Peptide antagonism and T cell receptor interactions with peptide-MHC complexes

We describe antagonist peptides that specifically inhibit cytolytic activity of T cell clones and lines that express the antigen-specific receptor of CD8+ T lymphocyte clone 2C, which recognizes peptides in association with syngeneic (Kb) and allogeneic (Ld) MHC proteins. Addition of an antagonist peptide that can bind to Kb on 2C cells decreased the tyrosine phosphorylation of CD3 zeta chains elicited by prior exposure of the cells to an agonist peptide-Kb complex. Contrary to previous agonist-antagonist comparisons, the 2C T cell receptor had higher affinity for an antagonist peptide-Kb complex than for a weak agonist peptide-Kb complex. This difference is considered in light of evidence that antigen-specific receptor affinity values can be substantially higher when determined with the receptor on live cells than with the receptor in cell-free systems.     

Fine specificity of the myelin-reactive T cell repertoire: implications for TCR antagonism in autoimmunity

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1-9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1-9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1-9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.     

Critical role for the common cytokine receptor gamma chain in intrathymic and peripheral T cell selection

The common cytokine receptor gamma chain (gammac), which is a functional subunit of the receptors for interleukins (IL)-2, -4, -7, -9, and -15, plays an important role in lymphoid development. Inactivation of this molecule in mice leads to abnormal T cell lymphopoiesis characterized by thymic hypoplasia and reduced numbers of peripheral T cells. To determine whether T cell development in the absence of gammac is associated with alterations of intrathymic and peripheral T cell selection, we have analyzed gammac-deficient mice made transgenic for the male-specific T cell receptor (TCR) HY (HY/gammac- mice). In HY/gammac- male mice, negative selection of autoreactive thymocytes was not diminished; however, peripheral T cells expressing transgenic TCR-alpha and -beta chains (TCR-alphaT/betaT) were absent, and extrathymic T cell development was completely abrogated. In HY/gammac- female mice, the expression of the transgenic TCR partially reversed the profound thymic hypoplasia observed in nontransgenic gammac- mice, generating increased numbers of thymocytes in all subsets, particularly the TCR-alphaT/betaT CD8+ single-positive thymocytes. Despite efficient positive selection, however, naive CD8+ TCR-alphaT/betaT T cells were severely reduced in the peripheral lymphoid organs of HY/gammac- female mice. These results not only underscore the indispensible role of gammac in thymocyte development, but also demonstrate the critical role of gammac in the maintenance and/or expansion of peripheral T cell pools.     

The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model

The mechanism of self-tolerance in the CD4(+) T cell compartment was examined in a double transgenic (Tg) model in which T cell receptor (TCR)-alpha/beta Tg mice with specificity for the COOH-terminal peptide of moth cytochrome c in association with I-Ek were crossed with antigen Tg mice. Partial deletion of cytochrome-reactive T cells in the thymus allowed some self-specific CD4(+) T cells to be selected into the peripheral T cell pool. Upon restimulation with peptide in vitro, these cells upregulated interleukin (IL)-2 receptor but showed substantially lower cytokine production and proliferation than cells from TCR Tg controls. Proliferation and cytokine production were restored to control levels by addition of saturating concentrations of IL-2, consistent with the original in vitro definition of T cell anergy. However, the response of double Tg cells to superantigen stimulation in the absence of exogenous IL-2 was indistinguishable from that of TCR Tg controls, indicating that these self-reactive cells were not intrinsically hyporesponsive. Measurement of surface expression of Tg-encoded TCR alpha and beta chains revealed that cells from double Tg mice expressed the same amount of TCR-beta as cells from TCR Tg controls, but only 50% of TCR-alpha, implying expression of more than one alpha chain. Naive CD4(+) T cells expressing both Tg-encoded and endogenous alpha chains also manifested an anergic phenotype upon primary stimulation with cytochrome c in vitro, suggesting that low avidity for antigen can produce an anergic phenotype in naive cells. The carboxyfluorescein diacetate succinimidyl ester cell division profiles in response to titered peptide +/- IL-2 indicated that expression of IL-2 receptor correlated with peptide concentration but not TCR level, whereas IL-2 production was profoundly affected by the twofold decrease in specific TCR expression. Addition of exogenous IL-2 recruited double Tg cells into division, resulting in a pattern of cell division indistinguishable from that of controls. Thus, in this experimental model, cells expressing more than one alpha chain escaped negative selection to a soluble self-protein in the thymus and had an anergic phenotype indistinguishable from that of low avidity naive cells. The data are consistent with the notion that avidity-mediated selection for self-reactivity in the thymus may lead to the appearance of anergy within the peripheral, self-reactive T cell repertoire, without invoking the induction of hyporesponsiveness to TCR-mediated signals.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56(Lck) and p59(Fyn). Studies of mutated TCR and chimeric CD4-CD3gamma molecules demonstrated that CD3gamma did not contain a recycling signal in itself. In contrast, the only sorting information in CD3gamma was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3gamma molecules. Finally, we found a correlation between the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed.     

The role of anergy in peripheral T cell unresponsiveness

When a T cell's encounter with specific antigen results in good signaling through the T cell antigen receptor yet does not lead to a proliferative response, the T cell enters a state of nonresponsiveness, or anergy. Anergy induction can result from a number of different situations, including antigen presentation by costimulation-deficient or "non-professional" antigen presenting cells, pharmacological blocking of T cell proliferation, or chronic stimulation of the T cell receptor by antigen. Anergy is a long-lived but temporary state characterized by a profound inability of the T cell to produce IL-2. Other effector functions may be affected to variable degrees. Anergy has been characterized most carefully under in vitro conditions, but several experimental models have demonstrated that T cells can also become anergic in vivo. This mechanism for tolerance induction may help to ensure that any mature autoreactive T cells which escape thymic deletion are unable to respond to host tissues. Furthermore, an understanding of the mechanism of anergy induction will most certainly lead to beneficial clinical applications, including improving graft acceptance and avoiding such deleterious immune responses as autoimmunity and allergy.     

T cell targeting of TAG72+ tumor cells by a chimeric receptor with antibody-like specificity for a carbohydrate epitope

BACKGROUND & AIMS: Chimeric receptors with specificity for defined tumor antigens are valuable tools for targeting cytolytic T cells specifically to tumor cells. The aim of this study, for the situation of gastrointestinal cancer, was to investigate the generation of a chimeric T cell receptor that specifically binds the tumor antigen TAG72 (CA72-4) and transmits a signal for cellular activation. METHODS: A single-chain antibody (scFv) was derived from the monoclonal anti-TAG72 antibody B72.3 by phage display techniques (B72.3-scFv) and fused to the signaling unit of the Fc epsilon-RI receptor gamma chain, resulting in a chimeric signaling receptor, B72.3-scFv-gamma. RESULTS: The B72.3-scFv and the chimeric B72.3-scFv-gamma receptor bound specifically to the TAG72 antigen. After transfection, T cells expressing the chimeric B72.3-scFv-gamma specifically recognized TAG72 positive cells. Cross-linking of the chimeric receptor with antigen resulted in interleukin 2 release and cytolytic activity against TAG72 positive tumor cells in vitro. CONCLUSIONS: T cells equipped with the chimeric anti-TAG72 receptor can be specifically activated to target and lyse TAG72 positive gastrointestinal tumor cells.     

The role of macrophages in antigen presentation and T cell tolerance

Bone marrow derived cells (dendritic cells, macrophages and B cells) are involved in antigen presentation and T cell tolerance. However, the precise functions of each cell type remain unclear. To determine the role of macrophages we produced transgenic mice expressing I-E molecules only on macrophages, by introducing the hybrid gene containing the colony stimulating factor-1 (CSF-1) receptor promoter region and the structural gene encoding E alpha d into C57BL/6 mice. In these mice I-E restricted antigen presentation and T cell priming were impaired. With respect to T cell tolerance, I-E reactive T cells were anergized but not clonally deleted. These results clearly demonstrate that macrophages by themselves are defective in efficient I-E restricted antigen presentation, so that T cells exposed to antigens expressed on macrophages are led to anergy.     

Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation

The T cell receptor (TCR) alphabeta heterodimer interacts with its ligands with high specificity, but surprisingly low affinity. The role of the zeta component of the murine TCR in contributing to the fidelity of antigen recognition was examined. With sequence-specific phosphotyrosine antibodies, it was found that zeta undergoes a series of ordered phosphorylation events upon TCR engagement. Completion of phosphorylation steps is dependent on the nature of the TCR ligand. Thus, the phosphorylation steps establish thresholds for T cell activation. This study documents the sophisticated molecular events that follow the engagement of a low-affinity receptor.     

Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis

Immunization of C57BL/6 mice with AChR provokes symptoms similar to those seen in the disease myasthenia gravis. To elucidate the structural requirements for T cell recognition of AChR and to identify TcR features which might provide targets for immunotherapy, a panel of T cell hybridomas was generated after immunization of mice with the immunodominant peptide of the AChR alpha chain. The TcR genes expressed by these hybridomas were sequenced. TcR-V beta 6 was preferentially employed, but other V beta genes were also observed. A conserved acidic residue was present in all CDR3 regions, regardless of the V beta. The TcR-V alpha repertoire was somewhat skewed with three V alpha families accounting for 82% of the sequences. The utilization of multiple T cell receptor V beta genes may contribute to the inability to inhibit EAMG by elimination of V beta 6+ T cells.     

The role of Tec protein-tyrosine kinase in T cell signaling

The Tec protein-tyrosine kinase family includes Btk, Itk/Tsk/Emt, Tec, Rlk/Txk, and Bmx which are involved in signals mediated by various cytokines or antigen receptors. Itk is expressed primarily in T cells and activated by TCR/CD3, CD28, and CD2. However, the defect in T cell signaling in itk-deficient mice is very modest. Thus, we looked for other Tec family kinases that could be expressed in lymphoid cells and involved in T cell signal transduction. Here, we demonstrate that Tec, expressed in T cells, is activated following TCR/CD3 or CD28 ligation and interacts with CD28 receptor in an activation-dependent manner. This interaction involves the Tec SH3 domain and the proline-rich motifs in CD28. We also show that Tec can phosphorylate p62(dok), one CD28-specific substrate, whereas Itk cannot. Overexpression of Tec but not Itk can enhance the interleukin-2 promoter activity mediated by TCR/CD3 or CD28 stimulation and introduction of a kinase-dead Tec but not Itk can suppress interleukin-2 expression, indicating that Tec is directly involved in T cell activation. Altogether, these data demonstrate that Tec kinase is an integral component of T cell signaling and that the two Tec family kinases, Tec and Itk, have distinct roles in T cell activation.     

T cell antigen receptor signal transduction

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.     

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab')2 reagent (CD3fos-F(ab')2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab')2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages.     

Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140

We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor-alpha (ER) to region E in vivo. In this system, the cDNAs corresponding to the A-D, E, E/F, A-E (deltaF), and full-length (wtER) domains of the human ER were each cloned into the yeast two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations in yeast harboring a GAL1-lacZ reporter. The reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity. We found that the interaction of E or E/F domains of the ER with full-length ER is estradiol dependent and estrogen responsive element independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions. In the presence of F domain, this activity is reduced 10-fold. The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region. We propose that the full-length ER contains intrinsic dimerization restraints contributed by regions outside domain E that are released upon binding hormone agonist. In addition, we have demonstrated that coactivator RIP140 is able to interact with the ER in vivo at the E domain of the receptor in the presence of estrogen. Yeast two-hybrid analysis shows that RIP140 does not homodimerize in the presence or absence of estrogens. We present evidence showing that the ER has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the ER itself that are present outside of the E domain compromise this ability.     

In vivo antagonism of a T cell response by an endogenously expressed ligand

3.L2 T cell receptor transgenic T cells are activated by the 64-76 peptide of the mouse hemoglobin d beta chain [Hb(64-76)], and their response is antagonized by the position 72 alanine substitution of this peptide (A72). To test the effect of this altered peptide ligand (APL) on 3.L2 T cell function in vivo, a transgene expressing A72 in major histocompatibility complex II positive cells (A72tg) has been introduced into mice. We demonstrate that 3.L2 T cells, when transferred to A72tg+ mice show a dramatically reduced proliferative response to Hb(64-76). Identical decreased responses were observed using T cells that developed in either A72tg+ or A72tg- hosts. This affect was not attributable to diminished precursor frequency, anergy, or competition for binding to I-Ek molecules. These results unequivocally demonstrate in vivo antagonism by an endogenous APL and characterize a class of self-peptides that, although inefficient in causing deletion in the thymus, effectively modulate T cell responses in the periphery.     

Antigen-specific T cell receptor antagonism by antigen-presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.