Structural analysis of the gene for the beta chain of the antigen-specific receptor of mammalian T-lymphocytes

The antigen specific receptor of T cells (TCR) is composed of alpha and beta chains and is normally present on the T cell surface complexed with the components which make up T3. In the case of beta chain, multiple somatic DNA rearrangements bring together V beta (variable), D beta (diversity) and J beta (joining) gene segments before a mature messenger RNA can be transcribed. So far beta chain genes have been extensively studied in the human and in the mouse system and we have very little information on other mammals. Our aims were to obtain information that may provide a structural basis for understanding developmental as well as evolutionary aspects of the TCR gene system in mammals. In this study we compare the hybridization pattern between a human cDNA probe coding for the beta chain constant region and restricted genomic DNA extracted from lymphocytes deriving from human as well as from rat and lamb. The comparison of the hybridization data represent a first piece of information about the variation of the structure of the TCR beta chain genes in mammals.     

Cutting edge: recombinase-activating gene expression and V(D)J recombination in CD4+CD3low mature T lymphocytes

The recombinase-activating genes, RAG-1 and RAG-2, can be expressed by a subset of B cells within germinal centers, where they mediate secondary V(D)J rearrangements. This receptor revision mechanism could serve either receptor diversification or tolerance-induced functions. Alternatively, it might rescue those cells the receptors of which have been damaged by somatic mutation. Less is known about the occurrence of similar mechanisms in T cells. Here we show that mature T cells with defective TCR surface expression can express RAG genes and are capable of initiating secondary V(D)J rearrangements. The possibility that a cell rescue mechanism based on the generation of a novel Ag receptor might be active in peripheral T cells is envisaged.     

The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors

T cells express either of the two forms of antigen-specific receptors, the alpha/beta and gamma/delta heterodimers. Their structure closely resembles that of immunoglobulins, and the variable part of the receptor molecule is created by somatic assembly of variable, diversity, and joining regions. The genetic structure of T cell receptor (TCR) genes and their rearrangement in T cell development have been elucidated in great detail in recent years. The human genes for the gamma and beta subunits are located on the short and long arms of chromosome 7, respectively, whereas the delta- and alpha-chain genes are located in tandem on the centromeric half of the long arm of chromosome 14. Expression of either alpha/beta or gamma/delta TCR complexes on T cells in the developing thymus is likely to proceed in an ordered fashion and results in the appearance of distinct T cell subpopulations. The process of DNA rearrangements required for the generation of functional variable region genes also predisposes lymphoid cells to aberrant DNA rearrangements, which can be detected as chromosomal abnormalities such as translocations and inversions. Molecular analysis of such aberrant rearrangements has shown that rearranging loci are fused to loci unrelated to antigen receptor genes. Furthermore, the breakpoint structures represent nonproductive intermediates in the hierarchy of physiological rearrangements. Accordingly, T cell tumors arising early in T cell development often carry chromosomal abnormalities involving the delta-chain locus, whereas tumors generated later in T cell development tend to show aberrations in the alpha-chain gene. This pattern seems to reflect the stage-specific accessibility of TCR loci for rearrangement by the recombinase machinery. This enzyme is guided by specific recombination signals that can sometimes also be found at the site of breakage on the participating locus in chromosomal abnormalities. Although some features of the mechanism of aberrant rearrangements are known, their biological consequences are less well understood. However, molecular analysis of the mechanism of chromosomal aberrations in T cell tumors suggests that their biological consequences may vary. Firm evidence for the pathogenic significance is missing for most of these lesions. This provides a challenge to molecular immunology to determine how chromosomal abnormalities are involved in tumor pathogenesis.     

