among DP cells following TCR{alpha} gene rearrangement and expression of the mature {alpha}ßTCR that selects self-MHC-restricted and self-tolerant DP cells for maturation to CD4 or CD8 single-positive (SP) thymocytes (reviewed in 7,8).

NK1.1+ {alpha}ßT (NKT) cells represent a minor subset of lymphoid cells expressing {alpha}ßTCR as well as markers of the NK cell lineage (reviewed in 9,10). Two types of murine NKT cells have been distinguished: (i) NKT cells that are predominantly found in thymus and liver are dependent on the monomorphic MHC class I molecule CD1d, and are characterized by a highly restricted repertoire of {alpha}ßTCR, and (ii) NKT cells in spleen and bone marrow are CD1d independent and exhibit a more diverse TCR repertoire (11). The present work deals with NKT cells present in the thymus, representing a small subpopulation of thymocytes partially residing in the DN1 (CD44+CD25) subset of DN thymocytes, whereas a proportion expresses CD4 (9,12). The repertoire of {alpha}ßTCR of these NKT cells is dominated by a single TCR{alpha} chain (V{alpha}14–J{alpha}281) (13) and a limited choice of TCRß chain families (Vß8 family, mostly Vß8.2, and Vß7, Vß2), whereas other Vß families such as Vß5 are under-represented (12,14,15). NKT cells bearing the V{alpha}14 TCR recognize {alpha}-galactosylceramide presented by CD1d (16) and exhibit a diverse set of functions (reviewed in 9) including, among others, tumor cell cytotoxicity and the secretion of cytokines involved in Th1/Th2 polarization (1722).

NKT cell development is impaired in mice bearing a variety of genetic defects, including the V{alpha}14 gene (21), CD1d (2325) and ß2-microglobulin (12,26,27), as well as the CD3-associated signaling molecules CD3{zeta} (28), p56lck (Lck) and p59fyn (29,30). The requirement of MHC class I as well as of the TCR V{alpha}14 gene for normal NKT development clearly points to a role of positive selection of MHC-restricted {alpha}ßTCR in NKT cell development, equivalent to repertoire selection of mainstream {alpha}ßT cells. The dependence on molecules associated with CD3 complex signaling is consistent either with a requirement of positive repertoire selection in NKT cell development or with a pre-TCR-dependent checkpoint equivalent to ß selection of mainstream {alpha}ßT cells. The latter possibility is supported by the absolute requirement of pre-T{alpha} for NKT development (31). However, in pre-T{alpha}-deficient mice, impaired positive selection due to reduced numbers of CD1d-expressing DP cells may have contributed to the failure of NKT cells to develop (31,32). Moreover, the developmental stage at which NKT cell development is arrested in pre-T{alpha}-deficient mice has not been characterized. Together, a role of ß selection in the development of NKT cells has not been unequivocally demonstrated.

Here we investigate the putative role of ß selection in NKT cell development, using mice deficient for CD3{zeta}/{eta} and/or Lck (3335), collectively referred to as CD3 signaling-deficient mice, and TCR{alpha}-deficient mice (36). CD3 signaling-deficient mice generate ~1/10th of the CD44+NK1.1+ thymocytes observed in wild-type mice, without expression of {alpha}ßTCR at the cell surface. These cells appear to contain NKT cell precursors arrested at a pre-NKT cell-like stage, as suggested by the following parameters: (i) rearranged TCRß VDJ genes are produced, (ii) pre-T{alpha} mRNA is expressed and (iii) V{alpha}14–J{alpha}281 rearrangements are undetectable. Remarkably, TCRß mRNA and intracellular TCRß polypeptide chains are not detected in CD44+NK1.1+ thymocytes of CD3 signaling-deficient mice. Whereas in wild-type NKT cells TCRß VDJ junctions are selected for productive Vß8 and against productive Vß5 rearrangements, both Vß families are non-selected in pre-NKT cells of CD3 signaling-deficient mice. CD44+NK1.1+ thymocytes of TCR{alpha}-deficient mice exhibit {gamma}{delta}TCR at the cell surface and a significant proportion displays intracellular TCRß protein. Both Vß8 and Vß5 rearrangements are selected for in-frame VDJ joints in the TCRß+ NKT cell subset of TCR{alpha}-deficient mice. The results suggest that CD3 signals regulate initial TCRß gene expression prior to ß selection in developing pre-NKT cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
C57Bl/6 (B6) mice, TCR{alpha}-deficient mice (36), CD3{zeta}/{eta}- deficient mice (33) and p56lck (Lck)-deficient mice (34), all on a B6 background, were bred in the specific pathogen-free animal facilities of the Max-Planck-Institute. CD3{zeta}/{eta}-deficient mice and Lck-deficient mice were intercrossed as previously described (35). Typical experiments employed four types of mice derived by crossing CD3{zeta}/{eta}–/–Lck+/– with CD3{zeta}/{eta}+/–Lck–/– mice: mice bearing wild-type alleles for both genes (referred to as wild-type mice), mice single-deficient for CD3{zeta}/{eta} ({zeta}-sd mice), mice single-deficient for Lck (Lck-sd mice), and mice double-deficient for Lck and CD3{zeta}/{eta} ({zeta}/Lck-dd mice). No distinction was made between mice homozygous or heterozygous for the wild-type alleles, as no consistent differences were seen. Mice were analyzed at 4–8 weeks of age.

