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Celiac disease (CD) patients who fail to respond to a gluten-free diet suffer from refractory celiac disease (RCD). A marked expansion of intraepithelial lymphocytes (IEL) lacking surface TCR/CD3 expression characterizes the RCD subtype II. In up to 50% of RCDII patients these so-called aberrant IEL (a-IEL) develop into lymphoma and can disseminate into other tissues. Elevated levels of Interleukin-15 (IL-15) in the intestine of CD and RCD patients likely contribute to the expansion of a-IEL. Here, we investigated if interactions with other cells might also influence a-IEL expansion. Similar to IL-15, cells from the monocyte lineage, particularly mature dendritic cells (DCs), promoted proliferation, prevented apoptosis and induced IFNγ secretion of a-IEL derived from RCDII biopsies (RCDII cell lines), which in turn induced CXCL10. In contrast to IL-15, mature DCs did not induce proliferation of regular TCR(+)IEL lines, generated from CD biopsies and IL-15-blocking antibodies did not inhibit DC-induced proliferation of RCDII cell lines. Furthermore, proliferation was dependent on cell-cell contact, but independent of the HLA-genotype of the stimulating cells. Our results suggest that contact with DC, either in the epithelium or upon dissemination, contributes to uncontrolled expansion of a-IEL in RCDII, independent of HLA-genotype and IL-15., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.

SCID, a syndrome characterized by the absence of T cells and adaptive immunity, can result from mutations in multiple genes that encode components of the immune system. Three such components are cytokine receptor chains or signaling molecules, five are needed for antigen receptor development, one is adenosine deaminase--a purine salvage pathway enzyme, and the last is a phosphatase, CD45. In this issue of the JCI, a report describes how complete deficiency of the CD3epsilon chain of the T cell antigen receptor/CD3 complex causes human SCID.

We investigated the molecular mechanism underlying a severe combined immunodeficiency characterized by the selective and complete absence of T cells. The condition was found in 5 patients and 2 fetuses from 3 consanguineous families. Linkage analysis performed on the 3 families revealed that the patients were carrying homozygous haplotypes within the 11q23 region, in which the genes encoding the gamma, delta, and epsilon subunits of CD3 are located. Patients and affected fetuses from 2 families were homozygous for a mutation in the CD3D gene, and patients from the third family were homozygous for a mutation in the CD3E gene. The thymus from a CD3delta-deficient fetus was analyzed and revealed that T cell differentiation was blocked at entry into the double positive (CD4+CD8+) stage with the accumulation of intermediate CD4-single positive cells. This indicates that CD3delta plays an essential role in promoting progression of early thymocytes toward double-positive stage. Altogether, these findings extend the known molecular mechanisms underlying severe combined immunodeficiency to a new deficiency, i.e., CD3epsilon deficiency, and emphasize the essential roles played by the CD3epsilon and CD3delta subunits in human thymocyte development, since these subunits associate with both the pre-TCR and the TCR.

The contribution of CD3gamma to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3gamma is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3gamma-deficient (gamma(-)) individuals. In contrast to gamma(-) mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural gamma(-) T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3gamma. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3gamma-dependent, as it was restored by retroviral transduction of CD3gamma. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3gamma contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of I kappa B kinase (IKK) complex and, consequently, to NF-kappa B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK alpha in both Jurkat and primary CD4(+) T cells. Vav-1/IKK alpha association is mediated by their helix-loop-helix domains, does not involve IKK beta, and is functionally relevant in that Vav-1-associated IKK alpha kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-kappa B activation is augmented by both IKK alpha and Vav-1, but not IKK beta. Confocal microscopy showed that endogenous Vav-1 and IKK alpha, but not IKK beta, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-kappa B pathway by targeting IKK alpha in the T cell membrane and favoring its activation in response to CD28 stimulation.

