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Keywords:

  • NK cells;
  • Tcells;
  • TCR;
  • γδ-T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

In this issue of the European Journal of Immunology (EJI), Stewart et al. report about a population of γδ-T cells expressing an extensive repertoire of NK cell receptors, and the presence of non-rearranged germline TCRδ transcripts in conventional NK cells. These findings and other recent studies highlight the similarities of NK cells and T cells and the problems in discriminating between these two lineages.

See accompanying article: http://dx.doi.org/10.1002/eji.200737354

Abbreviations:
GFP:

green fluorescent protein

NCR:

NK cytotoxicity receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

The relationship between NK cells and T cells has been a topic of considerable interest since the first description of NK cells in the 1970′s. NK cells were discovered based on functional criteria – the ability to kill certain tumor cells and thymocytes in vitro1. This activity was still detected in mice lacking a thymus and in mice impaired in their ability to rearrange T cell and B cell antigen receptors, demonstrating that neither the thymus nor gene rearrangement is necessary for the development of effector cells with “NK activity”. However, once the T cell receptor had been discovered, it became clear that T cells, in some circumstances, also mediate “NK activity”. In 1986, we published an opinion article in the Journal of Immunology entitled, “Natural Killer Cells: Definition of a Cell Type Rather Than a Function” 2. At its conclusion, we proposed: “NK cells mediate the vast majority of non-MHC-restricted cytotoxicity in an unimmunized host. They do not recognize targets via a CD3/Ti complex, do not rearrange T cell antigen receptor genes, and apparently have a distinct progenitor cell.”

An article in this issue of the European Journal of Immunology has re-addressed the relationship between NK cells and T cells, highlighting the difficulties in discriminating these two cell types. In this study, Stewart et al. 3 analyzed the lymphocytes in mice expressing a green fluorescent protein (GFP) inserted into the T cell receptor (TCR)-δ constant region gene. As expected, mature γδ-TCR+ T cells in these mice expressed high amounts of GFP and a CD3/TCR-γδ complex was readily detected on the cell surface of these T cells. Thymocytes with an intermediate level of GFP expression were detected and were characterized as cells undergoing differentiation into the TCR-γδ T cell lineage. An unexpected finding was that the CD3-NK1.1+ population in the spleen and other peripheral lymphoid organs was green, with varying intensities of fluorescence; however, B cells and myeloid cells were not fluorescent. The majority of CD3-NK1.1+ cells expressed GFP in low to intermediate amounts, but a minor fraction expressed high amounts of GFP, equivalent to the level observed on mature CD3+γδ-TCR+ T cells. Further analysis revealed that these GFP-bright CD3-NK1.1+ cells were absent in Rag1-deficient mice and athymic mice and expressed CD127, the IL-7 receptor α chain. A more careful analysis revealed that these cells did, in fact, express CD3 on the cells surface in very low amounts and they expressed abundant CD3ϵ intracellularly. Similar difficulties have been encountered in studying the αβ-TCR-bearing “NKT cells”, which when activated down-regulate expression of their cell surface CD3/TCR complex – hence, making them almost impossible to distinguish from conventional NK cells. The observations reported by Stewart et al. 3, underline the problem of discriminating between NK cells and T cells, and prompt reconsideration about what is known about CD3, TCR, and NK receptor expression in NK cells and T cells.

TCR rearrangement and expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

Mature T cells can be unambiguously defined as lymphocytes that rearrange and express TCR genes and express a functional CD3/TCR complex on their cell surface. T cells express either αβ-TCR heterodimers or γδ-TCR heterodimers, both of which assemble with a signaling complex containing the CD3γ, δ, ϵ, and ζ chains. Early studies from several laboratories established that mouse and human NK cells do not rearrange the TCR genes; however, germline transcription of TCRβ and TCRδ in NK cells has been reported previously 46. The presence study in this issue of EJI by Stewart et al.3 provide an elegant demonstration of germline transcription of the TCRδ locus in most resting, mature mouse NK cells in spleen, lymph nodes, and liver. Expression of germline transcripts of TCR and Ig loci have been documented in several cell types, and likely just represent accessibility of the gene region rather than having functional significance or developmental consequences.