Flow cytometry
Four-color analyses employed a FACSCalibur (Becton Dickinson, Mountain View, CA). The following mAb, labeled with either FITC, phycoerythrin (PE), allophycocyanin (APC) or biotin (B), were purchased from PharMingen (San Diego, CA): anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-TCR-ß (H57-597), anti-TCR{delta} (GL3), anti-CD44 (IM7), anti-CD25 (7D4), anti-CD3{varepsilon} (500A2), anti-CD69 (H1.2F3) and anti-NK1.1 (PK136). Streptavidin–PerCP was used for B-labeled mAb. Whenever possible, a minimum of 2000 events per smallest analyzed subset were collected. In most analytical experiments, as well as in preparative flow cytometry using a MoFlow (Cytomation Bioinstruments, Freiburg, Germany), thymocytes were stained with antibodies to CD44 and NK1.1, labeled as appropriate for the color combination used. Cells gated as CD44+ and NK1.1+ were further characterized as indicated for each experiment. In some experiments negative gating for CD8 was employed. Intracellular staining was done, after blocking of the corresponding surface molecules with unlabeled mAb, using cells fixed with paraformaldehyde and permeabilized with saponin (35,37).

Semiquantitative analysis of V{alpha}14–J{alpha}281 rearrangement and of TCRß rearrangement, and sequencing of cloned TCRß and {gamma} V(D)J junctions
CD44+NK1.1+ thymocytes were sorted using a MoFlow cell sorter. DNA was prepared from the sorted populations as described (38). DNA concentrations were first determined by spectrophotometric determination and preparations were adjusted to similar concentrations. Fine adjustments were made by PCR of the insulin gene in serial dilutions, as previously described (39,40). TCR Vß8DJß2 and Vß5DJß2 rearrangements were amplified using primers described in (41,42), and V{alpha}14–J{alpha}281 rearrangements were amplified using primers described in (43), by PCR in serial dilutions as previously described (39,40). V{gamma}1J{gamma}4 and V{gamma}2J{gamma}1 rearrangements (for nomenclature, see 44) were amplified using primers described in (45). PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide or were cloned in the pCR-XL-TOPO vector (Invitrogen, San Diego, CA) and subjected to automated DNA sequencing using a ABI 310 genetic analyzer (Perkin-Elmer, Applied Biosystems, Weiterstadt, Germany). To identify in-frame and out-of-frame rearrangements, sequences were compared to database Vß and Jß reading frames. Groups of identical sequences were counted as single sequences.

Semiquantitative RT-PCR for TCRß and pre-T{alpha} mRNAs
CD44+NK1.1+ thymocytes were sorted using a MoFlow cell sorter. RNA was isolated using the PAN-RNA-Kit I (Pan-Biotech, Aichenbach, Germany) as directed by the manufacturer. cDNA was prepared from total RNA using the Display-Thermo-RT-Kit (Display Systems Biotech, Vista, CA) as directed by the manufacturer. Quantification of cDNA was done essentially as described in (35). Estimation of expression levels of mRNAs for pre-T{alpha} by RT-PCR was done using the following primers (5') 5'-CTTCTGGGCGTCAGGT-3'; (3') 5'-TAGGTGAAGGCGTCTAGGG-3', at 94°C for 20 s, 63°C for 20 s (five cycles), 61°C for 30 s (31 cycles) and 72°C for 30 s, for 36 cycles, yielding a 203-bp fragment. Expression levels of TCR Vß8 and Vß5 families were estimated by RT-PCR using primers described in (46) at 94°C for 40 s, 64°C for 1 min and 72°C for 2 min, for 30 cycles. Reactions were done in a volume of 25 µl using 0.25 µl cDNA Polymerase Mix (Clontech, Palo Alto, CA). Then 10 µl of the materials was subjected to gel electrophoresis on 1.3% agarose gels, which were stained with ethidium bromide and inversely photographed.