IL-12 is mainly produced by CD8alpha(+) dendritic cells (DCs) and induces Th1 polarization of the immune response. We investigated the influence of lymphocytes on splenic DC (SDC) and thymic DC (TDC) development and on their IL-12 production capacity. First, CD3epsilon(-/-) mice, lacking T cells, and RAG-2(-/-) mice, lacking T and B cells, possess numbers of SDCs, TDCs, and CD8alpha(+) SDCs similar to wild-type (WT) mice. Second, SDCs and TDCs from CD3epsilon(-/-) mice do not secrete IL-12 in vitro after different stimulations, whereas DCs from pTalpha(-/-) mice, possessing reduced T cell number, and RAG-2(-/-) mice, produce an IL-12 level similar to that of WT DCs. We show that T lymphocytes restore the capacity of DCs to produce IL-12 after stimulation in vivo by reconstitution of CD3epsilon(-/-) mice with WT T cells and in vitro by coculture of CD3epsilon(-/-) DCs with WT T cells. The regulation of IL-12 production occurred at the transcriptional level, with an increase of IL-12p35 transcripts and a decrease of IL-12p40 transcripts. Although IL-4 restores IL-12 production by CD3epsilon(-/-) SDCs, anti-IL-4 Abs inhibited only partially the IL-12 production in coculture of CD3epsilon(-/-) DCs and WT T cells. Taken together, these data show that T lymphocytes potentiate IL-12 production by DCs and that IL-4 is not solely involved in this regulation. In conclusion, B and T cells exert balanced actions on DCs by respectively inhibiting or promoting IL-12 production.

The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta 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 TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 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 TCR beta VDJ genes but before expression of TCR beta 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, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.

Background & Aims: T-cell receptor alpha mutant (TCRalpha(-/-)) mice spontaneously develop chronic colitis mediated by CD4(+) TCRalpha(-)beta(+) T cells. The aim of this study was to analyze the mechanisms of expansion of these cells by characterization of the TCRbeta repertoire., Methods: TCRbeta repertoire was analyzed by reverse-transcription polymerase chain reaction/Southern blot and DNA sequencing. Clonality of T cells was examined in the lymphoid tissues and colons of TCRalpha(-/-) mice and interleukin 4-deficient TCRalpha(-/-) mice. In addition, an in vitro culture system using syngeneic colonic epithelial cells as antigens was used., Results: The clonal expansion of a restricted subset of Vbeta8.2(+) T cells was characterized by conservation of a single negatively charged amino acid residue in the second position of the complementarity-determining region 3 (CDR3). These T cells were observed in the diseased colon and appendix (cecal patch) of TCRalpha(-/-) mice, but not germfree TCRalpha(-/-) mice. Culture of polyclonal T cells from young TCRalpha(-/-) mice with colonic epithelial cells under T helper 2 conditions resulted in the survival of Vbeta8.2(+) T cells characterized by the same CDR3 pattern. In addition, the transfer of the cultivated T cells induced mild colitis in recombination-activating gene 1 mutant mice., Conclusions: In the TCRalpha(-/-) mice, the development of colitis is associated with the presence of a restricted diversity of Vbeta8. 2(+) T-cell subsets characterized by a specific TCR motif. The limited diversity of lamina propria T cells that are derived from naive T cells expanded by reacting with luminal bacterial antigens is likely caused by the survival of these T cells after stimulation with self-antigens in the presence of a T helper 2 environment.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

During alphabeta thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRbeta genes. On expression of a TCRbeta polypeptide chain, the pre-TCR is assembled, and TCRbeta locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRbeta locus allelic exclusion in mice single-deficient or double-deficient for CD3zeta/eta and/or p56(lck). These mice display defects in the expression of endogenous TCRbeta genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRbeta VDJ (variable, diversity, joining) rearrangements by a functional TCRbeta transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25(+) double-negative (DN) thymocytes of the mutant mice failed to express the TCRbeta transgene, suggesting defective expression of the TCRbeta transgene similar to endogenous TCRbeta genes. In the mutant mice, a proportion of CD25(+) DN thymocytes that failed to express the transgene expressed endogenous TCRbeta polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRbeta chains or more than one endogenous TCRbeta chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRbeta locus by inducing the expression of rearranged TCRbeta genes in CD25(+) DN thymocytes.

