T cell antigen receptors in autoimmunity

Three mAb to variable region determinants of the alpha/beta-chain TCR were used to detect discrete populations of peripheral blood T cells. T cells sharing a TCR determinant defined by such an antibody presumably use the same or similar TCR V or J genes for their alpha- or beta-chains. Thus analysis with these mAb provides a tool to investigate TCR gene usage and expression. Since autoantigen specific T cells may play an important role in initiating autoimmune diseases, TCR were analyzed in different autoimmune diseases and control groups including rheumatoid arthritis, Graves disease, idiopathic thrombocytopenic purpura, psoriasis, SLE, insulin-dependent diabetes mellitus, and in nonautoimmune control diseases and normals. Purified T cells were stained by indirect immunofluorescence with three mAb to TCR variable regions: mAb S511 stains 1.8 +/- 0.9% (mean +/- 2 SD), mAb C37 stains 3.4 +/- 1.5% and mAb OT145 stains from 0 to 6% of T cells from normal donors. Several individuals were identified with expanded subsets of positive T cells. One patient with adult ITP followed during a 12-mo period consistently had elevated percentages of T cells staining with the mAb OT145 (15.9 to 24.5%). These cells were found to be exclusively CD8+. By Southern blotting DNA prepared from these OT145+, CD8+ cells, but not DNA from the patient's OT145- T cells, revealed a clonal rearrangement using a beta-chain C region probe. Thus this patient had a monoclonal expansion of CD8+, OT145+ cells. Hyperexpression of a TCR variable region, as defined by the available mAb, could not be associated with any of the diseases studied. Examination of T cells at the site of autoimmunity, such as T cells from rheumatoid arthritis synovial fluid, revealed normal percentages of cells staining with these mAb. Immunoperoxidase staining of psoriatic lesional skin showed no striking enrichment of T cells bearing one or the other TCR type.     

Ontogeny of T cell receptors in the chicken thymus

A panel of murine mAb against chicken TCR and associated molecules was used to study the ontogeny of T cells. The intrathymic maturation of the TCR-gamma delta, (TCR-1) and TCR-alpha beta (TCR-2) sublineages was the focus of these studies employing immunoperoxidase staining of tissue sections and immunofluorescence analysis of cell suspensions. The first CD3+ cells appeared in the thymus on embryonic day 9 (E9) when the CD3 Ag was restricted to the cytoplasm. In tissue sections, both TCR-1+ and TCR-2+ cells were observed on E12, whereas only the TCR-1 cells were identifiable by surface immunofluorescence. On the next day, when a discrete thymic medullary region was first recognizable, the TCR-1 cells were present in both cortex and medulla. Two days later (E15), TCR-1 cells were found in the spleen. Surface TCR-2+ cells did not appear until E14, began to migrate in to the medulla on E17, and appeared in the spleen on E19. The first TCR-1 cells thus move quickly through this maturational pathway, whereas TCR-2 cells undergo a prolonged developmental period in the cortex. While most TCR-1+ cells were CD4-CD8-, a minor subpopulation (5 to 15%) were CD4-CD8+, and less than 1% were CD4+CD8+. In contrast, immature TCR-2+ thymocytes in the cortex were predominantly CD4+CD8+, whereas cells expressing a higher density of the CD3/TCR-2 complex were either CD4+CD8- or CD4-CD8+ and were localized in the thymic medulla. In the medulla of the mature thymus, the TCR-1+ cells preferentially occupy the cortico-medullary junction and form small aggregates around vessels. TCR-2+ cells were less frequent in these areas of TCR-1 accumulation. The thymic ontogeny and, by implication, the selection of the receptor repertoire thus differs substantially for these two TCR isotypes.     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.     

Growth and characterization of T cell colonies from human thymus

A semisolid microculture system was used to study T cell colonies grown from human thymocytes. Colony growth was absolutely dependent upon media conditioned by peripheral blood leukocytes (PBL) in the presence of phytohemagglutinin. Plating efficiency was further enhanced by the addition of a non-T, adherent, radiation-resistant (7500 rad) PBL subpopulation, but was not enhanced by culture supernatants of these cells. The T colony precursor cell in the thymus occurred with a frequency of 8.0 X 10(-3) and had a surface receptor for the OKT3 monoclonal antibody. Thymocyte colony cells were functionally distinct from PBL and the major thymocyte population. The colony cells proliferated in response to T cell mitogens, but only in the presence of exogenous growth factors. The cells stimulated normal PBL in mixed leukocyte culture (MLC), but did not respond to alloantigens in MLC or in assays of spontaneous cytotoxicity. This culture system should prove helpful in the study of human thymocyte differentiation.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