Single-cell RT-PCR for pre-T{alpha} mRNA
Thymocyte subpopulations were sorted on a MoFlow using the single-cell deposit mode into individual tubes containing 25 ml lysis buffer (PAN kit I reagents were used). Adsorbin (3 µl) was added, and after vortexing and 5 min incubation on ice the mixture was centrifuged at 10,000 r.p.m. for 1 s. The pellet was washed twice with 150 µl of wash buffer and then dryed in a thermomixer at 65°C for 1–3 min. RNA was eluted for 5 min at 65°C with 5 µl of diethylpyrocarbonate (Sigma, St Louis, MO)-treated bidistilled water and the eluted material was transferred into fresh tubes containing 5 µl RT reaction mix (consisting of 2 µl display Thermo-RT 5xbuffer,1 µl dNTP mix, 1 µl T25V primer and 1 µl display Thermo-RT terminator mix; Display Systems Biotech, Vista, CA), and incubated at 42°C for 40 min and 65°C for 10 min. PCR reactions were done with 1 µl aliquots, using the primers and conditions described above for pre-T{alpha}, except that 42 cycles were used.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD3 signaling-deficient mice develop CD44+NK1.1+thymocytes without {alpha}ßTCR at the cell surface
We have previously studied mainstream {alpha}ßT cell development in mice single-deficient (sd) and double-deficient (dd) for CD3{zeta}/{eta} and Lck. While the {zeta}-sd and Lck-sd mice exhibited an incomplete block in the DN to DP transition, {alpha}ß thymocyte development in {zeta}/Lck-dd mice was completely blocked at the pre-T cell stage, i.e. TCRß VDJ rearrangements were normal (39), TCRß mRNA and polypeptide chains were generated at low level (39,40), pre-T{alpha} mRNA was strongly expressed and clonotypic TCR{alpha} mRNA could not be detected (35). In the present work we focused on the NKT cells in the thymi of these mice. Thymocytes were analyzed in four-color flow cytometry experiments for CD44 and NK1.1, in combination with other markers typically positive or negative on NKT cells (i.e. CD4, CD8, c-kit, IL-2Rß, HSA, CD69, B220 and CD2, data not shown) or in combination with TCR components. In the experiment in Fig. 1Go, CD44+ thymocytes were analyzed for NK1.1 and cell surface TCRß and CD3{varepsilon} expression. In this experiment, thymocytes were negatively gated for CD8 to exclude recently described populations of CD8+NK1.1+ {gamma}{delta} T cells and CD8+NK1.1+ {alpha}ß T cells not enriched for V{alpha}14 (47). As previously shown (reviewed in 9,10), most CD44+NK1.1+ cells of wild-type mice display TCRß and CD3{varepsilon} at the cell surface, albeit at low levels. In contrast, the majority of CD44+NK1.1+ thymocytes of CD3 signaling-deficient mice lack TCRß/CD3{varepsilon} surface (sf) expression. Whereas {zeta}/Lck-dd mice rarely possess any TCRßsf+CD44+NK1.1+ thymocytes, some {zeta}-sd and Lck-sd mice may have a small percentage of TCRßsf+CD44+NK1.1+ thymocytes. Lck-sd mice may also have a small percentage of surface CD3{varepsilon}+CD44+NK1.1+ thymocytes that bear {gamma}{delta}TCR (not shown). Absolute numbers of CD44+NK1.1+ cells per thymus in CD3 signaling-deficient mice are ~10-fold lower than in wild-type mice (B6, 3.0–6.3x105; {zeta}-sd, 0.2–0.7x105; Lck-sd, 0.3–0.7x105; {zeta}/Lck-dd, 0.4–1.0x105). Taken together, and in agreement with previous reports (2830), these results confirm that the development of NKT cells in the thymus is dependent on CD3 complex signaling.



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Fig. 1. CD3 signaling-deficient mice produce CD44+NK1.1+ thymocytes without {alpha}ßTCR at the cell surface. Total thymocytes of wild-type mice, {zeta}-sd mice, Lck-sd mice and {zeta}/Lck-dd mice were stained with anti-CD8 (PE), anti-CD44 (APC), anti-NK1.1 (B + PerCP) and either anti-TCRß (FITC) or anti-CD3{varepsilon} (FITC). CD8CD44+ cells were gated (top panels) and analyzed for NK1.1 versus TCRß or CD3{varepsilon} at the cell surface (sf). Quadrants were set using isotype-matched control antibodies labeled with the same fluorochromes. Percentages of subpopulations are given in the insets in each frame. Data are from individual representative mice derived by cross-breeding {zeta}-sd mice to Lck-sd mice as described in Methods; +, wild-type allele present; –, no wild-type allele present.

 
The data in Fig. 1Go also show that CD8CD44+ thymocytes in wild-type mice comprise a population of NK1.1 cells expressing surface TCRß/CD3{varepsilon} at higher levels than NKT cells. Surface TCRß/CD3{varepsilon} expression in these cells is drastically reduced in CD3 signaling-deficient mice, suggesting that the generation of these cells is also dependent on CD3 signals. This population my represent the physiological counterpart of the TCR{alpha}ß+CD4CD8 thymocytes that occur abundantly in some TCR transgenic mice, the origin of which is controversial (48,49). These cells have not been further studied in the present context.

CD44+NK1.1+ thymocytes of CD3-signaling-deficient mice contain TCRß VDJ re-arrangements and pre-T{alpha} mRNA, but lack V{alpha}14–J{alpha}281 rearrangements
Next, we investigated whether CD44+NK1.1+ thymocytes of CD3-signaling-deficient mice contain precursors of NKT cells. Mainstream pre-{alpha}ßT cells exhibit TCRß VDJ rearrangements and express pre-T{alpha} mRNA, but do not possess TCR{alpha} rearrangements. CD44+NK1.1+ thymocytes were purified by preparative flow cytometry from wild-type and CD3 signaling-deficient mice to >90% purity (Fig. 2Go), and analyzed for the three parameters of mainstream pre-{alpha}ßT cells. TCRß locus rearrangements were analyzed by semiquantitative PCR using primers specific for Vß8 and Vß5, together with a primer that hybridizes 3' of Jß2.6. The data in Fig. 3Go show that both TCR Vß gene families have rearranged in the CD44+NK1.1+ populations of wild-type mice as well as in that of CD3 signaling-deficient mice. Quantitatively, rearrangements are similar or only slightly reduced in sd mice compared to wild-type mice. In {zeta}/Lck-dd mice banding patterns dilute out earlier, consistent with ~2- to 3-fold reduced numbers of CD44+NK1.1+ cells bearing VßDJ rearrangements. In addition, we observe incomplete representation of the bands for Jß2.1–5, with different incomplete sets of bands in different experiments (data not shown). To assess Jß2 usage in CD44+NK1.1+ cells of {zeta}/Lck-dd mice PCR amplified VDJ junctions were randomly cloned and sequenced. With the exception of Vß5DJß2.1 joints, all Jß2 segments were found in combination with both Vß genes tested (Table 1Go). The shortest PCR product is favored resulting in over-representation of Jß2.6, as is also seen to varying extend in other NKT preparations (see Fig. 3Go). The results suggest that CD44+NK1.1+ populations in CD3 signaling-deficient mice contain a random collection of TCRß VDJ rearrangements. The level of rearrangements seems to be reduced compared to the mature NKT cells of wild-type mice, suggesting that not all of the CD44+NK1.1+ cells in CD3 signaling-deficient mice contain TCRß VDJ rearrangements. These results are consistent with the hypothesis that CD44+NK1.1+ cells in CD3 signaling-deficient mice contain precursors of NKT cells as well as other cells that do not rearrange TCRß genes, e.g. NK lineage cells.