Idiopathic CD4+ lymphocytopenia (ICL) is a rare syndrome affecting adults and defined by a stable loss of CD4+ T cells in the absence of any known cause of immune deficiency. Defective T-cell proliferations to mitogens and antigens have been described in some of such patients displaying clinical signs of immune deficiency such as opportunistic infections. We investigated here the hypothesis that T-cell depletion and dysfunction could be due to biochemical defects of the CD3-TCR pathway in CD4+ and/or CD8+ subsets from three patients with severe stable ICL (below 150 CD4+ T cells/microliter) and opportunistic infections. Patient 1 had a general T lymphocytopenia, whereas patients 2 and 3 displayed a selective loss of CD4+ T cells. We observed in all patients a major reduction of the proliferative response to CD3-TCR stimulation that affected only the depleted T-cell subpopulation. Moreover, in two cases, impaired early biochemical events of the CD3-TCR pathway were detected. In patient 1 and 3, we found a defect (of distinct intensity) of CD3-induced protein tyrosine phosphorylation in CD4+ cells compared to control cells, whereas this process was normally induced in CD4+ T cells from patient 2. Taken together, this study reveals that the heterogeneity of the ICL syndrome was situated at the cellular level, and involved in two cases abnormalities of transducing molecules of the CD3-TCR pathway.

CD3 gamma, delta, epsilon, and zeta proteins together with the pre-TCR alpha-chain (pT alpha) and a rearranged TCR beta-chain assemble to form the pre-TCR that controls the double negative (DN) to double positive (DP) stages of thymopoiesis. The CD3 proteins are expressed before pT alpha and TCR beta-chains in prothymocytes and are expressed intracellularly in precursor NK cells, suggesting that the CD3 complex may function independent of pT alpha and TCR beta. In this report, both the role of CD3 epsilon exclusively, and the role of CD3 proteins collectively, in thymocyte and NK cell development were examined. In a mouse strain termed E delta P, a neomycin cassette inserted within the CD3 epsilon promoter abolishes CD3 epsilon and delta expression and also abolishes CD3 gamma expression in all but a small minority (< or =1%) of prothymocytes. These prothymocytes became deficient in CD3 epsilon alone upon reconstitution of CD3 delta expression and were severely, but not completely, arrested at the DN stage, as small numbers of double positive thymocytes were detected. In de facto CD3 gamma delta epsilon zeta(null) mice generated by crossing the epsilon delta P mice with CD3 zeta-/- mice, thymopoiesis were arrested at the CD44-CD25+ DN stage as observed in RAG-/- mice, DJ and VDJ recombination at the TCR beta locus was functional, and normal numbers of NK cells were detected. Together, the findings demonstrate that during thymocyte development, the CD3 complex collectively is not essential until the critical CD44-CD25+ DN stage in which pre-TCR begins to function, whereas CD3 epsilon is critical for the assembly of pre-TCR. Moreover, CD3 proteins are dispensable for NK cell development.

The activation, function, and development of peripheral T lymphocytes are dependent on the ability to signal properly through the surface T-cell antigen receptor (TCR)-CD3 complex. Transmission of such signals requires the activation of specific cytoplasmic protein tyrosine kinases (PTK) associated with the TCR. Recently, mutations in one such PTK, called ZAP-70, have been shown to be responsible for a rare, autosomal recessive form of severe combined immunodeficiency syndrome (SCID) in humans. This distinctive SCID syndrome is characterized by the selective absence of peripheral CD8+ T cells and by the presence of circulating CD4+ T cells that do not respond to TCR-mediated stimuli in vitro. T-cell immunodeficiency syndromes that arise as a consequence of inherited mutations in either the CD3epsilon or CD3gamma subunit proteins have also been described in rare patients. Absence of these TCR components results in severely decreased expression of the surface TCR-CD3 complex and defective signal transduction through the TCR. In this report, the clinical, laboratory, and molecular findings of these immunodeficiency disorders are described, insights are provided by these inherited defects into the pathways of TCR signal transduction, and T-cell development is discussed.

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.

Signalling through the TCR is mediated by the cytoplasmic tails of the CD3 complex. Deficiencies in the expression of different CD3 components have lead to dramatic, yet dissimilar, effects on T-cell development and to selective deficits in peripheral T-cell subsets. Recent studies of human patients and animal models with CD3 deficiencies are providing insights into the redundant and unique roles of these molecules.

Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3- (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin-2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 gamma, delta and epsilon homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32-kDa dimer that may represent a heterodimer of different CD3 constituents. TCR alpha and beta gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor.