Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR αβ lymphocytes

Authors


Abstract

A subset of CD8+ T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8+ T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7+ memory cells and CCR7 effector cells. Since NKG2A can only be induced on naive CD8+ T cells while CD94 memory cells are refractory, it is likely that commitment to the CD94:NKG2A+ subset occurs during the first encounter with antigen. CCR7+CD94:NKG2A+ T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-γ. These cells occur as a separate CD94:NKG2A+ T cell lineage with a distinct TCR repertoire that differs from that of the other CD8+CD94 T cells activated in situ.

1 Introduction

Major histocompatibility complex class I-specific killer cell receptors have originally been described on NK cells 13. They include killer cell immunoglobulin-like receptors (KIR), leukocyte immunoglobulin-like receptors 4 and C-type lectin domain containing proteins such as CD94 or Ly49. CD94 forms heterodimers with NKG2A (CD94:NKG2A) or with NKG2C (CD94:NKG2C). Binding of the receptor to its ligand HLA-E conveys an inhibitory signal when CD94 is heterodimerized with NKG2A that harbors an ITIM-motif 5, 6. By contrast, the signal through CD94:NKG2C of which the latter molecule binds to the ITAM-motif harboring adaptor molecule DAP-12 is co-stimulatory 79.

TCRαβ CD8+ lymphocytes may express the same type of killer cell receptors. In mice, many of the Listeria monocytogenes-specific CD8+ T cells express CD94 10, 11. CD94 is also induced during LCMV infection and the ensuing memory cells remain CD94 positive 10, 12. Furthermore, polyoma virus-specific CD8+ T cells up-regulate CD94:NKG2A during acute infection 13 which results in down-regulation of their antigen-specific cytotoxicity. In humans, the situation may be quite different. The only CD94+ T cells with a known antigen specificity described to date recognize tumor-associated tissue differentiation antigens 6, 1417, whereas CD8+ T cells specific for herpes B 18, CMV 19, or for EBV during acute mononucleosis 18, 20 do not express CD94. Interestingly, many intraepithelial T lymphocytes express CD94:NKG2A 9, suggesting that its expression is induced after recognition of particular antigens and/or under particular conditions of antigenic stimulation. These conditions may, at least partially, be mimicked in vitro. It has been shown that CD8+ T cells express CD94 when stimulated in the presence of IL-15, TGF-β or IL-12 2123. However, we found that the induction of CD94 in vitro merely reflects the potential of naive CD8+ T cells to up-regulate CD94 but that, in contrast to CD94:NKG2A+ or CD94:NKG2C+ T cells in vivo, this expression is transient. Furthermore, once naive CD8+ T cells have differentiated into CD94 memory-effector cells in vivo, they become refractory to CD94 induction. This suggests strongly that CD94:NKG2A+, CD94:NKG2C+ as well as CD94-negative memory-effector T cells are distinct populations that are committed to their respective CD94 phenotypes.