With respect to the CD3 subunits, Stewart et al.3 did not detect CD3ϵ proteins in the cytoplasm of mouse NK cells. However, human NK cell clones established from fetal liver, which do not rearrange TCRβ, TCRγ, or TCRδ genes, expressed CD3δ, CD3γ, and CD3ϵ proteins in their cytoplasm, but not on their cell surface, and CD3ϵ is transcribed by IL-2 activated human peripheral blood NK cells 7. Both mouse and human NK cells express CD3ζ, which associates with NKp46 8, and in humans with CD16 (FcγRIII) 9 and NKp30 10. Collectively, these prior studies have established that NK cells that do not rearrange their TCR genes can express germline TCR transcripts and can express CD3 subunit proteins.

Expression of “NK receptors” on T cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

A number of cells surface glycoproteins have been referred to as “NK receptors” either for historical reasons (they were first identified on NK cells) or because they are predominantly expressed on NK cells. Included in this “NK receptor” category are members of the Ly49, KIR, CD94, NKG2, 2B4 (CD244), DX5 (CD49b), CD16, CD56, NKR-P1 (CD161), and NK Cytotoxicity Receptor (NCR) families 11. However, all of these “NK receptors” can be expressed on subsets of T cells or other cell types. Although most of these “NK receptors” are not expressed on resting, naïve αβ-TCR+ T cells, they are frequently acquired after activation, predominantly on CD8+ T cells, but also on some CD4+ T cells. By contrast, “NK receptors” are often detected on γδ-TCR+ T cells, even without deliberate activation of these cells. In the present study, Stewart et al.3 detected expression of NK1.1, 2B4, NKG2D, NKG2A, and Ly49 on mouse γδ-TCR+ T cells. In addition, NKp46, which is considered one of the receptors most selectively expressed on NK cells, was found on ∼60% of these NK1.1+ γδ-TCR+ T cells (Fig. 1). These investigators noted an inverse correlation between the cell surface density of the CD3/γδ-TCR complex and NK1.1 (and other “NK receptors”) on this subset; therefore, unless very sensitive methods of detection are used to analyze the expression of CD3 or TCR, these cells might easily be inappropriately identified as NK cells, rather than γδ-TCR+ T cells. A similar problem is encountered when NK1.1+ “NKT cells” expressing the invariant Vα14 TCR are activated – they down-regulate the CD3/TCR complex rendering them essentially indistinguishable from NK cells 12. Studies of the intraepithelial lymphocytes isolated from the small intestine of patients with celiac disease have uncovered oligoclonal populations of αβ-TCR+ CD8+ T cells displaying lower amounts of TCR/CD3 on the cell surface and an abundance of “NK receptors”, suggesting that these cells have been “re-programmed” to acquire receptors enabling them to mediate functions usually mediated by NK cells 13.

thumbnail image

Figure 1. Rearrangement of TCR genes and of “ NK receptors” in T cells and NK cells.

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Recent studies have also described a minor population of NK1.1+ cells lacking cells surface expression of CD3 in the thymus and spleen of wild-type and TCRβ–/–δ–/– mice that express in-frame rearranged TCRγ genes 14. These cells are absent in nude mice, implying a requirement for the thymus for their development. This minor subset of cells may represent aberrant γδ-T cells that are unable to generate a functional TCRδ chain and persist rather than die. A similar population of thymic-dependent NK1.1+ cells (also expressing NKG2D, CD94, and DX5) has also been described by Vosshenrich and colleagues 15. Whether these cells express in-frame rearranged TCRγ genes was not reported; however, unlike conventional NK cells these thymic-dependent NK1.1+ cells required Gata-3 and IL-7 for their development. This cell type has been proposed as a novel thymic-dependent NK cell subset, but might instead be failed δγ-T cells, whose functional relevance is as yet unknown.

Bona fide” NK cells and NK-like T cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

The shared repertoire of receptors expressed by NK cells and T cells blurs the distinction between these cell types. The only unambiguous difference between NK cells and T cells is rearrangement of the TCR loci – genomic rearrangement is irreversible and a heritable trait and clonal marker in all the progeny of these T cells, even if they acquire “NK receptors” after activation. T cells rearrange TCR genes; NK cells do not. Without this definition, the two cell type's cannot be distinguished.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. TCR rearrangement and expression
  5. Expression of “NK receptors” on T cells
  6. Bona fide” NK cells and NK-like T cells
  7. Acknowledgements

L.L.L. is an American Cancer Society Research Professor and supported by NIH grants AI066897, AI068129, CA095137, and CA105379. I thank Hergen Spits and members of my lab for helpful discussion.

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    WILEY-VCH

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