A variable immunoreceptor in a subpopulation of human neutrophils

The TCRαβ Complex Is Expressed by a Neutrophil Subpopulation.

A series of control experiments prompted us to test the hypothesis that human neutrophils express components of the TCR machinery. To authenticate the presence of the TCRαβ complex in neutrophils in situ, we performed immunocytochemistry using monoclonal antibodies against the TCR α-subunit and β-subunit. We consistently found positive staining in a subset of freshly obtained peripheral blood neutrophils indicating that significant quantities of the TCRαβ complex are expressed by a distinct neutrophil subpopulation (Fig. 1A). No staining was observed in control experiments in the absence of primary or secondary antibodies (Fig. 7, which is published as supporting information on the PNAS web site). A survey among healthy donors (n = 5) revealed that, on average, a 5–8% fraction of circulating neutrophils displayed TCRαβ expression (data not shown). To obtain detailed information on the subcellular localization of the TCRαβ complex in neutrophils we performed ImmunoGold electron microscopy. Double-labeling for simultaneous detection of the TCR α- and β-subunits revealed specific staining in circulating neutrophils (Fig. 1B) and also in infiltrating tissue neutrophils in a case of acute appendicitis (Fig. 1C). Comparable to Jurkat leukemic T cells (positive controls) (Fig. 8, which is published as supporting information on the PNAS web site), gold particles were frequently grouped in pairs or in larger clusters consistent with the presence of TCRαβ heterodimers and higher-order TCR complexes (5). TCRαβ expression was most abundant on the cell surface of TCR-expressing neutrophils but also detectable in vesicular compartments and in the perinuclear region. These sites are in agreement with the biosynthetic pathway and the intracellular recycling route of the TCR in T cells (6).

The human leukemia cell line HL-60 can be induced to differentiate in vitro into neutrophil-like cells by all-transretinoic acid (ATRA) (7). Immunoblot demonstrated the presence of the TCRβ constant chain in native and differentiating HL-60 cells (Fig. 1D Top). In unstimulated neutrophils, a band was detected that was slightly larger than 47 kDa, consistent with the presence of a previously reported differentially glycosylated TCR variant (8) (Fig. 1D Bottom). Monoclonal antibodies to CD3 and CD28 (9) are commonly used to mimic activation of the TCR. Activation with CD3 and, to a lesser degree, CD28 antibodies triggered induction of TCRβ. These results are in line with the finding that human neutrophils express CD3ζ (Fig. 4 B and C) and CD28 (10).

Circulating Human Neutrophils Express Individual-Specific TCR Vαβ mRNA Repertoires.

We next determined whether HL-60 cells and neutrophils exhibit TCR Vα and Vβ repertoire diversity. RT-PCR expression profiling of 32 TCR Vα mRNAs revealed isolated expression of TCR Vα3 mRNA in HL-60 cells (Fig. 1E Upper). Complementarity-determining region 3 (CDR3) spectratyping of the Vα3 chain revealed oligoclonality (11), consistent with the clonal nature of HL-60 cells (Fig. 1E Upper Left). To assess TCR Vα and Vβ repertoire diversity in neutrophils, TCR Vαβ mRNA expression profiling was performed on neutrophil and T cell fractions from healthy individuals (n = 6). We found expression of a multitude of TCR Vαβ chain mRNAs in the neutrophil fraction (Fig. 1 E and F). Control experiments to exclude contamination are shown in Fig. 9, which is published as supporting information on the PNAS web site. Significant differences of TCR Vαβ gene expression patterns between neutrophils and T cells from the same individuals were observed (Fig. 10, which is published as supporting information on the PNAS web site). Representative analysis of the Vα14 and Vβ14 CDR3 spectratypes revealed Gaussian and skewed (Vβ14 Neutrophils 1) CDR3 length distribution profiles indicative of complex Vαβ repertoires as they are observed in T cells (12). TCR Vαβ expression profiles in neutrophils differed interindividually (Fig. 1 E and F and Fig. 10). Together, these data indicate that peripheral blood neutrophils express complex and individual-specific TCR Vαβ repertoires.

