The many important facets of T-cell repertoire diversity

In the thymus, a diverse and polymorphic T-cell repertoire is generated by random recombination of discrete T-cell receptor (TCR)-alphabeta gene segments. This repertoire is then shaped by intrathymic selection events to generate a peripheral T-cell pool of self-MHC restricted, non-autoaggressive T cells. It has long been postulated that some optimal level of TCR diversity allows efficient protection against pathogens. This article focuses on several recent advances that address the required diversity for the generation of an optimal immune response.     

Molecular and genetic parameters defining T-cell clonal selection

Clonal selection of T cells occurs in the thymus and is responsible for generating a useful and functional repertoire of T cells. Aberrations in clonal selection result in altered T-cell homeostasis in the secondary lymphoid organs ranging from an absence of T cells to an overabundance of autoreactive T cells. The advent of new technologies facilitating the manipulation of the mouse genome has helped refine our understanding of the molecular and genetic pathways involved in clonal selection and has also revealed a high degree of complexity. Herein, we attempt to review recent advances in thymic selection processes, achieved mostly through genetic manipulations.     

Sonic hedgehog signalling in T-cell development and activation

The production of mature functional T cells in the thymus requires signals from the thymic epithelium. Here, we review recent experiments showing that one way in which the epithelium controls the production of mature T cells is by the secretion of sonic hedgehog (SHH). We consider the increasing evidence that SHH-induced signalling is not only important for the differentiation and proliferation of early thymocyte progenitors, but also for modulating T-cell receptor signalling during repertoire selection, with implications for positive selection, CD4 versus CD8 lineage commitment, and clonal deletion of autoreactive cells. We also review the influence of hedgehog signalling in peripheral T-cell activation.     

Generating intrathymic microenvironments to establish T-cell tolerance

Alphabeta T cells pass through a series of lymphoid tissue stromal microenvironments to acquire self tolerance and functional competence. In the thymus, positive selection of the developing T-cell receptor repertoire occurs in the cortex, whereas events in the medulla purge the system of self reactivity. T cells that survive are exported to secondary lymphoid organs where they direct first primary and then memory immune responses. This Review focuses on the microenvironments that nurture T-cell development rather than on T cells themselves. We summarize current knowledge on the formation of thymic epithelial-cell microenvironments, and highlight similarities between the environments that produce T cells and those that select and maintain them during immune responses.     

The molecular basis for public T-cell responses?

Public T-cell responses, in which T cells bearing identical T-cell receptors (TCRs) are observed to dominate the response to the same antigenic epitope in multiple individuals, have long been a focus of immune T-cell repertoire studies. However, the mechanism that enables the survival of a specific TCR from the diverse repertoire produced in the thymus through to its involvement in a public immune response remains unclear. In this Opinion article, we propose that the frequency of production of T cells bearing different TCRs during recombination has an important role in the sharing of TCRs in an immune response, with variable levels of 'convergent recombination' driving production frequencies.     

T-Cell Tolerance: Central and Peripheral

Somatic recombination of TCR genes in immature thymocytes results in some cells with useful TCR specificities, but also many with useless or potentially self-reactive specificities. Thus thymic selection mechanisms operate to shape the T-cell repertoire. Thymocytes that have a TCR with low affinity for self-peptide–MHC complexes are positively selected to further differentiate and function in adaptive immunity, whereas useless ones die by neglect. Clonal deletion and clonal diversion (Treg differentiation) are the major processes in the thymus that eliminate or control self-reactive T cells. Although these processes are thought to be efficient, they fail to control self-reactivity in all circumstances. Thus, peripheral tolerance processes exist wherein self-reactive T cells become functionally unresponsive (anergy) or are deleted after encountering self-antigens outside of the thymus. Recent advances in mechanistic studies of central and peripheral T-cell tolerance are promoting the development of therapeutic strategies to treat autoimmune disease and cancer and improve transplantation outcome.T cells recognize pathogen fragments in the context of surface MHC molecules on host cells. As such, they have the potential to do enormous damage to healthy tissue when they are not appropriately directed, that is, when they respond to self-antigens as opposed to foreign antigens. T lymphocyte tolerance is particularly important, because it impacts B-cell tolerance as well, through the requirement of T cell help in antibody responses. Thus, failure of T-cell tolerance can lead to many different autoimmune diseases. The tolerance of T cells begins as soon as a T-cell receptor is formed and expressed on the cell surface of a T-cell progenitor in the thymus. Tolerance mechanisms that operate in the thymus before the maturation and circulation of T cells are referred to as “central tolerance.” However, not all antigens that T cells need to be tolerant of are expressed in the thymus, and thus central tolerance mechanisms alone are insufficient. Fortunately, additional tolerance mechanisms exist that restrain the numbers and or function of T cells that are reactive to developmental or food antigens, which are not thymically expressed. These mechanisms act on mature circulating T cells and are referred to as “peripheral tolerance.”     

