T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity

The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.     

Differential sensitivity to X-ray of chromosomes of blood T-lymphocytes and B- and T-cell lines

Summary Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a significantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia. Supported in part by USPHS grants CA-14413 and CA-16935.     

Differential sensitivity to x-ray of chromosomes of blood T-lymphocytes and B-and T-cell lines.

Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a singificantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia.     

Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s.

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.     

Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly

The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER.     

Human immunodeficiency virus type 1 Nef does not alter T-cell sensitivity to antigen-specific stimulation.

We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1.     

T-cell protein tyrosine phosphatase regulates bone resorption and whole-body insulin sensitivity through its expression in osteoblasts

Insulin signaling in osteoblasts contributes to whole-body glucose homeostasis in the mouse and in humans by increasing the activity of osteocalcin. The osteoblast insulin signaling cascade is negatively regulated by ESP, a tyrosine phosphatase dephosphorylating the insulin receptor. Esp is one of many tyrosine phosphatases expressed in osteoblasts, and this observation suggests that other protein tyrosine phosphatases (PTPs) may contribute to the attenuation of insulin receptor phosphorylation in this cell type. In this study, we sought to identify an additional PTP(s) that, like ESP, would function in the osteoblast to regulate insulin signaling and thus affect activity of the insulin-sensitizing hormone osteocalcin. For that purpose, we used as criteria expression in osteoblasts, regulation by isoproterenol, and ability to trap the insulin receptor in a substrate-trapping assay. Here we show that the T-cell protein tyrosine phosphatase (TC-PTP) regulates insulin receptor phosphorylation in the osteoblast, thus compromising bone resorption and bioactivity of osteocalcin. Accordingly, osteoblast-specific deletion of TC-PTP promotes insulin sensitivity in an osteocalcin-dependent manner. This study increases the number of genes involved in the bone regulation of glucose homeostasis.     

T-cell therapies for T-cell lymphoma

T-cell lymphomas represent a subpopulation of non-Hodgkin lymphomas with poor outcomes when treated with conventional chemotherapy. A variety of novel agents have been introduced as new treatment strategies either as first-line treatment or in conjunction with chemotherapy. Immunotherapy has been demonstrated to be a promising area for new therapeutics, including monoclonal antibodies and adoptive cellular therapeutics. T-cell therapeutics have been shown to have significant success in the treatment of B-cell malignancies and are rapidly expanding as potential treatment options for other cancers including T-cell lymphomas. Although treating T-cell lymphomas with T-cell therapeutics has unique challenges, multiple targets are currently being studied both preclinically and in clinical trials.     

Defective T-cell activation is associated with augmented transforming growth factor Beta sensitivity in mice with mutations in the Sno gene

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo.     

Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell Leukemia virus type 1 protease

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.     

T-cell hybridomas recognizing the envelope proteins of Semliki forest virus: their sensitivity to endo/lysosomal protease and the antigenicity.

Eight T-cell hybridomas were established from the draining lymph node of C3H mice immunized with Semliki forest virus (SFV). Six of them showed specificity toward viral-structure protein E2, while the remaining two clones included one with specificity to an other structural protein E1 and the other with specificity to C. The production of IL-2 by the E2 protein-specific T-cell hybridomas in the presence of SFV was suppressed by treating the antigen-presenting cells (APC) with ammonium chloride raising pH of the acidic compartments. It was found also that treatment of APC with a thiol protease inhibitor, leupeptin or E64, resulted in a reduced response of some of the E2-specific T-cell hybridomas. The E2 protein of SFV proved to be resistant at pH 7.0, and sensitive at pH 5.0 to in vitro cathepsin B treatment. In contrast, the E1 and C proteins proved to be resistant to both pH values. These results indicate that the thiol protease, probably cathepsin B, works as one of the enzymes group involved in antigen processing.     

T-cell anergy and peripheral T-cell tolerance

The discovery that T-cell recognition of antigen can have distinct outcomes has advanced understanding of peripheral T-cell tolerance, and opened up new possibilities in immunotherapy. Anergy is one such outcome, and results from partial T-cell activation. This can arise either due to subtle alteration of the antigen, leading to a lower-affinity cognate interaction, or due to a lack of adequate co-stimulation. The signalling defects in anergic T cells are partially defined, and suggest that T-cell receptor (TCR) proximal, as well as downstream defects negatively regulate the anergic T cell's ability to be activated. Most importantly, the use of TCR-transgenic mice has provided compelling evidence that anergy is an in vivo phenomenon, and not merely an in vitro artefact. These findings raise the question as to whether anergic T cells have any biological function. Studies in rodents and in man suggest that anergic T cells acquire regulatory properties; the regulatory effects of anergic T cells require cell to cell contact, and appear to be mediated by inhibition of antigen-presenting cell immunogenicity. Close similarities exist between anergic T cells, and the recently defined CD4+ CD25+ population of spontaneously arising regulatory cells that serve to inhibit autoimmunity in mice. Taken together, these findings suggest that a spectrum of regulatory T cells exists. At one end of the spectrum are cells, such as anergic and CD4+ CD25+ T cells, which regulate via cell-to-cell contact. At the other end of the spectrum are cells which secrete antiinflammatory cytokines such as interleukin 10 and transforming growth factor-beta. The challenge is to devise strategies that reliably induce T-cell anergy in vivo, as a means of inhibiting immunity to allo- and autoantigens.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

T-cell activation

Activation of T lymphocytes results in immediate intracellular biochemical changes, including increases in cytosolic Ca(2+) levels, stimulation of protein kinase C (PKC) and regulation of protein tyrosine kinases (PTKs). This review describes recent advances in the study of the signalling steps downstream of PKC and PTKs in T cells. A model is presented in which the GTP-binding protein p21(ras) acts as an integrator of the signal transduction pathways controlled by the T-cell antigen receptor.