Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.     

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

Inefficient positive selection of T cells directed by haematopoietic cells.

Intrathymic differentiation of alpha beta TCR+ T cells depends on positive selection of CD4+CD8+ thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows the maturation of only those T cells capable of restricted antigen recognition in the context of the hosts' MHC alleles. Studies of normal or T-cell receptor-transgenic mice engrafted with MHC-different bone marrow or thymuses support the conclusion that positive selection is directed by MHC molecules expressed on non-haematopoietic cells, presumably thymic epithelial cells. Here we, present contrary evidence that class I MHC molecules expressed by haematopoietic cell types direct positive selection of CD8+ T cells, though at a reduced rate compared with positive selection directed by thymic epithelial cells. The identity of cell types that direct positive selection bears directly on mechanistic models of the process, including the idea that thymic epithelial cell MHC molecules uniquely present specialized peptides that mediate positive selection, and the notion that thymic epithelial cells express unique differentiation-inducing cell surface molecules.     

Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.     

MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8.

Interactions between major histocompatibility complex (MHC) molecules and the CD4 or CD8 coreceptors have a major role in intrathymic T-cell selection. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay, and a binding site for CD8 on class I has been identified. Here we demonstrate that a region of the MHC class II beta-chain beta 2 domain, structurally analogous to the CD8-binding loop in the MHC class I alpha 3 domain, is critical for function with both mouse and human CD4.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Major histocompatibility complex-linked specificity of gamma delta receptor-bearing T lymphocytes

Several recent studies have identified a distinct subset of CD3(T3)+CD4-CD8-T lymphocytes that express a CD3-associated heterodimer made up of the protein encoded by the T-cell receptor (TCR) gamma-gene and a second glycoprotein termed TCR delta (refs 1-4). TCR gamma delta is expressed on CD3+ thymocytes during fetal ontogeny before the appearance of TCR alpha-beta (alpha beta) (refs 5-7), on CD3+CD4-CD8- adult thymocytes, and on a subset (1-10%) of CD3+ cells in adult peripheral lymphoid organs and the peripheral blood. TCR gamma delta-expressing T cells probably represent a distinct mature T-cell lineage with the capacity to proliferate in response to receptor-mediated signals, and to display non-major histocompatibility complex (MHC)-restricted cytolysis. Critical to understanding the function of this T-cell subset is the identification of the ligand(s) recognized by TCR gamma delta. Here we describe an alloreactive CD3+CD4-CD8-TCR gamma delta-expressing, TCR alpha beta-negative, T-cell line that manifests MHC-linked recognition specificity for both proliferation and cytotoxicity. Our results suggest that T cells expressing TCR gamma delta are capable of self-non-self MHC discrimination and that they can undergo MHC-influenced selection during differentiation like TCR alpha beta-expressing T cells.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Positive selection of CD4-CD8+ T cells in the thymus of normal mice

The diversification of the repertoire of T-cell antigen receptor (TCR) specificities is influenced by at least two selection processes which occur in the thymus. One of these, termed 'negative selection', is required to install a state of tolerance to self-antigens in the T-cell repertoire and is often achieved by clonal deletion. The second type of selection operating in the thymus results in preferential differentiation of T cells that have restriction specificity for thymic major histocompatibility complex glycoproteins, but the mechanisms leading to this selective process are not yet clear. One model used to describe this 'positive selection' proposes that only those T cells with sufficient avidity for the MHC glycoproteins expressed in the thymus are allowed to acquire functional competence. Here we directly investigate the generation of TCR specificities by following the fate of developing V beta 17+ CD4-CD8+ T cells under conditions where one of the main class I-MHC molecules, either H-2K or H-2D, was specifically blocked by in vitro monoclonal antibody treatment. The results show that development of V beta 17+ CD4-CD8+ T cells in the SJL H-2s mouse strain is selectively abrogated by blocking class I-Ks molecules but is unaffected by blocking class I-Ds molecules. These data directly demonstrate that generation of CD4-CD8+ T cells expressing a particular TCR V beta segment can be correlated with the expression of a particular class I-MHC molecule, thereby providing evidence for positive selection.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.     

Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4+8+ and CD4+8- T cells, but have no mature CD4-8+ T cells and are defective in CD4-8+ T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta+ CD4-8+ T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

CD1b restricts the response of human CD4-8- T lymphocytes to a microbial antigen.

Molecules encoded by the human CD1 locus on chromosome 1 (ref. 33) are recognized by selected CD4-8- T-cell clones expressing either alpha beta or gamma delta T-cell antigen receptors. The known structural resemblance of CD1 molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHC) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that CD1 may represent a family of antigen-presenting molecules separate from those encoded in the MHC. Here we report that the proliferative and cytotoxic responses of human CD4-8- alpha beta TCR+ T cells specific for Mycobacterium tuberculosis can be restricted by CD1b, one of the four identified protein products of the CD1 locus. The responses of these T cells to M. tuberculosis seemed not to involve MHC encoded molecules, but were absolutely dependent on the expression of CD1b by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class II-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of CD1 molecules and suggest that the CD1 family plays a role in cell-mediated immunity to microbial pathogens.     

Progression of autoimmune diabetes driven by avidity maturation of a T-cell population

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the 'avidity maturation' of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2Kd class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide-MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.