CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.     

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.     

Modification of the T cell antigen receptor (TCR) complex by UDP-glucose:glycoprotein glucosyltransferase. TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha, beta, gamma, delta, epsilon, and zeta molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.     

Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post-translational limitations on receptor assembly.     

TCR beta chain influences but does not solely control autoreactivity of V alpha 14J281T cells.

CD1d-dependent accumulation of alphabeta T cells bearing a canonical Valpha14Jalpha281 alpha-chain (Valpha14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Valpha14+ T cells do not exhibit significant autoreactivity despite high reactivity to alpha-galactosylceramide presented by CD1d (alpha-GalCer/CD1d). To assess the role of TCRbeta chain in determining the alpha-GalCer/CD1d vs autoreactive specificity of Valpha14+ T cells, we conducted TCRalpha or TCRbeta chain transduction experiments. In this study we demonstrate, by combining different TCRbeta chains with the Valpha14 alpha-chain in retrovirally transduced T cell lines, that the Valpha14 alpha-chain plays a primary role, necessary but not sufficient for imparting alpha-GalCer/CD1d recognition. beta-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Valpha14+ T cells, since transduction of TCRalphabeta chains from a high CD1d autoreactive Valpha14+ T cell line conferred the alpha-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Valpha14+ T cell reactivity is due to both beta-chain diversity and control mechanism(s) beyond primary TCR structure.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

Blockade of transgenic gamma delta T cell development in beta 2-microglobulin deficient mice.

The gamma delta T cell receptor (TCR) of the hybridoma KN6 recognizes the self molecule encoded by a class I gene which maps within the TL region of the major histocompatibility complex (MHC) of H-2b mice. Mice transgenic (Tg) for this TCR were crossed with mice genetically deficient in beta 2-microglobulin (beta 2m). No mature Tg gamma delta T cells were detected in the thymus or the spleen of the beta 2m- gamma delta Tg mice. We conclude that interaction between the Tg gamma delta TCR and a beta 2m-associated molecule (probably an MHC class I molecule) is required for the generation of mature Tg gamma delta T cells.     

T cell antigen receptor (TCR) transmembrane peptides colocalize with TCR,not lipid rafts,in surface membranes

We have previously shown that a synthetic peptide termed core peptide (CP), which corresponds to a sequence within the transmembrane domain of the alpha chain of the T cell antigen receptor (TCR), can inhibit IL-2 production in antigen-stimulated T cells and can suppress inflammation in several T cell-mediated animal models of disease. As the first step in determining the mechanism of CP action, we examined the association of CP with the plasma membrane of human T cells using confocal microscopy. A homogeneous distribution of CP was observed in the plasma membrane of human T cells. This membrane localization was dependent on the presence of positive charges in the CP sequence. CP analogs, containing either neutral or negatively charged amino acids in place of the positive amino acid charges, did not localize within TCR membranes. Following antibody-induced TCR clustering, there was specific colocalization of CP with surface TCR. No association was observed with other cell surface receptors when similarly clustered. Since TCR activation leads to an increased movement of the receptor complex to cholesterol/glycosphingolipid (GSL) plasma membrane microdomains (rafts) we examined whether the association of CP with TCR was raft-driven. TCR clustering led only to a partial colocalization of TCRs with raft GSL, ganglioside GM1, and a complete colocalization of CP with TCRs. We conclude that CP associates specifically with plasma membrane TCRs and not raft lipids.     

Tissue distribution and appearance in ontogeny of alpha/beta T cell receptor (TCR2) in chicken

We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.     

Leptospira interrogans activation of human peripheral blood mononuclear cells: preferential expansion of TCR gamma delta+ T cells vs TCR alpha beta+ T cells

Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-gamma, IL-12, and TNF-alpha. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6-8 days, and the majority of cells contained in these cultures were gamma/delta T cells. These cultures showed a 10- to 50-fold expansion of gamma/delta T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce gammadelta T cell or NK cell expansion, but induced significant alphabeta T cell expansion. Vgamma9/Vdelta2 were expressed on all gamma/delta T cells expanded by exposure of PBMC to Leptorspira: Leptospira stimulation of purified TCRgammadelta(+) T cells, obtained from 8-day cultures of Leptospira-stimulated PBMC, induced high levels of IFN-gamma production, but no cell proliferation, suggesting that such stimulation of gammadelta T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCRgammadelta(+) T cells. These results indicate that Leptospira can activate gammadelta T cells and alphabeta T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.     

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

Antigen specific and MHC nonrestricted cytotoxicity of T cell receptor alpha beta+ and gamma delta+ human T cell clones isolated in IL-4

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.