2 Results

2.1 Expression of CD94, NKG2C and NKG2A by TCRαβ CD8+ lymphocytes

A minor subpopulation of T cells express CD94. We found that in ten healthy individuals studied, an average of 4.7±3.8% of the TCRαβ CD8+ T cells expressed CD94 heterodimerized with NKG2A while 2.1±1.8% expressed CD94 heterodimerized with NKG2C. In addition, 0.8–42% of cells expressed CD94 at a low level without expressing NKG2A or NKG2C. This wide range was caused by the fact that the bulk of CD8+CCR7RA+ effector T cells of which the presence in normal individuals varies considerably according to age and CMV status 24 were CD94lowNKG2ANKG2C. Fig. 1 shows the combinations of CD94, NKG2A and NKG2C that TCRαβ CD8+ T cells may express, as well as the transcription of these molecules in the cells with the respective phenotypes. To be able to identify TCRαβ CD8+ T cells by FACS by one parameter only, we gated on CD8high cells. This efficiently excludes NK cells and TCRγδ T cells that express either no, or only low levels of CD8 and CD8high cells in peripheral blood are >99% TCRαβ-positive. As reported by others 14, CD94 expression appeared higher in CD94:NKG2A+ than in CD94:NKG2C+ cells (Fig. 1a, b). However, because many of the CD94lowNKG2C cells expressed CD94 at levels comparable to that of CD94lowNKG2C+ cells, the latter could not be discriminated by the level of CD94 expression; nor could CD94:NKG2C+ T cells be identified on the basis of the NKG2C-PCR, which has been the only tool available until now. Whereas in vivo (Fig. 1c), transcripts for CD94 and NKG2A were only present in cells expressing the protein at the cell surface, NKG2C transcripts were present in CD8+CD94+NKG2C as well as in CD8+CD94 T cells. Moreover, after CD94 had been induced by stimulation with anti-CD3 in the presence of IL-15 (Fig. 1d, e) the correlation between transcription and surface expression was loose for all three molecules. Most noticeably, in spite of an abundant transcription of NKG2C (Fig. 1f), only CD94:NKG2A heterodimers were expressed at the cell surface. Hence, only the recently produced NKG2C-specific monoclonal antibody demarcated the CD94:NKG2C+ cells precisely.

Figure 1.

CD94 and NKG2A/C expression by CD8+ T cells. (a–b) CD94, NKG2A and NKG2C expression by cells gated on CD8high (99.7% TCRαβ CD8+ lymphocytes for this particular individual). (c) Quantitative real-time PCR of CD94, NKG2C, NKG2A of 3.103 CD94, CD94:NKG2A+ and CD94:NKG2C+ CD8+ T cells. (d–e) CD94, NKG2A and NKG2C expression of immobilized anti-CD3 stimulated CD8high cord blood cells after a 5-days culture in the presence of IL-15. (f) Quantitative real-time PCR of CD94, NKG2C, NKG2A of 3×103 CD94, CD94low and CD94:NKG2A+ cultured cells. Results of the PCR's are expressed as percent of GAPDH transcripts.

Cell surface expression of NKG2C or NKG2A was a prerequisite for the modulation of the TCR-mediated cytotoxicity of CD94+ T cell clones. It is shown in Fig. 2 that anti-CD94 antibodies modulate the anti-CD3-induced redirected lysis of P815 target cells only when CD94 is dimerized with an NKG2 molecule at the cell surface. CD94:NKG2C+ clones lysed their targets more efficiently when costimulated by the anti-CD94 antibody (Fig. 2a). In addition, the anti-CD94 antibody induced some lysis without the anti-CD3 antibody, a phenomenon that appeared to be characteristic of a given clone because it was reproducible with some, but not with other CD94:NKG2C+ T cell clones (data not shown). By contrast, no effect of the anti-CD94 antibody was observed in the CD94+NKG2C clones that were NKG2C+ by RT-PCR only (Fig. 2b) while in the CD94:NKG2A+ clones the anti-CD94 antibody inhibited the anti-CD3-mediated killing of P815 (Fig. 2c).

Figure 2.

Modulation of the CD3-redirected killing of 51Cr-labeled P815 cells by (a) CD94:NKG2C+ T cell clones, (b) NKG2C-PCR-positive CD94+NKG2C clones and (c) CD94:NKG2A+ clones triggered by OKT3 alone (– – • – –), OKT3 plus anti-CD94 (— ○ —), anti-CD94 (— ⊳ —) or without antibodies (— x —). Results, representative of at least five other clones with the same phenotype are expressed as the mean of triplicates ± SD.