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Fig. 2. Preparative flow cytometry of CD44+NK1.1+ cells. Total thymocytes were stained with anti-CD44 (APC) and anti-NK1.1 (PE) and the DP populations were sorted using a MoFlow cell sorter (top panels). Sorted populations were reanalyzed for purity directly after sorting without restaining (bottom panels). Sorting gates and percentages of cells within the gates are shown in each frame. Sorts have been performed at different occasions so that differences in NK1.1 staining intensity should be disregarded. Sorts were done with pooled thymocytes of two to four mice derived by cross-breeding {zeta}-sd mice to Lck-sd mice as described in Methods; +, wild-type allele present; –, no wild-type allele present.

 


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Fig. 3. Analysis of TCRß VDJ rearrangements in CD44+NK1.1+ thymocytes. CD44+NK1.1+ thymocytes were purified by preparative flow cytometry as shown in Fig. 2Go. DNA was prepared and adjusted to similar concentrations using spectrophotometric determination and PCR amplification at 2-fold dilutions of the insulin gene which displays as two bands (bottom panels). Vß8DJß2 and Vß5DJß2 junctions were amplified at the indicated concentrations. PCR products were separated on agarose gels, stained with ethidium bromide and inversely photographed. Rearranged TCRß VDJ genes display as six bands corresponding to Jß2.1 to Jß2.6 from top to bottom. The additional band seen just above the Jß2.6 band in {zeta}-sd and {zeta}/Lck-dd mice is variable and presumably a PCR artifact, as no unaccountable products were detected by cloning and sequencing (see Tables 1 and 2GoGo). The germline configuration is not amplified by this method. Data are from pools of two to four mice derived by cross-breeding {zeta}-sd mice to Lck-sd mice as described in Methods; +, wild-type allele present; –, no wild-type allele present. B6Thy, total thymocytes of strain B6.

 

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Table 1. Usage of Jß2 segments in TCRß VDJ rearrangements of CD44+NK1.1+ thymocytes of {zeta}/Lck-dd micea
 
In mainstream {alpha}ßT cell development pre-T{alpha} is strongly expressed at the DN stages but expression continues into the DP stage, declining as rearranged TCR{alpha} genes become expressed (50). Pre-T{alpha} mRNA levels in CD44+NK1.1+ thymocytes of B6 wild-type mice and CD3 signaling-deficient mice were determined by semi-quantitative RT-PCR. CD44+NK1.1+ cells of B6 mice were sorted into surface TCRß+ and TCRß subpopulations (Fig. 4AGo), because pre-T{alpha} expression would suggest the presence of immature NKT cell precursors in the minor TCRßsf subset. CD44+NK1.1+ cells of {zeta}/Lck-dd mice were sorted as shown in Fig. 2Go. As shown in Fig. 4(B)Go, pre-T{alpha} mRNA was detected in CD44+NK1.1+ populations of both wild-type and CD3 signaling-deficient mice. Significantly, CD44+NK1.1+TCRßsf cells of both wild-type and CD3 signaling-deficient mice exhibit pre-T{alpha} mRNA. In wild-type mice, pre-T{alpha} mRNA continues to be expressed in the further developed TCRßsf+ NKT cells. We considered the possibility that the pre-T{alpha} signals in sorted CD44+NK1.1+ cells were caused by the ~5% of cells that did not appear in the CD44+NK1.1+ gate and that may contain mainstream {alpha}ß thymocytes particularly in wild-type mice. To exclude this possibility single-cell RT-PCR were performed. The results in Fig. 4(C)Go show that >70% of individually sorted TCRßsf and TCRßsf+CD44+NK1.1+ cells of wild-type mice exhibited pre-T{alpha} mRNA, similar to DN3 cells. The real percentage of pre-T{alpha}+ cells may well be higher because some of the negative results in single-cell RT-PCR may have technical reasons. The data exclude the possibility that the pre-T{alpha} signals stem from NK1.1 cells that are present at ~5% in our sorted CD44+NK1.1+ populations (Figs 2 and 4AGoGo). Together with the TCRß VDJ rearrangements, the presence of pre-T{alpha} mRNA supports the hypothesis that the TCRßsf CD44+NK1.1+ cells of wild-type and of CD3 signaling-deficient mice contain precursors of NKT cells.



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Fig. 4. Analysis of pre-T{alpha} mRNA by RT-PCR in sorted CD44+NK1.1+ thymocytes of wild-type and {zeta}/Lck-dd mice. (A) Purification by flow cytometry of TCRßsf+ and TCRßsf subsets of CD44+NK1.1+ thymocytes of B6 mice. Total thymocytes were stained for CD44 versus NK1.1 (a) and the DP population gated for TCRßsf expression (b). Sorted TCRßsf+ (e and f) and TCRßsf (c and d) populations were reanalyzed for TCRßsf (c and e) and for CD44 and NK1.1 (d and f). (B) RNA and cDNA were prepared from the sorted cell populations and cDNAs were adjusted to similar concentrations by PCR at several dilutions of the HPRT gene. B6 Thy, total thymocytes. Pre-T{alpha} mRNA was amplified by PCR in the indicated dilutions. (C) TCRßsf+ and TCRßsf CD44+NK1.1+ thymocytes, and CD44NK1.1CD4CD8CD3{varepsilon}CD25+ (DN3) cells were collected from B6 mice by single-cell deposit mode into individual tubes. Preparation of cDNA and PCR reactions were performed as described in Methods. The results of 12 randomly tested cells of each type are shown. Pre-T{alpha} mRNA was detected in >70% of individual cells in each of the three cell populations tested. PCR products were separated on agarose gels, stained with ethidium bromide and inversely photographed. The PCR primers used for pre-T{alpha} amplify a 203-bp fragment (see Methods).

 
Next, CD44+NK1.1+ thymocytes of wild-type mice, {zeta}-sd mice, Lck-sd mice and {zeta}/Lck-dd mice were analyzed by semiquantitative PCR for V{alpha}14–J{alpha}281 rearrangements. V{alpha}14–J{alpha}281 rearrangements are not found in mainstream {alpha}ß thymocyte development (51). As a result, unsorted B6 thymocytes exhibit only a faint PCR signal under the conditions used, whereas a strong signal was seen in CD44+NK1.1+ thymocytes of wild-type mice (Fig. 5Go). Significantly, CD44+NK1.1+ cells of {zeta}/Lck-dd mice never exhibited a positive PCR signal for V{alpha}14–J{alpha}281 rearrangement, whereas {zeta}-sd mice and Lck-sd mice either exhibited faint signals or were negative. Taken together, the results described so far suggest that NKT cell development in CD3 signaling-deficient mice proceeds to a stage in which TCRß VDJ rearrangements occur and pre-T{alpha} mRNA is expressed. However, development is arrested before TCR{alpha} locus rearrangement or before selection of cells bearing V{alpha}14–J{alpha}281 TCR.



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Fig. 5. Analysis of V{alpha}14–J{alpha}281 rearrangements in CD44+NK1.1+ thymocytes. CD44+NK1.1+ thymocytes were purified by preparative flow cytometry as shown in Fig. 2Go. DNA was prepared and adjusted to similar concentrations using spectrophotometric determination and PCR amplification at serial dilutions of the insulin gene which displays as two bands (bottom panels). V{alpha}14–J{alpha}281 junctions were amplified at the indicated DNA concentrations. PCR products were separated on agarose gels, stained with ethidium bromide and inversely photographed. Data are from pools of two to four mice derived by cross-breeding {zeta}-sd mice to Lck-sd mice as described in Methods; +, wild-type allele present; –, no wild-type allele present. B6 Thy, total thymocytes of strain B6.

 
Differential regulation of TCRß gene expression in NKT cells of wild-type mice, CD3 signaling-deficient mice and TCR{alpha}-deficient mice
As previously shown, mainstream pre-{alpha}ßT cells of CD3 signaling-deficient mice express 5- to 10-fold lower levels of TCRß mRNA (39,52) and intracellular TCRß polypeptide chains (39,40,53), compared to wild-type mice. We therefore analyzed intracellular and cell surface TCRß protein levels in NKT cells of wild-type and CD3 signaling-deficient mice. As shown in Fig. 6Go, the vast majority of NKT cells of wild-type mice express TCRß both intracellularly (ic) and at the cell surface (sf). In contrast, CD44+NK1.1+ cells of {zeta}-sd and Lck-sd mice reveal two populations with respect to TCRßic expression. Whereas the majority of the cells lack intracellular TCRß, a minority of the cells has TCRßic levels similar to wild-type NKT cells. About half of the TCRßic+ cells of {zeta}-sd and Lck-sd mice display TCRß also at the cell surface. Significantly, CD44+NK1.1+ cells of {zeta}/Lck-dd mice are almost entirely devoid of intracellular TCRß protein.



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Fig. 6. Flow cytometry analyses of intracellular (ic) and surface (sf) TCRß polypeptide chains in CD44+NK1.1+ cells. Total thymocytes were stained with anti-CD44 (APC), anti-NK1.1 (B + PerCP), and with anti-TCRß first at the cell surface (FITC) and subsequently by intracellular staining (PE). CD44+NK1.1+ populations were gated (top panels) and analyzed for TCRßsf versus TCRßic (bottom panels). Data are from individual representative mice derived by cross-breeding {zeta}-sd mice to Lck-sd mice as described in Methods; +, wild-type allele present; –, no wild-type allele present. Percentages of subpopulations are given in the insets.