The evolution of vertebrate antigen receptors: a phylogenetic approach

Classical T cells, those with alpha beta T-cell receptors (TCRs), are an important component of the dominant paradigm for self-nonself immune recognition in vertebrates. alpha beta T cells recognize foreign peptide antigens when they are bound to MHC molecules on the surfaces of antigen-presenting cells. gamma delta T cells bear a similar receptor, and it is often assumed that these T cells also require specialized antigen-presenting molecules for immune recognition, which we term "indirect antigen recognition." B-cell receptors, or immunoglobulins, bind directly to antigens without the help of a specialized antigen-presenting molecule. Phylogenetically, it has been assumed that T-cell receptors and the genes that encode them are a monophyletic group, and that "indirect" antigen recognition evolved before the split into two types of TCR. Recently, however, it has been proposed that gamma delta-TCRs bind directly to antigens, as do immunoglobulins (Ig's). This calls into question the null hypothesis that indirect antigen recognition is a common characteristic of TCRs and, by extension, the hypothesis that all TCR gene sequences form a monophyletic group. To determine whether alternative explanations for antigen recognition and other historical relationships among TCR genes might be possible, we performed phylogenetic analyses on amino acid sequences of the constant and variable regions which encode the basic subunits of TCR and Ig molecules. We used both maximum-parsimony and genetic distance-based methods and could find no strong support for the hypothesis of TCR monophyly. Analyses of the constant region suggest that TCR gamma or delta sequences are the most ancient, implying that the ancestral immune cell was like a modern gamma delta T cell. From this gamma delta-like ancestor arose alpha beta T cells and B cells, implying that indirect antigen recognition is indeed a derived property of alpha beta-TCRs. Analyses of the variable regions are complicated by strong selection on antigen-binding sequences, but imply that direct antigen binding is the ancestral condition.     

Analysis of T cell receptor transcripts using the polymerase chain reaction

The immune system is composed of two major types of lymphocytes, called B and T cells, that recognize foreign antigens. Recognition of antigens is accomplished through the generation of a large repertoire of different cell surface receptors, called immunoglobulins (Igs) on B cells and T cell receptors (TCRs) on T cells. The elucidation of Ig structure and molecular genetics preceded that of the TCR because of the greater abundance of Ig protein and mRNA. Although studies of TCRs have recently shed light on many of the issues of T cell recognition, the process of examining TCR gene structure has been tedious. Such analyses are also difficult because of the time required for the production, maintenance, and culturing of T cell clones. This report describes several strategies that use the polymerase chain reaction (PCR) to analyze very rapidly the structure of TCRs. Specific manipulations of the amplified material are discussed, as are the advantages of using the PCR to study TCR diversity.     

Quaternary structure and assembly process of the T cell receptor.

The human immune system contains T and B lymphocytes which respond in an antigen-specific manner to foreign antigens. These foreign antigens are recognized by multimeric receptors expressed on the cell surface of T and B lymphocytes. The subunits that make up the T and B cell receptor complexes have been identified, but their stoichiometries and positions in the complex remain to be resolved. Elucidation of the quaternary structures is necessary to understand the molecular basis of signal transduction events which follow antigen recognition and will contribute to the design of drugs that can modulate T and B cell responses. Here, I will discuss recent insights into the quaternary structures of the TCR and BCR and the striking similarities between the two, both in the structures of the subunits and in the overall quaternary structures. In addition, the intracellular assembly processes of these receptor complexes will be discussed, as well as the recent realization that these processes appear to be mediated by house-keeping proteins that transiently bind to partial TCR and BCR complexes. Similar to the role of BiP which mediates assembly processes of B cell immunoglobulins, a recently identified intracellular protein of 90 kD, called IP90, appears to play a role in TCR and BCR assembly processes. Analyses of the IP90 protein might contribute not only insight into the folding and assembly processes in lymphocytes, but also into those of newly synthesized proteins in many different cell types.     

Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.     

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.     

A concept of immune regulation of somatic cell differentiation

On the basis of several lines of experimental evidence a hypothesis is advanced on autoimmune regulation of somatic cell differentiation in an immunologically mature organism ("self-anti-self" hypothesis of differentiation). There are supposed to be clones of lymphocytes interacting via their antigen-recognizing receptors with autologous differentiation antigens on various target cells. This interaction would modify the genetically determined rate of cell differentiation. Some implications of the hypothesis are discussed in relation to immunological memory, tolerance etc. In particular, the new concept might imply similarity (or identity) of the genes coding for autologous differentiation antigens and those responsible for the idiotypes of antigen-recognizing lymphocyte receptors.     