2.2 CD94:NKG2A and CD94:NKG2C expression by naive, central memory and effector TCRαβ CD8+ lymphocytes

Naive, memory and effector T cells can be discriminated on basis of the expression of the chemokine receptor CCR7 and the different splicing variants of CD45 25. Naive and central memory cells express CCR7 that is required to home to secondary lymphoid organs. Compared to naive cells (CD45RAhighCCR7+), central memory cells express a lower level of CCR7 and are CD45RAlow or CD45RA. Effector-memory cells are characterized by the loss of CCR7. CD8+ effector cells may re-express CD45RA, which together with the loss of the CD28/CD27 molecules is considered characteristic of highly differentiated effector cells 26. It is shown in Fig. 3a that after gating on CD8+ T cells, all four populations were easily identified (an individual with a relatively high number of effector cells is shown). Furthermore, it is shown that CD94:NKG2A may be expressed during every stage of memory-effector differentiation (Fig. 3b) while CD94:NKG2C expression at the cell surface was clearly an exclusive trait of CCR7CD45RA+ effector cells (Fig. 3c). The CD8+CD94:NKG2C+ T cells also expressed more molecules characteristic of effector cells than the CD8+CD94:NKG2A+ T cells. Approximately 80% of the CD94:NKG2C+ expressed CD56, a marker shown to define effector cells 27 and 75% expressed CD57 but only 17% expressed CD27. By contrast, only 20% of the CD94:NKG2A+ cells were CD56+ and 35% were CD57+ and 81% were CD27+. Furthermore, FACS-sorted CD8+CD94:NKG2C+ T cells transcribed approximately 1.5–2 times more IFN-γ and granzyme B than the CD94:NKG2A+ cells. Hence, the expression of CCR7CD45RA+, CD27, CD56, CD57 and granzyme B of CD8+CD94:NKG2C+ T cells is comparable to that of effector T cells but none of these markers are expressed homogeneously. This effector cell-like phenotype was found in every individual tested and was always more pronounced than on CD8+CD94:NKG2A+ T cells. By contrast, naive cells never expressed CD94:NKG2A (Fig. 3b) nor CD94:NKG2C (Fig. 3c) nor CD94 heterodimers (data not shown) which confirms that CD94 is acquired after antigenic stimulation 28.

Figure 3.

CCR7 and CD45RA expression by CD94:NKG2A+ and CD94:NKG2C+ TCRαβ CD8+ lymphocytes. (a) CCR7 and CD45RA expression by CD8+ T cells demarcating naive (CCR7+CD45RAhigh), central memory (CCR7+CD45RAlow/neg), effector-memory (CCR7RA) and effector (CCR7RA+) CD8+ T cells. (b, c) CCR7 and CD45RA expression by CD8+CD94:NKG2A+ and CD8+CD94:NKG2C+ T cells. The data are from the same individual with a relatively high number of effector cells shown in Fig. 1. Identical phenotypes were found in ten normal controls.

2.3 Lymph node CD8+CD94:NKG2A+ T cells produce high amounts of IFN-γ and are clonally distinct from their CD8+CD94 counterparts

Fig. 4a shows the expression of CD94 on activated (CD69-positive) and CD69-negative CD8+ T cells in a lymph node. Five percent of the cells expressed high levels of CD94 (Fig. 4a), all of which was in conjunction with NKG2A (Fig. 4b). All NKG2A+ cells were CD3 positive (Fig 4c), corroborating that the lymph node CD94:NKG2A+ NK cells 29 had been efficiently excluded from the analysis by the gate on CD8high. No CD94:NKG2C heterodimers were detected (Fig. 4d). Fig 4e shows that except for the CD8+CCR7CD45RA+ effector cells, all other stages (naive, central memory and memory-effector) were present. Approximately half of the CD69+ T cells had lost CCR7 that had initially allowed the entry into the lymph node, strongly suggesting that both CD3+CD8+CD69+NKG2A+ as well as the CD3+CD8+CD69+CD94:NKG2A cells had been activated in situ (Fig. 4f and g). Cells of both phenotypes were granzyme-negative (Fig. 4h) showing that they had not yet advanced towards the stage of CD8+CCR7CD45RA effector memory cells in the periphery in which granzyme is easily detected by the same methods (data not shown). Interestingly, the activated CD69+NKG2A+ cells produced approximately ten times more IFN-γ than the CD69+NKG2A cells (Fig 4h).

Figure 4.

Expression of CD94 and NKG2 molecules and TCR BV-usage of CD8+ lymph node T cells. (a–g) Five-color FACS-analysis with anti-CD8, anti-CCR7, anti-CD45RA, anti-CD69, anti-CD94, anti-NKG2A and anti-NKG2C monoclonal antibodies. The phenotype data are representative of seven preparations of lymph node cells. (a) CD94 expression by CD8+CD69 (4%) and by CD8+CD69+ (7%) T cells. (b–d) NKG2A, CD3 and NKG2C expression of CD8+CD94+ lymph node T cells. (e) Naive (CCR7+CD45RAhigh), central memory (CCR7+CD45RAlow/neg) and effector-memory (CCR7RA) CD8+ lymph node T cells. (f–g) Loss of CCR7 by 50% of the CD8+CD69+NKG2A+ T cells and by 60% of CD8+CD69+NKG2A T cells. (h) Quantitative real-time PCR of NKG2A, IFN-γ and granzyme B of 3×103 cells with the depicted phenotype. Results are expressed as the percent of GAPDH transcripts. (i) Spectratypes of BV14, BV21 and BV22 of CD8+CD69+NKG2A+ (upper panel) and CD8+CD69+NKG2A T cells (lower panel) using primers specific for BC and the respective BV.

We argued that if the differences in NKG2A-expression and IFN-γ production would be linked to a maturation stage of the same clone, the TCR-usage of the CD69+CD94:NKG2A+ cells would be similar to that of the CD69+NKG2A cells. To test this hypothesis, we identified the T cell repertoire of the two populations through spectratyping 30 a method that measures the size heterogeneity of the TCR hypervariable CDR3 region. Fig. 4i shows that both populations had extremely limited repertoires (only BV14, BV21 and BV22 of the 17 BV that were used are shown). Many of the spectratypes of the 21 BV-families tested consisted of single peaks only. Of the 23 TCR present in the CD69+CD94:NKG2A+ population, 7 used the same BV with an identical CDR3-length as 1 of the 39 TCR in the CD69+CD94 population (1 in BV1, BV3, BV6 and 2 peaks in BV13 and BV17). Of these 7 potentially identical TCR, 4 used the same BJ but only 1 of these peaks had the same sequence (data not shown). Hence, only 1 of the 23 clones in the CD69+CD94:NKG2A+ population had a possible sister cell in the CD69+CD94 population and thus, both populations were not only functionally very distinct but also clonally unrelated.

2.4 Inducibility and stability of CD94:NKG2 expression in different subsets of CD8+ T cells

Triggering of the TCR may induce CD94-expression, which is enhanced in the presence of IL-15, TGF-β or IL-12 2123. Furthermore, intraepithelial T lymphocytes modulate CD94:NKG2A expression along with TCR-engagement 9. Fig. 5 shows that whether CD94 expression can be induced and whether it still can be modulated depends entirely on which differentiation stage the T cell is in. Naive CCR7+CD45RA+ CD8+ T cells mitogen-stimulated under limiting dilution conditions in the presence of IL-15 gave rise to clones with a low (n=23, not shown) or a variable (n=148, Fig 5a) expression of CD94 heterodimerized with NKG2A in the cells with the highest level of CD94 expression. Independently of the presence of IL-15 or IL-12, the intraclonal variation of CD94:NKG2A was always observed. Moreover, after sub-cloning of CD94:NKG2Ahigh cells from the clones with a variable expression, the same intraclonal variation in CD94:NKG2A expression was found (Fig 5b). Hence, although stimulation of virtually all naive CD8+ T cells in the presence of IL-15 induces CD94 of which most is in heterodimer with NKG2A, this expression is unstable. Interestingly, under the same conditions, CCR7+CD45RACD94 central memory cells were refractory to CD94 induction (n=60, Fig 5c, d). Similarly, CD8+CCR7CD94–/low T cells remained NKG2 negative after stimulation in IL-15 or IL-12 (data not shown). Although approximately 25% of the clones derived from CD94 non-naive cells expressed CD94 at low levels, CD94:NKG2-dimers were never expressed. Apparently, induction of CD94:NKG2A is restricted to naive T cells and once the cells have entered the memory-effector phase, it can no longer be induced under the same conditions.

Figure 5.

CD94-NKG2A/C expression in clones established from naive and from CD94 central memory cells. (a) Phenotypes of representative clones established from cells sorted with the gate on naive cells as shown in the left panel. For a number of clones with heterogenic CD94 expression we have confirmed their monoclonality by identification of their TCR-BV. b: phenotypes of subclones established from the CD94:NKG2Ahigh cells of the clone shown in (a). (c–d) Phenotypes of representative clones established from cells sorted with the gate on memory cells shown in the left panel.

Fig 6 shows that activated CD8+ lymph node T cells are committed to their CD94:NKG2A+ or CD94:NKG2C+ phenotype. CD8+CD69+CD94:NKG2A+ cells sorted from lymph nodes and mitogen-stimulated under limiting dilution conditions yielded clones with a stable CD94:NKG2A+ phenotype (n=48, Fig 6, upper panel). As in the periphery, the non-naive CD94 were refractory to CD94 induction. Furthermore, clones established from peripheral blood CD8+ T cells sorted on basis of expression of CD94:NKG2A (n=62, Fig. 6, middle panel) or CD94:NKG2C (n=35, Fig. 6, lower panel) showed stable CD94highNKG2A+ and CD94lowNKG2C+ phenotypes for at least 20 cell divisions. Hence, we believe that once CD8+ T cells have been activated in vivo, they are committed to either a CD94:NKG2A+, CD94:NKG2C+ or CD94 lineage.

Figure 6.

CD94-NKG2A/C expression in clones established from naive, CD94 central memory, CD94:NKG2A+ and CD94:NKG2C+ T cells. Upper panel (lymph nodes): phenotypes of representative clones established from cells sorted with a gate on either CD8+CD69+CD94:NKG2A+ T cells (R1) or on CD8+CD69+CD94:NKG2A T cells (R2). Middle panel (peripheral blood): phenotypes of representative clones established from cells sorted with a gate on CD94:NKG2A. Lower panel (peripheral blood): phenotypes of representative clones established from cells sorted with a gate on CD94:NKG2C. Although for the latter, also some clones with a homogeneous CD94-negative phenotype were obtained (n=11, data not shown), we believe that this simply reflects a few contaminating CD94 cells in the original sort that become apparent owed the low cloning efficiency of CD94:NKG2C+ T cells.