We next characterized the TCRαβ-expressing neutrophil subpopulation by flow cytometry. Ethanol-fixed neutrophil CD16^bright cells showed positive staining for TCRα for a subfraction of 5% cells (Fig. 2A). Neutrophils showed positive staining for CD16 but not for T cell markers (CD3, CD4, and CD8) (Fig. 11, which is published as supporting information on the PNAS web site). We also observed positive staining for TCRα in HL-60 cells (Fig. 2B). The identification of a CD16^bright TCRα⁺ subfraction by flow cytometry thus confirmed the presence of the TCR in neutrophils.

Murine Neutrophils Express TCR Vαβ Repertoires.

To test whether TCR expression in neutrophils is limited to the human immune system, purified splenic neutrophils from two normal mouse strains (CD1 and 129/SvJ) and two mutant strains lacking T cells (CD1 nude and C.B17 SCID) were analyzed for the presence of the TCRαβ. CD1 athymic mice have no T cells (13), and in C.B17 SCID mice the generation of TCRs is severely inhibited because of DNA repair defects, which leads to a failure of V(D)J recombination and combined T and B cell deficiency with minor “leakiness” (14). Immunocytochemistry showed positive staining of the TCRβ chain in a 5–7% subpopulation of neutrophils from three mouse strains investigated (Fig. 3A), and RT-PCR revealed mRNA expression of TCRα and TCRβ constant chains (Fig. 3B). TCR CDR3 spectratyping confirmed the presence of complex Vα5 and Vβ16 repertoires in the neutrophil fraction of normal 129/SvJ mice (Fig. 3C). Neutrophils from T cell-deficient CD1 nude mice also expressed TCR Vαβ repertoires, whereas no evidence for TCR Vαβ repertoire expression was found in neutrophils from recombination-defective C.B17 SCID mice.

To test whether and to which extent neutrophils express the TCR conditions of severe T cell deficiency, we assessed the expression of TCRαβ constant chains in peripheral blood neutrophils from an 8-week-old infant with X-linked SCID (X-SCID). X-SCID is a rare inherited immune system disorder with an incidence of 1:200,000 caused by mutations in the IL-2 receptor γ-chain (15). Affected individuals typically display dramatically reduced or absent T cells (T cell-negative/B cell-positive X-SCID) (16). At the time of blood draw the infant's WBC were as follows: 2,100 × 10³ neutrophils per microliter, 1,380 × 10³ lymphocytes per microliter, and 940 × 10³ monocytes per microliter. Importantly, RT-PCR analysis of purified X-SCID neutrophils revealed expression of both the TCRα and the TCRβ constant genes, strongly suggesting that TCR expression in human X-SCID neutrophils occurs in the virtual absence of T cells (Fig. 12, which is published as supporting information on the PNAS web site). Consistent with these results, we also found that neutrophils from an adult patient with severe iatrogenic T cell deficiency (acute graft rejection) express the TCR (Fig. 12).

Neutrophils Express Essential Components of the TCR Signaling Complex.

We next investigated whether HL-60 and neutrophils express TCR constant chains and components of the TCR signaling complex. RT-PCR showed expression of each of the TCR α, β, γ, and δ constant chains (TRAC-TRDC) in HL-60 cells and neutrophils (Fig. 4A and C). Both cell types also express the CD3ζ chain (17), ζ-chain-associated protein kinase 70 (ZAP70) (18), and linker for activation of T cells (LAT) (19). ATRA-mediated differentiation induced up-regulation of CD3ζ and ZAP70 mRNA levels in HL-60 cells but had no effect on expression of LAT and TCR constant chains. Moreover, CD3- or CD28-activating antibodies resulted in increased CD3ζ but not ZAP70 expression (Fig. 4B).

TCR Expression in Neutrophils Is Modulated by G-CSF in Vivo.

We tested whether G-CSF impacts expression of the TCR machinery in circulating neutrophils in vivo. For this, a single dose (30 μg) of recombinant human G-CSF was administered s.c. to two healthy probands, and peripheral blood neutrophils were collected at various time points after injection (Fig. 13, which is published as supporting information on the PNAS web site). G-CSF suppressed mRNA expression of all neutrophil TCR constant chains and components of the TCR signaling complex (CD4, CD3ζ, ZAP70, and LAT) during the first 48 h after injection (Fig. 4C). This effect was maximal at 12 h (individual 2; data not shown) and 48 h after injection (individual 1; Fig. 4C). Up-regulation of matrix metalloproteinase 25 (20) and CD64 indicated that the effect was not due to a global suppression of neutrophil gene expression. At 132 h after injection mRNA levels of all TCR constant chains and most signaling components had increased but were still below those levels found before G-CSF administration. These results suggest that G-CSF potently modulates gene expression of the neutrophil TCR complex in vivo.

Neutrophils Express the RAG1/RAG2 Recombinase and Flexible TCR Vαβ Repertoires.

The recombination-activating gene proteins RAG1 and RAG2 initiate genomic V(D)J recombination. Western blot analysis showed constitutive expression of both proteins in HL-60 cells and circulating neutrophils (Fig. 5A). Constitutive expression of RAG1/RAG2 by neutrophils indicates that these cells possess the specific molecular equipment for generation of antigen receptor diversity. Time course analysis revealed that G-CSF induced up-regulation of RAG1 for a period of 48 h, which was paralleled by a moderate down-regulation of generally high RAG2 levels. To explore the dynamics of neutrophil TCRαβ repertoire diversity over time and its responsiveness to G-CSF we characterized the CDR3 spectratypes of selected V chains for the duration of 7 months in the two probands who were administered G-CSF. Some CDR3 spectratypes changed from Gaussian to oligoclonal profiles (Fig. 5B), whereas others remained constant, indicating that TCRαβ repertoires in neutrophils are variable. Clonal analysis of the CDR3 repertoire changes confirmed these results on the nucleotide sequence level (Fig. 14, which is published as supporting information on the PNAS web site).

Stimulation of the TCR/CD3 Complex Induces Up-Regulation of bcl-x_L Expression, Inhibits Apoptosis, and Enhances IL-8 Secretion.

To explore a possible function of the neutrophil TCR in apoptosis, we determined the effect of the TCR agonists CD3 and CD28 on the expression of antiapoptotic factors and cell survival. Stimulation of neutrophils with both anti-CD3 and anti-CD3/CD28 antibodies induced up-regulation of bcl-x_L starting at 3 h (Fig. 6A). CD28 stimulation alone did not alter bcl-x_L expression. Engagement of CD3 had an antiapoptotic effect on neutrophils, as evidenced by TUNEL staining (Fig. 6B) and flow cytometry (data not shown). Furthermore, we examined whether CD3/CD28-mediated TCR engagement has an effect on neutrophil IL-8 secretion, an essential chemokine for early neutrophil recruitment. Indeed, time course analysis of CD3 and CD3/CD28 activation showed enhanced secretion of IL-8 by neutrophils within 24 h (Fig. 6C). This response was also observed when Fc γ-receptors were blocked by antibodies to CD16 and CD32 (Fig. 15, which is published as supporting information on the PNAS web site) or in the presence of autologous serum (data not shown), indicating that it is not mediated by nonspecific Fc receptor activation. Taken together, these data demonstrate that engagement of the CD3-dependent TCR signal transduction pathway inhibits apoptosis and triggers increased IL-8 secretion in neutrophils.

Cell Isolation and Cell Culture Experiments.

Neutrophils were isolated by dextrane sedimentation and density gradient centrifugation (FICOLL-Paque, Amersham Bioscience, Munich, Germany). Neutrophils were routinely >97% pure. T cells were enriched from peripheral blood mononuclear cells by positive selection using MACS CD3 MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) (average yields >93%). Peripheral blood from an 8-week-old male infant with X-SCID was drawn before stem cell transplantation. Venous blood from a male kidney transplant patient (triple immunosuppressive therapy) (Fig. 12B) and from two healthy volunteers who were administered recombinant human G-CSF (Filgrastim, Amgen, Thousand Oaks, CA) at a single dose of 30 μg s.c. was obtained at various time points. Written informed consent had been obtained from all individuals included in this study.

CD1-Foxn1^nu mice, CB17/Icr-Prkdc^scid/Crl mice (4 weeks old, male), and normal mice (129/SvJ and CD1) were purchased from Charles River Laboratories (Wilmington, MA) and maintained under specific pathogen-free conditions until the day they were killed. Neutrophils from pooled (n = 7) splenocyte cell suspensions were isolated by MACS using the Anti-Ly6G MicroBead kit (Miltenyi Biotec). The Ly6G negative mononuclear cell fraction was used as controls. Isolated neutrophils were 93% (129/SvJ and CD1 mice) and >98% pure (CD1 nude and C.B17 SCID mice), as assessed by flow cytometry (data not shown).

Promyelocytic HL-60 and Jurkat cells (DSMZ, Braunschweig, Germany) were cultivated in RPMI medium 1640 (10% FCS/1% MEM). Detailed information on cell activation experiments is provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Immunocytochemistry, Flow Cytometry, and ImmunoGold Electron Microscopy.

For immunocytochemistry and electron microscopy studies the cells were simultaneously stained with mouse anti-human monoclonal antibodies to TCRαF1 and TCRβF1 (Endogen, Bonn, Germany). Positive staining was visualized by using an Axio Imager ZI (Zeiss, Göttingen, Germany) and Axio Vision software. Flow cytometric analyses were performed on a FACSCalibur using CellQuest Pro software (Becton Dickinson Biosciences, San Jose, CA). For visualization of apoptotic neutrophils, TUNEL was performed on neutrophil cytospins using the In Situ Cell Death Detection POD Kit (Roche, Mannheim, Germany) followed by counterstaining with DAB (DakoCytomation, Hamburg, Germany). For ImmunoGold electron microscopy cells, human neutrophils, Jurkat cells, and sections of appendix tissue were examined in a LEO912AB electron microscope (Zeiss). For detailed description of immunocytochemistry, flow cytometry, and electron microscopy, see Supporting Materials and Methods.

RT-PCR, TCR CDR3 Size Spectratyping, and Assessment of CDR3 Sequence Diversity.

RNA from all cells was prepared with TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was made by using the First Strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). mRNA expression levels were quantitated with the Fast Start cDNA SYBR Green Kit (Roche Diagnostics) on the Lightcycler. Data were normalized against GAPDH.

TCR Vα and Vβ mRNA expression profiling and size spectratyping of the antigen-binding Vα and Vβ CDR3 regions were performed as previously reported (11, 12). CDR3 spectratypes were assessed on an ABI 310 Genetic Analyzer capillary sequencer (Applied Biosystems, Toroed, Norway). The sequences of additional PCR primers can be requested from the authors. Authenticity of all relevant PCR products was confirmed by sequencing. PCR runs were repeated at least twice.

To assess neutrophil TCR Vα3 CDR3 sequence diversity specific RT-PCR amplification products were excised from agarose gels, purified (QIAEX II Gel Extraction Kit; Qiagen, Valencia, CA) and cloned into a TA vector (pCR-TOPO; Invitrogen, ABI 310 Genetic Analyzer) by using standard protocols. For each time point, cDNA sequences of the Vα3 CDR3 were analyzed for a total of 40 randomly picked clones.

Immunoblotting and ELISA.

Immunoblot analyses were performed with standard techniques using the following antibodies: TCRβ chain (Endogen), CD3ζ (Serotec, Oxford, U.K.), ZAP70 (Cell Signaling Technology, Boston, MA), actin (Santa Cruz Biotechnology, Santa Cruz, CA), bcl-2 (DAKO, Hamburg, Germany), bcl-X_L (Santa Cruz Biotechnology), RAG1 (BD Biosciences, San Jose, CA), and RAG2 (BD Biosciences). Pilot immunoblotting experiments revealed that detection of RAG1 and RAG2 from peripheral blood leukocyte subpopulations was possible only when protein fractions were prepared with TRIzol reagent, which allows separation of cellular protein from DNA. Neutrophil IL-8 secretion was assessed by ELISA (Human IL-8 ELISA kit; Pierce Biotechnology, Rockford, IL).