 
In order to ascertain the lack of TCRß expression in CD44+NK1.1+ cells of {zeta}/Lck-dd mice we analyzed TCRß mRNA levels by RT-PCR. While CD44+NK1.1+ cells of wild-type mice contain mRNA for Vß8 but not for Vß5 family members, that of {zeta}/Lck-dd mice are devoid of either TCR Vß8 or Vß5 mRNAs (Fig. 7Go). Because we could clearly detect TCRß VDJ rearrangements in CD44+NK1.1+ cells of {zeta}/Lck-dd mice (see Fig. 3Go), these results suggest that NKT cell development is blocked between rearrangement and transcription of TCRß VDJ genes. The TCRßsf staining without intracellular TCRß in a few of the CD44+NK1.1+ cells of all analyzed mice (see Fig. 6Go) is therefore most likely due to non-specific background. Together, the results suggest that expression of TCRß VDJ genes in early NKT cell development requires factors in addition to productive TCRß VDJ rearrangement and is regulated at the mRNA level.



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Fig. 7. Analyses of TCR Vß8 and Vß5 mRNAs by RT-PCR in CD44+ NK1.1+ cells of wild-type and of {zeta}/Lck-dd mice. CD44+NK1.1+ thymocytes were purified by preparative flow cytometry as shown in Fig. 2Go. RNA and cDNA were prepared and cDNAs were adjusted to similar concentrations by PCR at several dilutions of the HPRT gene. Vß8DJCß and Vß5DJCß cDNAs were amplified by PCR in the indicated dilutions. Non-rearranged TCRß RNA species are not amplified by this method. PCR products were separated on agarose gels, stained with ethidium bromide and inversely photographed. B6Thy, total thymocytes of strain B6.

 
In TCR{alpha}-deficient mice NK{alpha}ßT cell development is blocked due to the inability to generate TCR{alpha} chains rather than as a result of a functionally incompetent CD3 complex. As shown in Fig. 8Go, TCR{alpha}-deficient mice generate CD44+NK1.1+ thymocytes without surface {alpha}ßTCR expression at ~10-fold reduced numbers similar to that of CD3 signaling-deficient mice. The majority of the CD44+NK1.1+ thymocytes of TCR{alpha}-deficient mice but only few of the CD44+NK1.1+ thymocytes in either wild-type or CD3 signaling-deficient mice express surface {gamma}{delta}TCR. Significantly, intracellular TCRß protein is seen at high levels in around one-fourth of the CD44+NK1.1+ thymocytes of TCR{alpha}-deficient mice, in contrast to CD44+NK1.1+ thymocytes of {zeta}/Lck-dd mice that lack TCRßic protein. These data suggest that the expression of intracellular TCRß protein in NKT cells of TCR{alpha}-deficient mice is induced by CD3 signals.



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Fig. 8. Analysis of intracellular TCRß expression in CD44+NK1.1+ cells of wild-type, TCR{alpha}-deficient and {zeta}/Lck-dd mice. Total thymocytes of wild-type mice, {zeta}/Lck-dd mice and TCR{alpha}-deficient were stained with anti-CD44 (APC), anti-NK1.1 (B + PerCP), together with anti-TCRßsf (FITC) and anti-TCRßic (PE) or anti-TCR{delta}sf (FITC). CD44+NK1.1+ cells were gated (top panels) and analyzed for TCRßic versus TCRßsf (middle panels) or TCR{delta} at the cell surface (sf). Percentages of subpopulations given in each frame.

 
Selection status of TCRß VDJ genes in NKT cells of wild-type mice, CD3 signaling-deficient mice and TCR{alpha}-deficient mice
We determined the proportions of productive and non-productive TCRß, TCR V{gamma}1 and TCR V{gamma}2 joints in sorted CD44+NK1.1+ populations of wild-type and {zeta}/Lck-dd mice, and in sorted TCRßic and TCRßic+ subpopulations of CD44+NK1.1+ cells of TCR{alpha}-deficient mice. Expected frequencies of in-frame TCRß VDJ junctions in selected and unselected populations are 71.4 and 33% respectively (42). As shown in Table 2Go, sorted CD44+NK1.1+ cells of wild-type mice contain 80% productive Vß8DJ rearrangements, whereas only 13% of the Vß5DJ rearrangements were productive. These data are consistent with the selection of mature NKT cells of wild-type mice predominantly for Vß8.2 TCR and against other TCR Vß families (reviewed in 9,10). In contrast to wild-type mice, the proportions of in-frame Vß8DJ and in-frame Vß5DJ junctions in CD44+NK1.1+ thymocytes of {zeta}/Lck-dd mice were not significantly different from the 33% expected in non-selected populations, consistent with the lack of expression of rearranged TCRß VDJ genes in these cells. Among CD44+NK1.1+ thymocytes of TCR{alpha}-deficient mice, TCRßic cells appear somewhat depleted of in-frame TCRß joints, indicating that expression of TCRß proteins takes place soon but not immediately after rearrangement. Most significantly, and in contrast to mature NKT cells of wild-type mice, the TCRßic+ subpopulation in TCR{alpha}-deficient mice is positively selected for both Vß8DJ and Vß5DJ joints, suggesting indiscriminate induction of TCRß VDJ genes. NKT cells of wild-type mice and pre-NKT cells of {zeta}/Lck-dd mice contain V{gamma}1J{gamma}4 and V{gamma}2J{gamma}1 rearrangements which appear unselected or slightly depleted of in-frame joints. CD44+NK1.1+ cells of TCR{alpha}-deficient mice are selected for productive V{gamma}1J{gamma}4 and against productive V{gamma}2J{gamma}1 joints, similar to NK {gamma}{delta}T cells described in wild-type mice (54). The results suggest that NKT cells of TCR{alpha}-deficient mice are selected by the {gamma}{delta}TCR and have thus experienced CD3 signals in their development.