Spontaneous T cell antigen receptor variants of a human T leukemia cell line

We have sought to address the question of clonal variation of TCR within a human T leukemia cell line, HPB-ALL. To do so, a panel of anti-idiotypic antibodies was produced and the cell line examined for variants. We isolated both spontaneous idiotype and receptor-negative variants without applying mutagens or any selective pressure other than sorting the cells. These sorted and cloned populations are all clonally related to each other as shown by their beta-TCR locus gene rearrangements. The idiotype variants have alpha-chains which are differentially glycosylated, but they have the same size core protein after treatment with peptide N-glycosidase F to remove their carbohydrate side chains. This probably accounts for their idiotypic difference, since the antibody that distinguishes them appears dependent upon glycosylation for its binding, as shown by immunoprecipitation in the presence versus the absence of tunicamycin, which inhibits glycosylation from occurring. The idiotype variants differed from one another in variable region sequences by only a single amino acid substitution in the beta-chain, which is likely not important for the idiotypic difference. The receptor-negative variant produces both alpha- and beta-mRNA and cytoplasmic protein for TCR, but fails to transport this protein to the cell surface. We conclude that idiotype and receptor-negative variants of a T cell clone can occur in the absence of appreciable somatic mutation.     

The interaction of nominal antigen with T cell antigen receptors. I. Specific binding of multivalent nominal antigen to cytolytic T cell clones

In this report, we describe an experimental strategy for analyzing the interaction of nominal antigen with antigen-specific T cell clones. Our approach was based on the notion that low affinity interactions between nominal antigen and T cell antigen receptors might be detected by using a highly multivalent form of the antigen in which a large number of identical, appropriately spaced epitopes are attached to a polymer backbone. Antigens of this kind should be capable of multivalent binding to receptors on the T cell, resulting in a marked enhancement of the overall avidity of the interaction. To examine this possibility, we established a series of murine cytolytic T cell (Tc) clones specific for the readily detectable hapten fluorescein isothiocyanate (FL). These clones lysed FL-conjugated target cells in an antigen-specific fashion and also showed specificity for target cell MHC gene products. The interaction of these clones with the nominal antigen FL was assessed by flow cytometry, using a series of water-soluble FL-conjugated polymers varying in polymer backbone and FL isomer. High m.w. (600 to 2000 Kd) polymers of acrylamide, dextran, or Ficoll conjugated with 300 to 800 FL groups/molecule bound specifically to anti-FL Tc clones. There was little binding to syngeneic spleen cells, thymocytes, noncytolytic T cell clones, or T cell clones specific for other haptens such as NIP. Polymer concentrations in the 1 to 10 micrograms/ml range produced readily detectable binding within minutes at 20 degrees C, and the binding approached plateau levels at polymer concentrations of between 100 and 300 micrograms/ml. Studies with closely related FL isomers showed that the same antigen fine specificity was operative in both lysis of FL-conjugated target cells and in binding of FL-conjugated polymers. The functional significance of the observed binding was assessed by measuring the effect of FL-conjugated polymers on lymphokine secretion by the clones. High m.w. FL-conjugated polymers caused a dose-dependent increase in the production of macrophage activation factor (MAF) by anti-FL Tc clones, but did not increase MAF production by an NIP-specific clone. In contrast, concanavalin A induced MAF production by both FL-specific and NIP-specific clones. Thus, the observed binding is both specific and functionally significant. These results suggest that soluble nominal antigen, in an appropriately multivalent form, can bind specifically to antigen receptors on Tc clones.     

Rational design of T cell receptors with enhanced sensitivity for antigen

Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3β stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3β loop, stabilizing it. G107 TRBV possess a gap in their CDR3β where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3β stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an ～10-1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2(+) TCR tested. Analysis of fine specificity indicated a preserved binding orientation. These results support the feasibility of developing high affinity antigen specific TCR for therapeutic purposes through the identification and manipulation of critical framework residues. They further indicate that amino acid variations within TRBV not directly involved in ligand contact can program TCR sensitivity, and suggest a role for CDR3 stability in this programming.     

Regulation of human T lymphocyte surface antigen mobility by purinergic receptors

The present study was undertaken to establish whether the capping mechanism of normal human T lymphocytes is regulated by a purinergic receptor. Interaction of T lymphocytes with adenosine significantly increased the mobility of the T3, T4, and T8 surface antigens. This enhanced rate of capping was reflected by a significant decrement in the time intervals to achieve half-maximal capping. T lymphocytes preincubated with theophylline or isobutylmethylxanthine did not exhibit accelerated capping or a decrease in the time required for half-maximal capping in response to adenosine, suggesting that these agents inhibited the binding of adenosine to its receptor. A role for a cAMP-dependent pathway in capping was suggested by the observation that the phosphodiesterase inhibitor RO-201724 caused a decrease in the concentration of adenosine required to accelerate the capping process. Moreover, exposure of T lymphocytes to the cyclic nucleotide derivatives 8-N3-cAMP and 8-Br-cAMP mimicked the effect of adenosine, significantly reducing the time to half-maximal capping. Photoaffinity labeling of intracellular cAMP receptors with 32P-8-N3-cAMP indicated that adenosine caused occupancy of the receptors. This effect of adenosine was inhibited by theophylline, a known purinergic receptor blocker. The data support the concept that the T cell capping mechanism is mediated by an adenylate cyclase-coupled purinergic receptor that activates a cAMP-dependent pathway, and that this pathway is functional in the T3+, T4+, (inducer) and T3+, T8+ (suppressor) subsets.     

Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen

The intestinal epithelium is comprised of a monolayer of intestinal epithelial cells (IEC), which provide, among other functions, a physical barrier between the high Ag content of the intestinal lumen and the sterile environment beyond the epithelium. IEC express a nonclassical MHC class I molecule known as the thymus leukemia (TL) Ag. TL is known to interact with CD8αα-expressing cells, which are abundant in the intestinal intraepithelial lymphocyte compartment. In this report, we provide evidence indicating that expression of TL by IEC modulates the cytokine profile of CD4(+) T cells favoring IL-17 production. We show in an adoptive transfer model of colitis that donor-derived cells become more pathogenic when TL is expressed on IEC in recipient animals. Moreover, TL(+)IEC promote development of IL-17-mediated responses capable of protecting mice from Citrobacter rodentium infection. We also show that modulation of IL-17-mediated responses by TL(+)IEC is controlled by the expression of CD8α on CD4(+) T cells. Overall, our results provide evidence for an important interaction between IEC and CD4(+) T cells via TL, which modulates mucosal immune responses.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Functional characteristics of T cell receptors during sensitization against histocompatibility antigens in vitro

Using the mixed leukocyte culture (MLC) reaction as a model system for the generation of cytotoxic effector lymphocytes, we have examined changes which occur in recognition-binding function during T cell sensitization. Properties of recognition-binding units on normal and immune T lymphocytes were assessed through the ability of T cells to bind to specific cellular immunoadsorbants at various stages of in vitro sensitization. While antigen-specific recognition-binding function was readily detected on fully cytotoxic effector cells, we were unable to detect functionally specific binding of unsensitized lymphocytes to cellular immunoadsorbants. The ability of cells undergoing sensitization in MLC to bind specifically to target cell monolayers appeared congruent in time with cytotoxic function. These results suggest that a fundamental membrane-associated change occurring during T cell sensitization may be the development of a strong and specific target-cell binding function.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Quantum dot fluorescence characterizes the nanoscale organization of T cell receptors for antigen

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.     

A novel pathway of human T cell activation via a 103 kD T cell activation antigen

A novel triggering signal for human proliferating and cytotoxic T lymphocytes defined by a 103 kD T cell-specific activation antigen (Tp103) is described. Tp103 is expressed on all proliferating normal T cells but is not present, or present only in low amounts, on resting peripheral blood T lymphocytes. Cross-linking of T cell and Fc receptor-positive accessory or target cells by an antibody against Tp103 leads to activation of the T cell. The proliferative response is due to an autocrine IL 2-dependent mechanism and can be inhibited by antibodies against the IL 2 receptor or by Cyclosporin A. Resting Tp103-positive T cells also respond to anti-Tp103. Although Tp103 is not linked to the antigen receptor/T3 complex, triggering via Tp103 can be inhibited by modulation of the T3 molecule. Thus, Tp103 defines a new antigen-independent pathway of T cell activation that can be regulated via other T cell surface structures.     

Prevalence of antigen receptor variants in human T cell lines and tumors

Previously, we have shown that a human T acute lymphoblastic leukemia cell line, HPB-ALL, exhibits clonal heterogeneity within its Ag receptor, as revealed by varying reactivity patterns with a panel of anti-idiotype mAb. We now extend these findings to another human T acute lymphoblastic leukemia cell line, SUP-T13, and to two fresh human chronic lymphocytic leukemias, JE and EF. In the two cell lines, two types of Ag receptor variants could be found: those that retained a receptor molecule but lost reactivity with an anti-idiotype mAb (idiotype variants), and those which had lost surface receptor expression completely (receptor-negative variants). The idiotype variants, at least in HPB-ALL, have differentially glycosylated receptor alpha-chains from the parent. The receptor-negative cells, in HPB-ALL as well as in SUP-T13, produce cytoplasmic receptor and CD3 proteins but do not transport them to the surface. Neither idiotype nor receptor-negative variants could be detected in either of the fresh tumors of chronic lymphocytic leukemias. The limit of sensitivity in these analyses was about 0.05%. We conclude that antigen receptor variants can spontaneously occur in cell lines derived from acute lymphoblastic leukemias, but are infrequent in chronic lymphocytic leukemias in vivo, and that therapy with anti-idiotype mAb may be a viable strategy for these malignancies.     

Development of alphabeta T cells in the human thymus

The thymus is the main producer of alphabeta T cells and is, therefore, crucial for a normal immune system. The intrathymic developmental pathway of human alphabeta T cells has now been delineated. The production of new T cells by the thymus decreases with age, and the thymus was thought to be redundant in adults once the peripheral T-cell pool has been formed early in life. However, recent work has shown that the thymus can function even at an advanced age. Research into the production of T cells in clinical settings that are associated with loss of T cells in the periphery has sparked renewed interest in the function of the human thymus.