Flanking V and J sequences of complementary determining region 3 of T cell receptor (TCR) δ1 (CDR3δ1) determine the structure and function of TCRγ4δ1

The γδ T cell receptor (TCR) differs from immunoglobulin and αβ TCR in its overall binding mode. In human, genes δ1, δ2, and δ3 are used for TCRδ chains. Previously, we have studied antigen binding determinants of TCRδ2 derived from dominant γδ T cells residing in peripheral blood. In this study we have investigated the critical determinants for antigen recognition and TCR function in TCRδ1 originated from gastric tumor-infiltrating γδ T lymphocytes using three independent experimental strategies including complementary determining region 3 (CDR3) of TCRδ1 (CDR3δ1)-peptide mediated binding, CDR3δ1-grafted TCR fusion protein-mediated binding, and TCRγ4δ1- and mutant-expressing cell-mediated binding. All three approaches consistently showed that the conserved flanking V and J sequences but not the diverse D segment in CDR3δ1 determine the antigen binding. Most importantly, we found that mutations in the V and J regions of CDR3δ1 also abolish the assembly of TCR and TCR-CD3 complexes in TCRγ4δ1-transduced J.RT3-T3.5 cells. Together with our previous studies on CDR3δ2 binding, our finding suggests that both human TCRδ1 and TCRδ2 recognize antigen predominately via flanking V and J regions. These results indicate that TCRγδ recognizes antigens using conserved parts in their CDR3, which provides an explanation for a diverse repertoire of γδTCRs only recognizing a limited number of antigens.     

Circulating CD4+ T lymphocytes with intracellular but no surface CD3 antigen in five of seven patients consecutively diagnosed with angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) accounts for less than 1% of all lymphatic malignancies. Oligoclonality or monoclonality for any of the T-cell receptor (TCR) chain genes can be demonstrated in the majority of the cases. During systematic screening for the presence of circulating lymphocytes with atypical coexpression of differentiation antigens in patients with T-cell lymphomas, we have discovered a minor population (accounting for 0.2% to 10.% of all lymphocytes) of atypical lymphocytes in the blood of five of seven patients consecutively diagnosed in 1997/1998 by lymph node histology to have AITL. The major distinguishing feature of these cells consists of the lack of the surface expression of the CD3 antigen, but not of the intracellular expression. These cells express the T-cell antigens CD2 and CD5 on their surface, but not CD7, and they express CD4 and CD45 at numbers of molecules per cell typical for T lymphocytes. Gene scan analyses for the TCR gamma chain revealed oligoclonality of these flow-sorted cells in one patient and monoclonality in two patients, the same patterns of TCR gamma chain gene as determined processing the respective diagnostic lymph nodes. Circulating CD4-expressing T lymphocytes with exclusively cytoplasmic expression of CD3 appear to represent the malignant population in patients with histologically diagnosed AITL.     

Antigen specificity of semi-invariant CD1d-restricted T cell receptors: the best of both worlds?

T lymphocytes are characterized by the use of structurally diverse TCR. The discovery of subsets of canonical T cells that have structurally homogeneous TCR presents an enigma: What antigens do these T cells recognize, and how does their antigen specificity relate to their functions? One subset of canonical T cells is restricted by CD1d, a non-classical antigen presenting molecule that presents lipids and glycolipids. Canonical CD1d-restricted T cells have semi-invariant TCR consisting of an invariantly rearranged TCR alpha chain, paired with diversely rearranged TCR beta chains. Most respond strongly to the unusual glycolipid alpha-galactosylceramide (alpha-GalCer), and can also respond to cellular antigens presented by CD1d. Mounting evidence indicates that alpha-GalCer responsive T cells are heterogeneous in their reactivities to cellular antigens, suggesting that an individual semi-invariant TCR may be capable of recognizing more than one ligand. Recent crystal structures of CD1b molecules with three different bound lipids indicate that the antigenic features of lipids may be localized over a smaller area than those of peptides, and that the positioning of the polar head group can vary substantially. A model that explains how CD1d-restricted T cells could possess both conserved and heterogeneous antigen specificities, is that different lipid antigens may interact with distinct areas of a TCR due to differences in the positioning of the polar head group. Hence, canonical CD1d-restricted TCR could recognize conserved antigens via the invariant TCR alpha chain, and have diverse antigen specificities that are conferred by their individual TCR beta chains.     

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.     

Immune Related Genes Underpin the Evolution of Adaptive Immunity in Jawless Vertebrates

The study of immune related genes in lampreys and hagfish provides a unique perspective on the evolutionary genetic underpinnings of adaptive immunity and the evolution of vertebrate genomes. Separated from their jawed cousins at the stem of the vertebrate lineage, these jawless vertebrates have many of the gene families and gene regulatory networks associated with the defining morphological and physiological features of vertebrates. These include genes vital for innate immunity, inflammation, wound healing, protein degradation, and the development, signaling and trafficking of lymphocytes. Jawless vertebrates recognize antigen by using leucine-rich repeat (LRR) based variable lymphocyte receptors (VLRs), which are very different from the immunoglobulin (Ig) based T cell receptor (TCR) and B cell receptor (BCR) used for antigen recognition by jawed vertebrates. The somatically constructed VLR genes are expressed in monoallelic fashion by T-like and B-like lymphocytes. Jawless and jawed vertebrates thus share many of the genes that provide the molecular infrastructure and physiological context for adaptive immune responses, yet use entirely different genes and mechanisms of combinatorial assembly to generate diverse repertoires of antigen recognition receptors.     