The role of polymorphic amino acids of the MHC molecule in the selection of the T cell repertoire

Allelic variants of MHC molecules expressed on cells of the thymus affect the selection and the specificity of the T cell repertoire. The selection is based on either the direct recognition by the TCR of the MHC molecules, or the recognition of a complex determinant formed by self-peptides bound to MHC molecules. In an analysis of the T cell repertoire in bone marrow chimeras that express allelic forms of MHC class II molecules in the thymus epithelium, we find that amino acid substitutions that are predicted to affect peptide binding influence the selection of the T cell repertoire during thymic selection.     

Thymic function and allogeneic T-cell responses in stem-cell transplantation

The thymus is the primary site of T-cell production early in life, and has now been shown to continue to function in both healthy and immunocompromised individuals late into life. Positive and negative selection occurring in the thymus are two of the most important processes that govern the development and specificity of peripheral T cells, including their restriction to self HLA and their ability to respond in an alloreactive manner. In the chimeric state that follows successful allogeneic stem-cell transplants, the specificity of alloreactive cells may be governed by either host- or recipient-derived cellular elements, as well as maturing lymphoid cells, which are, in turn, derived from donor stem cells or host cells surviving transplant conditioning. The ability to measure recent thymic emigrants via the detection of T-cell receptor excision circles has facilitated studies of thymic function in immunodeficient individuals, including HIV-1 infected subjects and recipients of autologous or allogeneic stem-cell transplant (SCT). These studies have now demonstrated that thymic function is likely to play a beneficial role in immune reconstitution in these settings, but have yet to clearly demonstrate what clinical variables are the most important determinants of thymic persistence. It is also not yet clear how much the degree of thymic function following allogeneic SCT influences the alloreactive T-cell repertoire, although studies in animal models and early clinical studies suggest that GvHD results in thymic injury and dysfunction. Future studies will further clarify how thymic function shapes the repertoire of T cells that mediate alloreactivity, as well as protective pathogen-specific immune responses, following SCT. Finally, these studies will also demonstrate whether endogenous mediators of thymic function could be selectively applied to regulate post-SCT thymic function and alloreactivity.     

Association of MHC and rheumatoid arthritis. Association of RA with HLA-DR4: the role of repertoire selection

Most patients with rheumatoid arthritis (RA) express HLA-DR4, HLA-DR1 or HLA-DR10. These alleles share a common amino acid motif in their third hypervariable regions: the shared epitope. In normals and patients with RA, HLA-DR genes exert a major influence on the CD4 alpha beta T-cell repertoire, as shown by studies of AV and BV gene usage and by BV BJ gene usage by peripheral blood CD4 alpha beta T-cells. However, the rheumatoid T-cell repertoire is not entirely under HLA-DR influence, as demonstrated by discrepancies in VB JB gene usage between identical twins discordant for RA and by contraction of the CD4 alpha beta T-cell repertoire in RA patients. Shared epitope positive HLA-DR alleles may shape the T-cell repertoire by presenting self peptides to CD4 T cells in the thymus. Peptides processed from HLA-DR molecules and encompassing the shared epitope may also be presented by HLA-DQ and select CD4 alpha beta T cells in the thymus. Thus, shared epitope-positive alleles impose a frame on the T-cell repertoire. This predisposing frame is further modified (by unknown factors) to obtain the contracted rheumatoid repertoire.     

Cell growth and gene rearrangement signals during the development of T lymphocytes within the thymus

The thymus provides signals that control the proliferation and differentiation of T lymphocytes and select the repertoire of T-cell specificities. Antibodies to CD3 molecules inhibit full rearrangement of T-cell receptor beta chain genes in organ cultures of early embryo mouse thymus. Whether this effect is mediated through gamma delta CD3 expressing cells, which are present in small numbers at this stage, or through low amounts of CD3 on alpha beta precursor cells is unclear. A requirement for special gene rearrangement signals within the thymus is supported also by the observations that growth factors such as IL-2 and IL-4, although stimulating proliferation of precursor cells removed from the thymus, do not induce full T-cell receptor gene rearrangements. Recent studies show that newly formed thymic lymphocytes expressing alpha beta CD3 receptors are targets for negative selection (deletion) as a means of removing autoreactive cells. Signalling to immature thymocytes via the alpha beta CD3 complex induces the activation of endogenous endonucleases that cleave DNA into oligonucleosomal fragments. We suggest that the activation of this mechanism is the means by which autoreactive cells are removed.     

The thymus and central tolerance

T-cell differentiation in the thymus generates a peripheral repertoire of mature T cells that mounts strong responses to foreign antigens but is largely unresponsive to self-antigens. This state of specific immunological tolerance to self-components involves both central and peripheral mechanisms. Here we review the process whereby many T cells with potential reactivity for self-antigens are eliminated in the thymus during early T-cell differentiation. This process of central tolerance (negative selection) reflects apoptosis and is a consequence of immature T cells receiving strong intracellular signalling through T-cell receptor (TCR) recognition of peptides bound to major histocompatibility complex (MHC) molecules. Central tolerance occurs mainly in the medullary region of the thymus and depends upon contact with peptide-MHC complexes expressed on bone-marrow-derived antigen-presenting cells (APCs); whether tolerance also occurs in the cortex is still controversial. Tolerance induction requires a combination of TCR ligation and co-stimulatory signals. Co-stimulation reflects interaction between complementary molecules on T cells and APCs and probably involves multiple molecules acting in consort, which may account for why deletion of individual molecules with known or potential co-stimulatory function has little or no effect on central tolerance. The range of self-antigens that induce central tolerance is considerable and, via low-level expression in the thymus, may also include tissue-specific antigens; central tolerance to these latter antigens, however, is likely to be limited to high-affinity T cells, leaving low-affinity cells to escape. Tolerance to alloantigens and the possibility of using central tolerance to promote acceptance of allografts are discussed.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

Theoretical considerations and probability models for the somatic development of the T-cell repertoire

A theory about the somatic development of the T-cell repertoire is described which explains the high proportion of alloreactive T lymphocytes as a side effect resulting from two biologically important selection processes. It is assumed that the T-cell receptors contain two binding sites X and Y similar to the two V regions of the B-cell receptors and immunoglobulin molecules, but with a different specificity repertoire as a result of the two selection processes. The first selection process is mediated by T-lineage inducer cells, which have already been postulated by Zinkernagel. They are believed to induce only cells with a binding site Y that can bind to major histocompatibility antigens (MHA) on the inducer cells. This selection forces the T-cell system to recognize preferentially cell-bound antigens; and it introduces under certain circumstances a high degree of self-MHA restriction and self-reactivity, and also a low degree of alloreactivity on the basis of cross-reactivity. The subsequent second selection process serves the purpose of self-tolerance induction. This process is expected under certain circumstances to result in a loss self-reactivity combined with a partial loss of self-restriction and selective enrichment of alloreactive T cells. Probability models are presented to illustrate how the two selection processes shape the T-cell repertoire and how the proportion of alloreactive cells is increased under special circumstances. It is possible that some T-cell subclasses are not affected by the second selection and maintain the primary repertoire. The theory is, in contrast to major competing theories, compatible with the assumption that the original V gene repertoires for the T-cell receptors are random.     

Association of MHC and rheumatoid arthritis: Association of RA with HLA-DR4 - the role of repertoire selection

Most patients with rheumatoid arthritis (RA) express HLA-DR4, HLA-DR1 or HLA-DR10. These alleles share a common amino acid motif in their third hypervariable regions: the shared epitope. In normals and patients with RA, HLA-DR genes exert a major influence on the CD4 αβ T-cell repertoire, as shown by studies of AV and BV gene usage and by BV BJ gene usage by peripheral blood CD4 αβ T-cells. However, the rheumatoid T-cell repertoire is not entirely under HLA-DR influence, as demonstrated by discrepancies in VB JB gene usage between identical twins discordant for RA and by contraction of the CD4 αβ T-cell repertoire in RA patients. Shared epitope positive HLA-DR alleles may shape the T-cell repertoire by presenting self peptides to CD4 T cells in the thymus. Peptides processed from HLA-DR molecules and encompassing the shared epitope may also be presented by HLA-DQ and select CD4 αβ T cells in the thymus. Thus, shared epitope-positive alleles impose a frame on the T-cell repertoire. This predisposing frame is further modified (by unknown factors) to obtain the contracted rheumatoid repertoire.     

The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Chimeric Thymus Rudiments: A Strategy for the Study of Tolerance Induction in Vitro

The techniques of fetal thymus organ culture have been widely utilized for the study of thymocyte differentiation under carefully controlled conditions. Recent results suggesting a role for dendritic cells (DC) in selection of a competent T-cell repertoire have prompted attempts to construct chimeric thymus rudiments in vitro. Here, we describe a novel approach based on the migratory properties of mature lymphoid DC. Purified DC from adult thymus or spleen were found to migrate into fetal thymus rudiments in culture and localize specifically within the medulla. This distribution closely mirrors the situation in vivo, underlining the physiological relevance of the resulting microenvironment. Recolonization was shown to be selective, excluding cell types not normally represented in the thymus. To assess the extent of repertoire selection in recolonized fetal thymi, chimeric rudiments in which I-E determinants were expressed exclusively on the surface of immigrant DC were constructed. The failure of such rudiments to recruit a population of CD4⁺8⁻Vβ6⁺ thymocytes, restricted to antigen recognition in the context of I-E, argued against a role for DC in positive selection, in contrast, the widespread deletion of potentially autoreactive CD4⁺8⁻Vβ17a⁺ cells suggested an active role for DC in negative selection of the T-cell repertoire. These conclusions are consistent with the findings of various in vivo studies, endorsing the suitability of such a model for the study of tolerance induction in vitro.     

Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.     

Journey through the thymus: stromal guides for T-cell development and selection

Lympho-stromal interactions in multiple microenvironments within the thymus have a crucial role in the regulation of T-cell development and selection. Recent studies have implicated that chemokines that are produced by thymic stromal cells have a pivotal role in positioning developing T cells within the thymus. In this Review, I discuss the importance of stroma-derived chemokines in guiding the traffic of developing thymocytes, with an emphasis on the processes of cortex-to-medulla migration and T-cell-repertoire selection, including central tolerance.     

Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire

We investigated the thymic and peripheral T-lymphocyte subsets in BALB/c mice undergoing acute or chronic Trypanosoma cruzi infection, in terms of expression of particular Vbeta rearrangements of the T-cell receptor. We first confirmed the severe depletion of CD4(+)CD8(+) thymocytes following acute T. cruzi infection. By contrast, the numbers of CD4(+)CD8(+) cells in subcutaneous lymph nodes increased up to 16 times. In subcutaneous lymph nodes, we found CD4(+)CD8(+) cells that expressed prohibited segments TCRVbeta5 and TCRVbeta12 (which are physiologically deleted in the thymus of BALB/c mice), as did some mature single-positive cells (CD4(+)CD8(-) and CD4(-)CD8(+)). In the thymus of infected animals, although higher numbers of immature cells bearing such Vbeta segments were seen, they were no longer detected in the mature single-positive stage, suggesting that negative selection occurs normally. We also found increased numbers of cells bearing the potentially autoreactive phenotype TCRVbeta5(+) and TCRVbeta12(+) in T-lymphocyte subsets from subcutaneous lymph nodes of T. cruzi chronically infected mice. In conclusion, our data indicate that immature T lymphocytes bearing prohibited TCRVbeta segments leave the thymus and gain the lymph nodes, where they further differentiate into mature CD4(+) or CD8(+) cells. Conjointly, these findings show changes in the shaping of the central and peripheral T-cell repertoire in both acute and chronic phases of murine T. cruzi infection. The release of potentially autoreactive T cells in the periphery of the immune system may contribute to the autoimmune process found in both murine and human Chagas' disease.