The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/ζ complex

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are thought to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56^lck SH2 domain with ζ-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/ζ complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8αβ and CD8αβ isoforms to the TCR. We demonstrate in vivo in association of CD8αα/p56^lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56^lck specifically impedes the association of ZAP-70 with CD8αα/p56^lck without affecting the ζ/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8αα or CD8αβ isoform with the TCR/CD3/ζ complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56^lck SH2 domain mediates recruitment of CD8/p56^lck to the activated TCR/CD3/ζ complex.     

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain,FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183^?EB6⁺ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4^? C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183⁺/EB6⁺ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and Fc?RI γ chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58⁺ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ-γ homodimers or in the form of ζ-γ heterodimers, while γ-γ homodimers were detectable in low amounts. However, p58⁺ clones displaying a unique association with γ-γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56^lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56^lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58-associated CD3 ζ, chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 ζ chains.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so-called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCRγδ cells were CD2⁺, suggesting an origin at a post-CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day-15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.     

Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice

Several cytokines including stem cell factor (SCF) and interleukin (IL)-7 are known to be required for development of γδ T cell receptor (TCR) intestinal intraepithelial lymphocytes (i-IEL) in mice. We show here the effects of IL-15 on the proliferation and maintenance of murine γδ i-IEL in vitro. γδ i-IEL constitutively expressed a high level of IL-15 receptor α mRNA and proliferated in response to IL-15 more vigorously than αβ i-IEL. Vγ/δ repertoire analysis revealed that IL-15, like IL-2, induced polyclonal expansion of γδ i-IEL, whereas γδ i-IEL responding to IL-7 showed a Vγ/δ repertoire skewed towards Vγ1/Vδ4, Vδ5. IL-15 efficiently prevented γδ i-IEL from apoptosis induced by growth factor deprivation. This rescue was accompanied by up-regulation of Bcl-2 expression. These results suggest that IL-15 plays important roles in proliferation and maintenance of γδ i-IEL.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

Expression of an avian CD6 candidate is restricted to αβ T cells,splenic CD8+ γδ T cells and embryonic natural killer cells

A candidate avian CD6 homolog is identified by the S3 monoclonal antibody. The S3 antigen exists in a phosphorylated glycoprotein form of 130 kDa and a nonphosphorylated form of 110 kDa. Removal of phosphate groups and N-linked carbohydrates indicates a 78-kDa protein core. During thymocyte differentiation, the γδ T cells do not express S3, whereas mature CD4⁺ and CD8⁺ cells of αβ lineage acquire S3 antigen. All αβ T cells in the blood and spleen express the S3 antigen at relatively high levels. In contrast, only the CD8⁺ sub-population of γδ T cells in the spleen expresses the antigen and neither αβ nor γδ T cells in the intestinal epithelium express the S3 antigen. The S3 antigen is also found on embryonic splenocytes with a phenotypic profile characteristic of avian natural killer cells. The biochemical characteristics and this cellular expression pattern imply that the S3 antigen is the chicken CD6 homolog.     

Selective activation of resting human γδ T lymphocytes by interleukin-2

In rheumatoid arthritis and other inflammatory diseases we and others have found that γδ T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate γδ T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the γδ but not the αβ T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the γδ T cells and a corresponding reduction of the fraction of αβ T cells. Limiting dilution analysis revealed that approximately 1/60 of the γδ T cells responded to IL-2 in contrast to only 1/250 of the αβ T cells. Comparison of the expression of the IL-2 receptor (IL-2R) a and P chains showed that there was a similar expression of the α chain on γδ and αβ T cells whereas the relative density of the β chain was more than twice as high on γδ T cells. Both the IL-2-induced proliferation of γδ T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2Rβ monoclonal antibody (mAb) but not by an anti-IL-2Rα mAb. Expression of CD69 on γδ T cells was dependent neither on the presence of B cells, monocytes, nor αβ T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of γδ T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on γδ T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated αβ T cells.     

The β2 integrin Mac-1 but not p150,95 associates with FcγRIIA

In this study we have compared the ligand binding activity of the two closely related β2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with FcγR, particularly FcγRIII, but K562 cells express only endogenous FcγRIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between FcγRIIA with Mac-1 and p150,95 in the absence of other FcγR. The main finding is that anti-FcγRII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.