3 Discussion

Our findings concerning the differential expression of CD94:NKG2A and CD94:NKG2C heterodimers on TCRαβ CD8+ peripheral blood and lymph node T cells may remodel our current views of these receptors quite substantially. Until to date, the presence of CD94 on memory-effector cells in variable stages of maturation and its swift induction by IL-12, IL-15 and TGF-β 2123 suggested somehow that CD8+ T cells could modulate CD94 expression during an immune response. Up-regulation of CD94:NKG2A inhibitory receptors could then serve as a control of cells displaying too much autoreactivity 11. Alternatively, the differential expression of inhibitory receptors within the same clone could inhibit activation-induced cell death thereby preserving some cells for entry into the memory pool 31. However, we found that in vivo, CD94, CD94:NKG2A+ and CD94:NKG2C+ appeared to be quite distinct populations, each committed to their respective CD94 phenotype. This was particularly evident when the discrimination was done on basis of NKG2C cell surface expression instead of transcription, the only method that has been available until now. In fact, the number of cells that expressed NKG2C at the cell surface represented only a minority of the cells that were positive by RT-PCR. This was the case in CD8+CD94 T cells in peripheral blood and even more evident in cells cultured in vitro (Fig. 1). In fact, all T cells clones that we have tested in this study, transcribed NKG2C independently of their phenotype (data not shown). By contrast, NKG2C cell surface expression never occurred in cells co-transcribing NKG2A and only when the level of transcription was significantly higher than that of CD94. If one of these conditions was not fulfilled, the formation of CD94 homodimers or of CD94:NKG2A heterodimers that was always found at the cell surface of NKG2A-RT-PCR+ cells when sufficient levels of CD94 were expressed prevailed. We do not know whether the NKG2C transcripts that did have the correct sequence (data not shown), are efficiently translated in surface-NKG2C cells. However, if a stable protein is produced without being expressed at the cell surface, these results possibly denote a preferential dimerization of CD94 with NKG2A or CD94 caused by a different affinity (NKG2A > CD94 >> NKG2C) of the CD94-monomer for the respective molecules.

In contrast to NKG2C that was expressed by CCR7CD45RA+ T cells that emerge only late in the immune response 19, NKG2A could be found at all differentiation stages occurring after the encounter with antigen. In fact, naive CD8+ T cells expressed NKG2A as early as 4–5 divisions after stimulation with IL-15 or IL-12. However, when these NKG2A+ cells were cloned under the same conditions, the intraclonal variation in NKG2A expression was considerable. It is notable that a similar heterogeneous expression in intraepithelial T cell clones has contributed to the idea that NKG2A expression depends on a recent engagement of the TCR 9. Although this might be true for cells that have up-regulated NKG2A in vitro, this does not seem to be the case for cells that have become NKG2A+ upon encounter of antigen in vivo. All clones established from the CD8+CD94:NKG2A+ T cells sorted ex vivo homogeneously expressed NKG2A for at least 20 cell divisions in vitro.

The different requirements for the in vitro induction of NKG2A and NKG2C as well as the fact that NKG2C is expressed exclusively by highly differentiated CD45RA+ effector cells suggest that CD94:NKG2C+ T cells are distinct from cells expressing CD94:NKG2A. We believe that the alternative explanation that CD94:NKG2A+ cells would express CD94:NKG2C during differentiation towards effector cells is unlikely, given the stability of the expression of the two molecules after cloning in vitro and the absence of CD94:NKG2A+NKG2C+ double positive cells in vivo. Moreover, we have compared the sequences of 15 different BV used by NKG2C+ T cell clones with 30 different sequences used by NKG2A+ clones from the donor of the analysis shown in Fig. 6 without identifying a common BV, which strongly suggests that there is little overlap between the two populations.

As for the KIR expressed by CD8+ effector T cells, we know very little of the conditions under which CD94:NKG2C is induced. It would make sense, however, if at the end of the immune response at the site of infection some CD8+ effector T cells would differentiate into cells expressing the costimulatory CD94:NKG2C receptor to deal with cells presenting the lowest quantities of antigen. By contrast, we do not believe that in humans, CD94:NKG2A expression is associated with a particular differentiation stage during an immune response. First, CMV and EBV-specific cells remain CD94 during acute and latent infection 1820. Secondly, NKG2A memory T cells are refractory to NKG2A induction by IL-15 or IL-12. Hence, CD94:NKG2A+ T cells appear to be a distinct population of lymphocytes that are committed to their lineage after the first encounter with antigen. This was most evident in lymph nodes where activated CD69+CD94 and activated CD69+CD94:NKG2A+ T cells used a different set of TCR and where clones established from the respective populations were stable with respect to their CD94 phenotypes.

The presence of CD69+CD94:NKG2A+ T cells in the lymph nodes does not mean that these receptors are induced in situ. In fact, we believe that these cells are a part of the CCR7-expressing CD8+CD94:NKG2A+ T cells in the peripheral pool (Fig. 1a) that, similarly to classical central memory cells, recirculate through lymph nodes. This is suggested by the presence in the lymph node of a comparable number of CD69CD94:NKG2A+ cells (Fig. 4a) as well as by the absence of cells expressing the intermediate levels of CD94:NKG2A that are expressed during induction in vitro (Fig. 1). Furthermore, in the seven lymph nodes analyzed, the percentage of CD8+CD94:NKG2A+ cells always fluctuated between 2–5%, which would be surprising if NKG2A were expressed upon activation of antigen-specific cells during an immune response.

We do not know whether the CD94:NKG2A+ T cells in the lymph nodes are activated by the same antigen that is recognized by the CD94 T cells. If not, their abundant production of IFN-γ, atypical for CD8+ T cells at this stage, could be an indication that these cells are the human counterparts of the CD8+ lymph node memory T cells in mice that provide an early non-antigen-specific source of IFN-γ in response to pathogen-derived products 32.

In conclusion, we believe that in humans, CD8+CD94+TCRαβ lymphocytes consist of two distinct populations of T cells that are characterized by a stable expression of NKG2C or NKG2A. CD94:NKG2C that costimulates TCR-mediated cytotoxicity is expressed by highly differentiated CD8+ effector T cells. CD94:NKG2A is present on effector cells as well as on memory cells that recirculate in lymph nodes. However, in humans NKG2A appears not to be expressed by virus-specific lymphocytes 1820 but rather by cells recognizing tissue differentiation antigens 6, 1417. One could argue that if the autoreactivity of these cells is controlled by the expression of the inhibitory CD94:NKG2A receptor 33, dendritic cells expressing costimulatory molecules induced by pathogen-derived products could break this tolerance. As a consequence, CD8+CCR7+CD94+TCRαβ lymphocytes would be activated in lymph nodes (our data) and produce much of the early source of IFN-γ without an interaction of the TCR with a pathogen-derived antigen 32. Such an until now unknown NK-like lineage of T cells able to produce cytokines supplementary to those produced by the CCR7+CD56+CD94:NKG2A+ IFN-γ producing lymph node NK-cells 34 would be an additional instrument to fine-tune the immune response.

4 Materials and methods

4.1 Cells, sorting and FACS analysis

Mononuclear cells (MNC) were obtained through Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation of peripheral blood, of cord blood or of single-cell suspensions prepared from mesenteric lymph nodes. Informed consent for the use for research purposes of the cord blood had been obtained from the mother of the children. The mesenteric lymph nodes had been sampled for HLA typing of organ donors according to the local procedures and the standards of the European Federation of Immunogenetics. Cells were analyzed/sorted using a FACS (FacsVantage SE, Becton Dickinson, Mountain View, CA) after staining with a combination of the following reagents: CD8-APC-Cy7, CD3-APC-Cy7 (Becton Dickinson); CD8-APC, CD94-PE (PharMingen, Basel, Switzerland); CD27-biotin, CD69-biotin (Ancell, Bayport, MN); CCR7-FITC, NKG2A, NKG2C (R&D systems, Oxon, UK); CD3-FITC, CD16-PE (Dako, Zug, Switzerland); Streptavidin-Red613, (PE-Texas Red) (Invitrogen); CD45RA-APC (Caltag laboratories, CA). The unlabeled NKG2A and NKG2C mAb were stained with the Ig subclass-specific goat anti-mouse antisera anti-IgG2a-FITC, anti-IgG2a-PE, anti-IgG2b-FITC or anti-IgG2b-PE (Southern Biotechnologies, Birmingham, AL). The purity of the cell-sorted populations was always higher than 97%.

4.2 In vitro cultures

Naive cord blood T cells (2×105) were stimulated with anti-CD3 bound to 96-well plates in 200 μl of RPMI 1640 medium containing 100 IU/ml penicillin, 100 mg/ml streptomycin, 1 mmol/l sodium pyruvate, nonessential amino acids, and 2 mmol/l L-Glutamine (Life Technologies), 10–5 mol/l β-mercaptoethanol (Sigma, St. Louis, MO) supplemented with 10% human AB serum and 10 ng/ml IL-15 (R&D Systems) or 0.5 ng/ml IL-12 (Genetics Institute, Cambridge, MA) where indicated. Cloning was performed by stimulating the sorted cells under limiting dilution conditions (0.3 cells/well) with phytohemagglutinin (Abbott-Murex, UK) in the presence of irradiated mononuclear cells.

4.3 cDNA synthesis and real-time quantitative PCR

cDNA was obtained by lysing 3×103 to 5×103 cells in 15 μl of buffer provided with the Moloney murine leukemia virus (M-MLV) reverse transcriptase kit (Invitrogen, Basel, Switzerland) supplemented with 0.12% Triton X-100 (Fluka, Buchs, Switzerland), 80 U/ml rRNasin (Promega, Madison, WI), 200 ng/ml oligo-dT (T)20 (Amplimmun, Madulain, Switzerland), 50 nM deoxynucleotide triphosphate (dNTP) (Invitrogen), and 3 μg t-RNA (Boehringer-Mannheim, Rotkreuz, Switzerland) followed by a 1-h incubation at 37°C. Quantitative PCR was performed with a LightCycler System (Roche Diagnostics AG, Rotkreuz, Switzerland) in microcapillary tubes containing 1.5 ml cDNA, 1× QuantiTect™ SYBR® Green PCR kit solution (Qiagen, Basel, Switzerland) and 0.45 mM of each primer pair in 20 μl final volume. We performed 50 PCR cycles (15 s at 95°C, 20 s at 55°C, 20 s at 72°C). A standard curve was done with six tenfold dilutions for each agarose gel-purified PCR product. The primer pairs used were the following: GAPDH: 5′-GGA CCT GAC CTG CCG TCT AG-3′, rev-5′-CCA CCA CCC TGT TGC TGT AG-3′; CD94: 5′-TGC TTC AGC TTC AAA ACA CAG A-3′, rev-5′-GCA TTT CCA TTT GGA TTA TAC GC-3′; NKG2A/B: 5′-ACT GAA CAG GAA ATA ACC TAT GCG-3′, rev-5′-ATG AGC TTC TCT GGA GCT GAT C-3′; NKG2C/E: 5′-ACC GAA CAG GAA ATA TTC CAA GTA-3′, rev-5′-AAT GCA AAT GAT TCC TAG GAC CT-3′; IFN-γ: 5′-GTG GAG ACC ATC AAG GAA GAC A-3′, rev-5′-TAT TGC TTT GCG TTG GAC ATT C-3′; granzyme B1: 5′-TGC AGG AAG ATC GAA AGT GCG-3′, rev-5′-GAG GCA TGC CAT TGT TTC GTC-3′. The conditions of the spectratyping have been published before 35.

4.4 Cytotoxicity assay

Cytotoxicity was assessed by standard 51Cr-release assay. Briefly, 3×10351Cr-labeled P815 cells were coincubated for 4 h with effector cells and the monoclonal antibodies indicated (anti-CD3 20 ng/mL, CD94 (HP-3D9) 2 μg/μl. Percentage specific lysis was calculated from the formula: percentage specific lysis =100×[(experimental counts-negative control (51Cr-labeled P815 alone))/(detergent control-negative control)]. Experiments were done in triplicates.

Acknowledgements

We thank Mrs. Solange Vischer and Colette Grand for expert technical assistance. Supported by a grant of the Swiss National Science foundation (# 3100–65′357.01) and by the “Dr Henri Dubois-Ferrière-Dinu Lipatti” Foundation.

Footnotes

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