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Table 2. Frequencies of in-frame TCR Vß and V{gamma} junctions in CD44+NK1.1+ thymocytesa
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NKT cells are generated in the thymus by a process that involves positive selection of cells with a highly restricted {alpha}ßTCR repertoire by CD1d MHC class I molecules (reviewed in 9,10). It is therefore to be expected that deficiencies in CD3 complex signaling result in impaired NKT cell development. However, it was so far not clear whether CD3-dependent selection equivalent to ß selection of mainstream {alpha}ß thymocytes takes place as well. This question has been difficult to address as a sequence of readily detectable surface phenotypes is not known for early NKT cell development. Precise stages at which NKT cell development is blocked in the absence of TCR or pre-TCR components have therefore not been previously determined.

The initial aim of the present work was to determine the developmental checkpoint at which NKT cell development is blocked in CD3 signaling-deficient mice. In contrast to wild-type mice, these mice appear to generate CD44+NK1.1+ cells in which TCRß VDJ genes rearrange, whereas no V{alpha}14–J{alpha}281 rearrangements and no surface {alpha}ßTCR/CD3 expression are detectable. The lack of detection of V{alpha}14–J{alpha}281 rearrangements may be due to an early block before TCR{alpha} rearrangements, but would also be consistent with inefficient positive selection of NKT cells with surface V{alpha}14-containing {alpha}ßTCR (43). However, a late developmental arrest due to lack of selection for the surface {alpha}ßTCR seems unlikely, as TCRß mRNA and polypeptide chains are not expressed in the CD44+NK1.1+ population of {zeta}/Lck-dd mice. In severely CD3-deficient mice, essentially all NKT cell development is arrested at this early TCRßsfCD44+NK1.1+ stage. In mice with residual CD3 signaling competence, such as the {zeta}-sd or Lck-sd mice, some NKT cells with {alpha}ßTCR at the cell surface are generated. The precursor progeny relationship between the TCRßsfCD44+NK1.1+ population in CD3 signaling-deficient mice and the mature NKT cells of wild-type mice is supported by our observation that pre-T{alpha} mRNA is expressed in the TCRßsfCD44+NK1.1+ population of {zeta}/Lck-dd mice, as well as in TCRßsfCD44+NK1.1+ populations and in more mature NKT cells of wild-type mice. With respect to these parameters, the developmental block in NKT cell development in CD3 signaling-deficient mice is similar to that observed for mainstream {alpha}ß thymocytes, suggesting that NKT cell development is arrested at a pre-NKT cell stage.

A pre-NKT cell-like stage has been invoked before on the basis of studies by Sato et al. on splenic NK1.1+ cells of Vß8.2 transgenic mice (55). These cells were positive for pre-T{alpha} and RAG-1/2 mRNAs, were negative for V{alpha}14–J{alpha}281 rearrangements, and exhibited TCR Vß8.2 expression at the cell surface without CD3{varepsilon}. They could be differentiated to TCRß+V{alpha}14+CD3{varepsilon}+ mature NKT cells by addition of granulocyte macrophage colony stimulating factor. Although there are similarities to the pre-NKT cells in CD3 signaling-deficient mice described here, we did not observe cells with the unusual TCRß+CD3{varepsilon} surface phenotype. It remains to be determined whether the pre-NKT cells described by Sato et al. represent a physiological intermediate in NKT cell development or may be generated as a consequence of the Vß8.2 transgene.

A further striking feature of pre-NKT cells became apparent upon determination of the intracellular TCRß polypeptide chain levels expressed in NKT cells of wild-type and CD3 signaling-deficient mice. Whereas 80–90% of wild-type NKT cells showed high levels of TCRßic staining, the pre-NKT cells of {zeta}/Lck-dd were essentially negative. As we could not detect TCRß mRNA by RT-PCR in {zeta}/Lck-dd pre-NKT cells, we think that little if any TCRß gene expression takes place in pre-NKT cells of severely CD3 signaling-deficient mice. In mice single-deficient for either Lck of CD3{zeta}, i.e. in mice with residual CD3 signaling competence, a small number of NKT cells expresses TCRß protein at the level of wild-type NKT cells, whereas the majority remain negative for TCRß protein expression similar to the {zeta}/Lck-dd mice. Together, the data are consistent with a developmental sequence in which pro-NKT cells first rearrange the TCRß genes, followed by induced expression of intracellular TCRß mRNA and protein. Cells that have expressed TCRß polypeptide chains proceed to rearrange TCR{alpha} genes and display {alpha}ßTCR at the cell surface, followed by selection of NKT cells with the canonical Va14–Ja281 TCR.

Is the induction of TCRß expression in pre-NKT cells dependent on CD3 signaling or not? NK {alpha}ßT cell development in TCR{alpha}-deficient mice is blocked due to the inability to rearrange TCR{alpha} genes rather than to a functionally incompetent CD3 complex. We found that nearly all of the NKT cells of TCR{alpha}-deficient mice express {gamma}{delta}TCR at the cell surface. Moreover, the selection for in-frame V{gamma}1J{gamma}4 and against in-frame V{gamma}2J{gamma}1 rearrangements suggests that NKT cells of TCR{alpha}-deficient mice have developed due to signals from a functionally competent CD3 complex. We found that NKT cells of TCR{alpha}-deficient mice contain a significant population with high TCRßic staining, in contrast to pre-NKT cells of CD3 signaling-deficient mice that fail to express intracellular TCRß mRNA and protein. The data therefore suggest that the expression of intracellular TCRß protein in NKT cell development is dependent on a functional CD3 complex. The possibility that induction of TCRß expression by CD3 signals is restricted to the NK {gamma}{delta}T cell lineage needs to be considered, but does not appear very likely. Our data on the mutual presence of TCR{gamma} and TCRß V(D)J rearrangements in both NK {alpha}ßT and {gamma}{delta}T cells suggests a bipotential pre-NKT precursor in which expression of TCRß, and possibly also TCR{gamma}{delta}, are differentially regulated.

As previously shown for mainstream {alpha}ßT cell development (39), CD3-dependent regulation of TCR gene expression may have a role in {gamma}{delta} lineage development. As previously shown, {gamma}{delta}T cell development appears to be dependent on CD3 signals (56). This may be the case not only for mainstream {gamma}{delta}T cells but also for NK {gamma}{delta}T cells, as suggested by the generation of NK {gamma}{delta}T cells in TCR{alpha}-deficient mice but not in {zeta}/Lck-dd mice. Similar to mainstream {alpha}ß versus {gamma}{delta} T cell development (56), dependence of NK {gamma}{delta}T cell development on CD3 signals appears to be less pronounced than that of NK {alpha}ßT cells, as NK {gamma}{delta}T cells but not NK {alpha}ßT cells arise in some CD3{zeta}-deficient mice (28). Nevertheless, the CD3 signals required for NK {gamma}{delta}T cells appear to be rather strictly defined, as suggested by the absence of NK {gamma}{delta}T cells in the CD3{zeta}/{eta}-deficient mice described here and the presence of these cells in the CD3{zeta}-deficient mice previously described (28). Whether or not the generation of NK {gamma}{delta}T cells is associated with CD3-dependent regulation of the expression of TCR{gamma} and/or {delta} genes remains to be determined.

Our analyses of the selection status of TCRß VDJ rearrangements suggests a two-step process of TCRß expression and selection in pre-NKT cells. TCRß VDJ junctions are unselected with respect to productive rearrangements in the CD44+NK1.1+ population of {zeta}/Lck-dd mice, consistent with the lack of expression of productive TCRß VDJ genes. In contrast, both TCR Vß8 and Vß5 joints are selected for in-frame rearrangements in NKT cells of TCR{alpha}-deficient mice, consistent with indiscriminate induction of productive TCRß VDJ genes that rearrange at random in pre-NKT cells. It is therefore likely that expression is followed by general and indiscriminate ß selection, based on survival and/or proliferation. As previously shown, selection for Vß8.2 occurs only after TCR{alpha} rearrangement and expression of a random repertoire of surface {alpha}ßTCR (14,26). Taken together, the data suggest an early developmental block in CD3 signaling-deficient mice, i.e. before expression of in-frame TCRß VDJ genes. In TCR{alpha}-deficient mice, the block in NK{alpha}ßT cell development and deviation to NK {gamma}{delta}T cells appears to occur later, i.e. after TCRß gene expression.

On the basis of these and other results (31), it is likely that pre-NKT cells express a structure similar to the pre-TCR of mainstream pre-{alpha}ßT cells (57). Signals generated by the pre-NK TCR may stimulate TCRßic+ pre-NKT cells to survive and proceed to TCR{alpha} gene rearrangements. However, it is so far not clear whether CIC are expressed on pro-NKT cells similar to early mainstream {alpha}ßT cells (6). CIC have previously been implicated in TCRß expression in mainstream pre-{alpha}ßT cells (39,40), a suggestion that is presently controversial (53). Although TCRß gene expression appears to be under the control of CD3 signals in both mainstream {alpha}ßT cells and NKT cells, we observe an interesting difference between the two lineages. While mainstream pre-{alpha}ßT cells exhibit a low CD3-independent level of TCRß gene expression, the total lack of TCRß mRNA and protein in pre-NKT cells suggests an absolute dependence on CD3 signals. NKT cell development may thus provide a particularly clear situation to study molecular mechanisms involved in TCRß gene regulation and it will be of interest to determine the role of CIC in this process.


    Acknowledgments
 
We thank Ingrid Falk for able technical assistance, Andreas Würch for valuable help with PCR techniques and flow cytometry, and Dr H. Mossmann and the animal house staff of the MPI for competent mouse breeding.


    Abbreviations
 
APC allophycocyanin
B biotin
CIC clonotype-independent CD3 complex
dd double-deficient
DN double negative
DP double positive
ic intracellular
PE phycoerythrin
Lck p56lck, Lck
sd single-deficient
sf surface

    Notes
 
Transmitting editor: S. H. E. Kaufmann

Received 29 November 2000, accepted 7 May 2001.


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 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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