A Quantitative Theory of Affinity-driven T Cell Repertoire Selection

Binding of the T cell antigen receptor (TCR) to peptides presented on molecules encoded by major histocompatibility complex (MHC) genes is the key event driving T cell development and activation. Selection of the T cell repertoire in the thymus involves two steps. First, positive selection promotes the survival of cells binding thymic self-MHC-peptide complexes with sufficient affinity. The resulting repertoire is self-MHC restricted: it recognizes foreign peptides presented on self, but not foreign MHC. Second, negative selection deletes cells which may be potentially harmful because their receptors interact with self-MHC-peptide complexes with too high an affinity. The mature repertoire is also highly alloreactive: a large fraction of T cells respond to tissues harboring foreign MHC. We derive mathematical expressions giving the frequency of alloreactivity, the level of self-MHC restriction, and the fraction of the repertoire activated by a foreign peptide, as a function of the parameters driving the generation and selection of the repertoire: self-MHC and self-peptide diversity, the stringencies of positive and negative selection, and the number of peptide and MHC polymorphic residues that contribute to T cell receptor binding. Although the model is based on a simplified digit string representation of receptors, all the parameters but one relate directly to experimentally determined quantities. The only parameter without a biological counterpart has no effect on the model's behavior besides a trivial and easily preventable discretization effect. We further analyse the role of the MHC and peptide contribution to TCR binding, and find that their relative, rather than absolute value, is important in shaping the mature repertoire. This result makes it possible to adopt different physical interpretations for the digit string formalism. We also find that the alloreactivity level can be inferred directly from data on the stringency of selection, and that, in agreement with recent experiments, it is not affected by thymic selection.     

Enhanced receptor expression and in vitro effector function of a murine-human hybrid MART-1-reactive T cell receptor following a rapid expansion

Peripheral blood lymphocytes (PBL) genetically modified to express T cell receptors (TCR) specific to known melanoma antigens, such as melanoma antigen recognized by T cells-1 (MART-1), and gp100 can elicit objective tumor regression when administered to patients with metastatic melanoma. It has also been demonstrated that modifications within the constant regions of a fully human TCR can enhance surface expression and stability without altering antigen specificity. In this study, we evaluated the substitution of murine constant regions for their human counterpart within the DMF5 MART-1-specific TCR. Unlike previous studies, all modified TCRs were inserted into retroviral vectors and analyzed for expression and function following a clinical transduction protocol. PBL were transduced with retroviral supernatant generated from stable packaging lines encoding melanoma-specific TCRs. This protocol resulted in high levels of antigen-specific T cells without the need for additional peptide stimulation and selection. Both the human and murinized TCR efficiently transduced PBL; however, the murinized TCR exhibited significantly higher tetramer binding, mean fluorescence intensity, as well as, increased in vitro effector function following our clinical transduction and expansion protocol. Additional TCR modifications including insertion of a second disulfide bond or the linker modifications evaluated herein did not significantly enhance TCR expression or subsequent in vitro effector function. We conclude that the substitution of a human constant region with a murine constant region was sufficient to increase receptor expression and tetramer binding as well as antitumor activity of the DMF5 TCR and could be a tool to augment other antigen-specific TCR.     

Treating cancer with genetically engineered T cells

Administration of ex vivo cultured, naturally occurring tumor-infiltrating lymphocytes (TILs) has been shown to mediate durable regression of melanoma tumors. However, the generation of TILs is not possible in all patients and there has been limited success in generating TIL in other cancers. Advances in genetic engineering have overcome these limitations by introducing tumor-antigen-targeting receptors into human T lymphocytes. Physicians can now genetically engineer lymphocytes to express highly active T-cell receptors (TCRs) or chimeric antigen receptors (CARs) targeting a variety of tumor antigens expressed in cancer patients. In this review, we discuss the development of TCR and CAR gene transfer technology and the expansion of these therapies into different cancers with the recent demonstration of the clinical efficacy of these treatments.